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What's the rationale behind freezing one's eggs if cells can be cloned?

What's the rationale behind freezing one's eggs if cells can be cloned?


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It seems to me there's a lot of discussions around the topic of somebody freezing her eggs.

What I don't understand though are the following:

  1. Why would you freeze your eggs when you know that the technology for transforming a somatic cell into an egg today exists, or doesn't it see here?

  2. Is it proven that freezing doesn't alter biological activity and hence not diminish the chance of giving birth to a malformed child?

To me it seems way easier and safer (and less expensive) to artificially produce female gametes than freezing them and, 10 years later, unfreeze and hope for them to be still biologically active.


Why Would You Freeze Your Eggs?

For 1, a woman may wish to freeze her eggs if she needs to delay childbirth. This page explores several reasons why, for example, "Women who want or need to delay childbearing in order to pursue educational, career or other personal goals.". Additionally, it explores some basic explanations of the process, if you are interested. If a woman has doubts about her fertility in 10 years time (using your final sentence as an example), such as if she is recently diagnosed with cancer, or needs to finish education but is ready to be committed to having a baby in more than 9 months' time, then she may decide to freeze her eggs for a time when she is more suited to have a baby, using a time when her body is more suited to producing eggs.

Does Freezing Have Adverse Effects?

As for your second question, the same page does mention successfully freezing an embryo for 10 years at -196 degrees Celsius, with no adverse effects on the eventual birth.

Regardless, exploring the risks reveals that the decision on whether the practice is seen as positive or negative is still on the fence - different parties argue for different opinions, as there appears to be no objective truth about proven adverse affects.

The risks described by this clinic are worth considering, albeit minimal compared to a risk of malformed children, however, it is still a popular choice, albeit put forward as 'experimental'. This study (2010, Rudick et al) showed how, in the US, cryopreservation was already being offered in 283 of 442 fertility clinics surveyed. 1/3 of the reasons for the choice was for women with cancer, and the other 2/3 was for women with 'advancing maternal age'.

The ACOG (American College of Obstetricians and Gynecologists), in 2013, stated that they do not endorse freezing of eggs 'for the sole purpose of circumventing reproductive aging in healthy women', however, the ASRM (American Society for Reproductive Medicine) lifted its label as being 'experimental' in 2012. The previously linked article describes a study conducted by the ASRM, which 'reviewed data from four randomized controlled trials and various observational studies that compared fertilization rates, embryo implantation rates and pregnancy rates of fresh eggs versus eggs that were frozen', and 'showed no increase in birth defects, developmental disorders or chromosomal abnormalities when in vitro fertilization cycles were conducted with frozen eggs, leading the society to declare the technique effective and safe'.

Conclusion

Your question is a slightly broad one, and can be answered by the subjective choice of the woman/women involved, however the ASRM still stresses that "we should proceed cautiously in using this as an elective technique", by making a clear line between 'need' and 'want'. However, there is no evidence to show that rates of abnormality are any higher using this method, but considering it is still a fairly new practice that is being marketed as a choice rather than a medical treatment, there is skepticism in the air around whether marketing scientific and medical designs as a mere 'choice' for older women will lead to this technology becoming better or worse.

Additionally, about your comment of it being 'less expensive', this website shows that the average cost of in vitro fertilization in the U.S. is currently about 10,000 - 20,000 USD, whereas the first website I linked (here) estimates the cost at about 10,000 USD to undergo an egg freezing cycle. Although, the prices will change from clinic to clinic. Regardless, the prices are still similar, and the choice lies in the woman choosing to pay and freeze her eggs if, for example, she has recently been diagnosed with cancer, or fears she will not be fertile in 5+ years time.

I hope this answer has been sufficient in answering your questions, and if you have any more questions, feel free to ask me :)

Sources:


Embryo Production

Lisette A. Maddison , . Wenbiao Chen , in Methods in Cell Biology , 2011

2 Screening for Gene-trap Events

Cross individual gene-trap injected fish to a wild-type fish.

Collect eggs from successful matings. i.

Hold the gene-trap fish in a separate small tank.

Screening for fluorescence 1.

At 24 hpf, 48 hpf, and 5 dpf. i.

Check for fluorescence on both red and yellow channels.

Keep embryos with fluorescent protein expression and raise them to maturity. Genomic DNA can be isolated from tailfin biopsies at 6 weeks of age for insert identification.

If more than 10 embryos with identical expression pattern are available from a single clutch, 1–3 embryos can be used for insert identification and the rest can be raised to maturity.


Human Genetic Engineering

Genetic technology harbors the potential to change the human species forever. The soon to be completed Human Genome Project will empower genetic scientists with a human biological instruction book. The genes in all our cells contain the code for proteins that provide the structure and function to all our tissues and organs. Knowing this complete code will open new horizons for treating and perhaps curing diseases that have remained mysteries for millennia. But along with the commendable and compassionate use of genetic technology comes the specter of both shadowy purposes and malevolent aims.

For some, the potential for misuse is reason enough for closing the door completely--the benefits just aren’t worth the risks. In this article, I’d like to explore the application of genetic technology to human beings and apply biblical wisdom to the eventual ethical quagmires that are not very far away. In this section we’ll investigate the various ways humans can be engineered.

Since we have introduced foreign genes into the embryos of mice, cows, sheep, and pigs for years, there’s no technological reason to suggest that it can’t be done in humans too. Currently, there are two ways of pursuing gene transfer. One is simply to attempt to alleviate the symptoms of a genetic disease. This entails gene therapy, attempting to transfer the normal gene into only those tissues most affected by the disease. For instance, bronchial infections are the major cause of early death for patients with cystic fibrosis (CF). The lungs of CF patients produce thick mucus that provides a great growth medium for bacteria and viruses. If the normal gene can be inserted in to the cells of the lungs, perhaps both the quality and quantity of their life can be enhanced. But this is not a complete cure and they will still pass the CF gene on to their children.

In order to cure a genetic illness, the defective gene must be replaced throughout the body. If the genetic defect is detected in an early embryo, it’s possible to add the gene at this stage, allowing the normal gene to be present in all tissues including reproductive tissues. This technique has been used to add foreign genes to mice, sheep, pigs, and cows.

However, at present, no laboratory is known to be attempting this well-developed technology in humans. Princeton molecular biologist Lee Silver offers two reasons. 1 First, even in animals, it only works 50% of the time. Second, even when successful, about 5% of the time, the new gene gets placed in the middle of an existing gene, creating a new mutation. Currently these odds are not acceptable to scientists and especially potential clients hoping for genetic engineering of their offspring. But these are only problems of technique. It’s reasonable to assume that these difficulties can be overcome with further research.

Should genetic engineering be used for curing genetic diseases?

The primary use for human genetic engineering concerns the curing of genetic disease. But even this should be approached cautiously. Certainly within a Christian worldview, relieving suffering wherever possible is to walk in Jesus’ footsteps. But what diseases? How far should our ability to interfere in life be allowed to go? So far gene therapy is primarily tested for debilitating and ultimately fatal diseases such as cystic fibrosis.

The first gene therapy trial in humans corrected a life-threatening immune disorder in a two-year-old girl who, now ten years later, is doing well. The gene therapy required dozens of applications but has saved the family from a $60,000 per year bill for necessary drug treatment without the gene therapy. 2 Recently, sixteen heart disease patients, who were literally waiting for death, received a solution containing copies of a gene that triggers blood vessel growth by injection straight into the heart. By growing new blood vessels around clogged arteries, all sixteen showed improvement and six were completely relieved of pain.

In each of these cases, gene therapy was performed as a last resort for a fatal condition. This seems to easily fall within the medical boundaries of seeking to cure while at the same time causing no harm. The problem will arise when gene therapy will be sought to alleviate a condition that is less than life-threatening and perhaps considered by some to simply be one of life’s inconveniences, such as a gene that may offer resistance to AIDS or may enhance memory. Such genes are known now and many are suggesting that these goals will and should be available for gene therapy.

The most troublesome aspect of gene therapy has been determining the best method of delivering the gene to the right cells and enticing them to incorporate the gene into the cell’s chromosomes. Most researchers have used crippled forms of viruses that naturally incorporate their genes into cells. The entire field of gene therapy was dealt a severe setback in September 1999 upon the death of Jesse Gelsinger who had undergone gene therapy for an inherited enzyme deficiency at the University of Pennsylvania. 3 Jesse apparently suffered a severe immune reaction and died four days after being injected with the engineered virus.

The same virus vector had been used safely in thousands of other trials, but in this case, after releasing stacks of clinical data and answering questions for two days, the researchers didn’t fully understand what had gone wrong. 4 Other institutions were also found to have failed to file immediate reports as required of serious adverse events in their trials, prompting a congressional review. 5 All this should indicate that the answers to the technical problems of gene therapy have not been answered and progress will be slowed as guidelines and reporting procedures are studied and reevaluated.

Will correcting my genetic problem, prevent it in my descendants?

The simple answer is no, at least for the foreseeable future. Gene therapy currently targets existing tissue in a existing child or adult. This may alleviate or eliminate symptoms in that individual, but will not affect future children. To accomplish a correction for future generations, gene therapy would need to target the germ cells, the sperm and egg. This poses numerous technical problems at the present time. There is also a very real concern about making genetic decisions for future generations without their consent.

Some would seek to get around these difficulties by performing gene therapy in early embryos before tissue differentiation has taken place. This would allow the new gene to be incorporated into all tissues, including reproductive organs. However, this process does nothing to alleviate the condition of those already suffering from genetic disease. Also, as mentioned earlier this week, this procedure would put embryos at unacceptable risk due to the inherent rate of failure and potential damage to the embryo.

Another way to affect germ line gene therapy would involve a combination of gene therapy and cloning. 6 An embryo, fertilized in vitro, from the sperm and egg of a couple at risk for sickle-cell anemia, for example, could be tested for the sickle-cell gene. If the embryo tests positive, cells could be removed from this early embryo and grown in culture. Then the normal hemoglobin gene would be added to these cultured cells.

If the technique for human cloning could be perfected, then one of these cells could be cloned to create a new individual. If the cloning were successful, the resulting baby would be an identical twin of the original embryo, only with the sickle-cell gene replaced with the normal hemoglobin gene. This would result in a normal healthy baby. Unfortunately, the initial embryo was sacrificed to allow the engineering of its identical twin, an ethically unacceptable trade-off.

So what we have seen, is that even human gene therapy is not a long-term solution, but a temporary and individual one. But even in condoning the use of gene therapy for therapeutic ends, we need to be careful that those for whom gene therapy is unavailable either for ethical or monetary reasons, don’t get pushed aside. It would be easy to shun those with uncorrected defects as less than desirable or even less than human. There is, indeed, much to think about.

Should genetic engineering be used to produce super-humans?

The possibility of someone or some government utilizing the new tools of genetic engineering to create a superior race of humans must at least be considered. We need to emphasize, however, that we simply do not know what genetic factors determine popularly desired traits such as athletic ability, intelligence, appearance and personality. For sure, each of these has a significant component that may be available for genetic manipulation, but it’s safe to say that our knowledge of each of these traits is in its infancy.

Even as knowledge of these areas grows, other genetic qualities may prevent their engineering. So far, few genes have only a single application in the body. Most genes are found to have multiple effects, sometimes in different tissues. Therefore, to engineer a gene for enhancement of a particular trait--say memory--may inadvertently cause increased susceptibility to drug addiction.

But what if in the next 50 to 100 years, many of these unknowns can be anticipated and engineering for advantageous traits becomes possible. What can we expect? Our concern is that without a redirection of the worldview of the culture, there will be a growing propensity to want to take over the evolution of the human species. The many people see it, we are simply upright, large-brained apes. There is no such thing as an independent mind. Our mind becomes simply a physical construct of the brain. While the brain is certainly complicated and our level of understanding of its intricate machinery grows daily, some hope that in the future we may comprehend enough to change who and what we are as a species in order to meet the future demands of survival.

Edward O. Wilson, a Harvard entomologist, believes that we will soon be faced with difficult genetic dilemmas. Because of expected advances in gene therapy, we will not only be able to eliminate or at least alleviate genetic disease, we may be able to enhance certain human abilities such as mathematics or verbal ability. He says, “Soon we must look deep within ourselves and decide what we wish to become.” 7 As early as 1978, Wilson reflected on our eventual need to “decide how human we wish to remain.” 8

Surprisingly, Wilson predicts that future generations will opt only for repair of disabling disease and stop short of genetic enhancements. His only rationale however, is a question. “Why should a species give up the defining core of its existence, built by millions of years of biological trial and error?” 9 Wilson is naively optimistic. There are loud voices already claiming that man can intentionally engineer our “evolutionary” future better than chance mutations and natural selection. The time to change the course of this slow train to destruction is now, not later.

Should I be able to determine the sex of my child?

Many of the questions surrounding the ethical use of genetic engineering practices are difficult to answer with a simple yes or no. This is one of them. The answer revolves around the method used to determine the sex selection and the timing of the selection itself.

For instance, if the sex of a fetus is determined and deemed undesirable, it can only be rectified by termination of the embryo or fetus, either in the lab or in the womb by abortion. There is every reason to prohibit this process. First, an innocent life has been sacrificed. The principle of the sanctity of human life demands that a new innocent life not be killed for any reason apart from saving the life of the mother. Second, even in this country where abortion is legal, one would hope that restrictions would be put in place to prevent the taking of a life simply because it’s the wrong sex.

However, procedures do exist that can separate sperm that carry the Y chromosome from those that carry the X chromosome. Eggs fertilized by sperm carrying the Y will be male, and eggs fertilized by sperm carrying the X will be female. If the sperm sample used to fertilize an egg has been selected for the Y chromosome, you simply increase the odds of having a boy (

90%) over a girl. So long as the couple is willing to accept either a boy or girl and will not discard the embryo or abort the baby if it’s the wrong sex, it’s difficult to say that such a procedure should be prohibited.

One reason to utilize this procedure is to reduce the risk of a sex-linked genetic disease. Color-blindness, hemophilia, and fragile X syndrome can be due to mutations on the X chromosome. Therefore, males (with only one X chromosome) are much more likely to suffer from these traits when either the mother is a carrier or the father is affected. (In females, the second X chromosome will usually carry the normal gene, masking the mutated gene on the other X chromosome.) Selecting for a girl by sperm selection greatly reduces the possibility of having a child with either of these genetic diseases. Again, it’s difficult to argue against the desire to reduce suffering when a life has not been forfeited.

But we must ask, is sex determination by sperm selection wise? A couple that already has a boy and simply wants a girl to balance their family, seems innocent enough. But why is this important? What fuels this desire? It’s dangerous to take more and more control over our lives and leave the sovereignty of God far behind. This isn’t a situation of life and death or even reducing suffering.

But while it may be difficult to find anything seriously wrong with sex selection, it’s also difficult to find anything good about it. Even when the purpose may be to avoid a sex-linked disease, we run the risk of communicating to others affected by these diseases that because they could have been avoided, their life is somehow less valuable. So while it may not be prudent to prohibit such practices, it certainly should not be approached casually either.

1 Lee Silver, Remaking Eden: Cloning and Beyond in a Brave New World, New York, NY: Avon Books, p. 230-231.

2 Leon Jaroff, Success stories, Time, 11 January 1999, p. 72-73.

3 Sally Lehrman, Virus treatment questioned after gene therapy death, Nature Vol. 401 (7 October 1999): 517-518.

4 Eliot Marshall, Gene therapy death prompts review of adenovirus vector, Science Vol. 286 (17 December 1999): 2244-2245.

5 Meredith Wadman, NIH under fire over gene-therapy trials, Nature Vol. 403 (20 January 1999): 237.

6 Steve Mirsky and John Rennie, What cloning means for gene therapy, Scientific American, June 1997, p. 122-123.

8 Edward Wilson, On Human Nature, Cambridge, Mass.: Harvard University Press, p. 6.


A recent poll on the Canadian Horse Journal website asked the question: Should equines be cloned?

Some 83 percent of respondents said no, not until more research has been done 15 percent said maybe, in special situations with strict parameters just two percent said yes, and that registration of clones should be allowed.

Once the darling concept of science fiction writers, cloning trotted onto the world stage on February 22, 1997 when it was announced that Dolly the sheep, a ewe cloned at the Roslin Institute in Midlothian, Scotland, had been born on July 5, 1996. Dolly was the first mammal to have been successfully cloned from an adult cell. She lived at the Institute until her death in 2003.

Dolly kicked the scientific barn doors down to open the way for all manner of cloned mammals. Along came “twins” of cats, rats, deer, cattle, fruit flies, rabbits, and others. Then, on May 4, 2003, the first equine and first mule, Idaho Gem, was born at the University of Idaho. He was quickly followed by two cloned “siblings,” Utah Pioneer born on June 9, and Idaho Star born on July 27.

Idaho Gem came along just a couple of weeks before the birth of the first cloned horse, Prometea, a Haflinger foal born May 28, 2003, at the Laboratory of Reproductive Technology, Cremona, Italy. The first cloned horse in North America, Paris Texas, was produced by Texas A&M University in 2005.

In 2006, the premier barrel racing gelding, Scamper, was cloned and his “twin” stallion became the first cloned horse to stand at stud in the US In 2010, a Criollo horse was born in Argentina, the first horse clone produced in South America.

But what really is cloning, and is the cloned horse literally identical to the original animal?

“We take advantage of two things,” explains Dr. Katrin Hinrichs, professor and Patsy Link Chair in mare reproductive studies at the College of Veterinary Medicine & Biomedical Sciences, Texas A&M University. “One, the nucleus of every cell in the body has the same DNA which codes for how to produce the entire animal. Two, the oocyte (egg) is ready to make an embryo, packed full of everything that will be needed for that embryo for the first three to four days of development. The basic procedure is just a variation on what happens normally at fertilization. Normally, the egg has half of a complement of DNA and the sperm enters the egg and brings in the other half of the needed DNA. Then, with a complete set of DNA, the egg starts to develop into an embryo. In cloning, we remove the DNA from the egg then introduce a cell [from the donor horse] that carries a full complement of DNA. Then we signal the egg to start to develop into an embryo.”

A cloned embryo that has developed to the stage at which it can be successfully transferred to the uterus of a recipient mare. It takes about seven to eight days of culture to get to this stage, and it typically has 100 to 500 cells at this stage. Photo courtesy Texas A&M University

Hinrichs explains that, while there are no changes to the genetics (DNA) of the cloned horse, the genes in the clone could be used differently – turned on more, or less, than they were in the original. Therefore, to a varying extent, the clone will differ somewhat from the original.

“Sometimes the placenta does not work as well as it should,” she adds, “and this can affect the health of the foal at birth, making it weak or having a large umbilical cord.”

As much as people might think that a cloned horse is a duplicate of the original, it does not necessarily follow that it can do what its original – often called its “twin” - can do. If the foal was weak at birth or if it was born with crooked legs (as may happen with any normal foal due to its position in the uterus or the pliability of membranes around it), the foal may not have the athletic qualities of the original horse. In addition, athletic ability is also a product of the animal’s environment, handling, management, and training.

Each horse has its own unique character, again shaped by its cumulative experiences, exposure, and handling. According to Hinrichs, there have been no studies to date on how similar the cloned horse is to its original twin. A misconception, she says, is that cloning will produce the same animal with the same memories and experiences. That simply isn’t possible. Cloning produces a genetically identical twin but, like any set of twins, each will have their own memories and experiences.

“We have found that each of our clients often has a unique reason for their cloning interest,” says Blake Russell, president of ViaGen in Cedar Park, Texas. “We are producing similar numbers from each gender and still reproduce genetics from great, proven stallions. There is a compelling case for cloning the elite ones, whether they spent their life as a mare, a gelding, or a stallion.”

ViaGen is a division of Trans Ova Genetics and offers cloning services and state-of-the-art technology for all non-primate species. It has offered its equine cloning service for 13 years. During that time it has produced 200 foals and the company has licensees in both Brazil and Argentina who are producing cloned foals, some for the polo market.

The most frequent reason for cloning is to preserve the unique genetics of an elite equine athlete whether in show jumping, polo, or barrel racing. Many of these horses are either geldings or mares. While mares can be bred, geldings of course cannot. If these valuable animals are cloned, their twin can be kept for exclusive breeding purposes.

“Cloned horses can breed,” says Hinrichs. “They are basically normal horses. Breeding is the main reason people are cloning horses [in order] to save valuable genetics so they can produce foals from those lines. A very common reason for cloning is because an excellent champion horse is a gelding. By cloning him, you can keep the cloned colt intact and then are able to ‘breed’ to the gelding through his clone.”

She adds, however, that cloning is a technically difficult procedure that has a huge requirement in terms of equipment costs, the need for expert personnel, and access to a supply of oocytes. In the early days when Prometea was foaled, the researchers experimented with 841 reconstructed eggs. Of these, 22 grew into transferrable embryos and, of 17 embryos transferred to recipient mares, only Prometea was carried to term and delivered.

Lynx Melody Too is the cloned foal of the AQHA mare, Lynx Melody, a National Cutting Horse Association World Champion. Photo courtesy of ViaGen

“The early challenges were focused around embryo care and transfer,” says Russell. “When ViaGen began offering equine cloning, equine embryo technology was still very much around the utilization of fresh embryos. ViaGen has successfully developed high quality embryo freezing technology, and today, 100 percent of our equine embryos are cryopreserved and shipped to our embryo transfer locations. ViaGen transfers most of our equine embryos in Texas to a veterinarian clinic that has worked with us for more than ten years.”

In ViaGen’s first year, they saw a few foals with contracted tendons and a few foals were lost for a variety of reasons, which led to changes in the technical process. Russell says that many of the research institutions were struggling with foal health immediately following birth. The most significant challenge that remains is improving the pregnancy rate. However, success rates now are high with healthy cloned foals being born that require no special care. The company guarantees a 60 day old, genetically verified, healthy and insurable foal for their clients.

Understandably, cloning has its squirm factor. Breakthroughs in science have put tantalizing breeding strategies at people’s fingertips. But just because you can, does it mean you should?

For some people, the argument is simply that it just doesn’t feel right to experiment with Mother Nature. There is a real fear that the backlash down the road is for the prevalence of disease to lead to genetic mutations and that repeated breeding for a specific line and/or discipline may lead to a genetic bottleneck putting the breed’s healthy diversity at risk.

Cloning is not for every breed organization either. The Jockey Club will not register a cloned Thoroughbred, nor in fact will it register any horse conceived by any method other than live cover.

The American Quarter Horse Association recently won a lawsuit after it was sued by an owner of cloned Quarter Horses and their offspring for antitrust activity.

“The owner prevailed in US Federal Court,” says Russell. “However, the AQHA did appeal and won a reversal in the appeals court. Therefore, AQHA is allowed to continue their restriction against the registration of cloned Quarter Horses and their offspring.”

Russell says, though, that most international registries have welcomed cloned horses and their offspring into their registries, and see cloning technology as simply another breeding tool to be responsibly used by breeders to advance their performance goals. “The AQHA’s decision doesn’t appear to be having much impact on the decisions of other registries around the world. ViaGen continues to provide cloning services to large numbers of AQHA owners and they are using the technology to reproduce the DNA of their elite AQHA horses. Most events other than horse racing welcome foals produced with advanced breeding techniques. ViaGen is also providing our services to owners of most other breeds around the world including Thoroughbreds used for purposes other than racing, Olympic sport horses, polo horses, barrel racing horses, cutting horses, etc.”

Cloning has a broader value in animal reproduction, especially in the preservation of genetic lines for wildlife protection.

“The main justification I see for cloning is to preserve genetics as in valuable geldings or in the case of rare or endangered species or breeds so that you can expand the gene pool,” says Hinrichs. “You could use cells from animals that died decades ago (if the cells were recovered before or at death and frozen) that are under-represented in the population today.”

The Frozen Zoo at San Diego Zoo Conservation Research has been cryopreserving genetic material of endangered species since 1976. According to their website, some 15,000 sperm samples from 1,232 individual males representing 309 species are currently stored in the facility. In addition, oocytes (eggs) of 381 females from 177 species are also cryopreserved for fertilization and embryo transfer. Properly managed, frozen cells remain viable for decades. As an example, their researchers were able to achieve successful fertilization by injecting the endangered southern white rhinoceros oocytes with sperm frozen for over 20 years.

On September 15, 2008, the French genetic bank, Cryozootech, announced the birth of the colt Gemini, a clone of the Thoroughbred gelding, Gem Twist, regarded as one of the best show jumpers in history. In 2012, the first foal of Gemini was born. Photo courtesy of ViaGen

In addition, frozen viable cell cultures from over 9,000 individual animals representing nearly 1,000 species are also in the collection. Banked DNA and tissue samples are kept for research studies that can provide valuable information to sustainably manage and preserve wild populations in the future. In the fight against illegal trade in wildlife parts, genetic barcodes make it possible to identify the species used in the manufacture of bags, shoes, or meat traded at urban markets.

Russell says that ViaGen offers a genetic preservation service to provide options to horse, livestock, and pet owners wanting to preserve the ability to consider cloning in the future.

“We have thousands of animals represented in our cryopreservation bank and we are often cloning horses that had their DNA preserved as long as 20 years ago.”

He adds that cloning has a value when breeders are faced with the challenge of a subfertile mare or stallion in their breeding program. A technology known as equine intracytoplasmic sperm injection (ICSI) can be useful when the frozen sperm supply is limited or the mare cannot produce a pregnancy on her own. But ICSI results are highly variable and expensive. He says that the cloning route opens up easier future methods of reproduction such as artificial insemination (AI) or natural service.

“It can make much more economic sense to reproduce a proven stallion that has passed versus investing in the incredible expense and challenge to utilize a specialized technology such as ICSI with every breeding,” he says. “We find a similar situation with aging, elite mares.”

Cloning is not cheap and should be considered a long term investment.

“We are currently offering our equine cloning service for $85,000,” says Russell. “This is guaranteed to produce a healthy, genetically verified foal and [able to] pass an insurance exam at 60 days of age. ViaGen allows the client to pick up the foal alongside the recipient mare at 60 days of age and later return the recipient mare when the foal is weaned. For those recipient mares and foals that leave the country, ViaGen allows the client to retain the recipient mare. ViaGen agrees to keep the foal and recipient mare for up to 60 days for inspection, but many of the clients choose to take them home sooner so that they can raise them in their own management system.”

ViaGen has proven to have a positive track record with each cell line over the last ten years. As cloned foals grow and make their own contribution to performance and production careers, they are seeing an increase in demand.

For breeders thinking about cloning, the first step is a simple biopsy sample taken by their veterinarian. ViaGen provides a genetic preservation biopsy kit. Cells are grown in culture from the biopsy sample and, once complete, they are cryopreserved for future use. This keeps options open potentially for decades, and there should not be a need to re-collect a biopsy sample from the horse. Tissue, however, cannot be taken from a horse that has been euthanized. The fee for the service is $1,600 and the annual storage fee is $150 per animal.

How ViaGen’s Blake Russell Got Started In Cloning

ViaGen President Blake Russell was raised in the Quarter Horse racing business and spent his formative years with horses. He joined the company over ten years ago. Together with a group of experienced horsemen, they put together a list of great performing horses that had elite pedigrees but were unable to breed. Champion racing Quarter Horse Tailor Fit was on the top of the list. He was gelded as a yearling as his owners were more interested in a career at the racetrack than a breeding career. His dam, Silk Skirt, was a proven outlier and his sire, Strawfly Special, was one of the greats.

Tailor Fit went on to win the AQHA world racing title twice and earned well over a million dollars and a speed index of 110. He represented the traits most desired in performance horses with tremendous conformation, a level of heart and determination that is rarely seen, and elite speed. Russell said one look at his pedigree showed that those traits did not appear by chance, but were the result of a superior set of genes.

“I approached the owner of Tailor Fit following his retirement and she requested that I move forward with cloning him since I was carrying the passion for building a breeding program around him. I made the investment, and it has been a journey that is difficult to describe with words.”

The result? Pure Tailor Fit.

“Pure Tailor Fit (the cloned stallion) has been healthy and normal in every way. His athleticism is off the charts, and his personality traits draw everyone to him as the star attraction. ‘Fit’s’ oldest crop of foals turned two in 2016, and I have 20 of them on our place. We are extremely enthusiastic about their future as they seem to carry the attributes that started this journey in the first place.”

Pure Tailor Fit is the cloned stallion owned by Blake Russell, President of ViaGen, Texas. Pure Tailor Fit was cloned from two-time world champion racing Quarter Horse gelding, Taylor Fit. Photo courtesy of ViaGen

Racing the colts in the AQHA is forbidden since they cannot be registered, but Russell’s program is designed around producing barrel racing horses and exceptional roping horses.

“Each day ‘Fit’ or one of his babies confirms our decision to bring this pedigree back into production. People are often confused by cloning and don’t understand the value of identical twins. Full siblings are not a good reflection of the power of cloning. Pure Tailor Fit carries exactly the same breeding value as Tailor Fit would have represented if he had been allowed to remain intact and breed. The ability to take a high performer with an elite pedigree and no genetic defects and establish a breeding program is the reason for cloning technology to exist.

ViaGen Equine is cloning elite horses for Olympians, world class breeders, and owners of the world’s top equine athletes. However, I can step out my front door every day and see with my own eyes the power of this technology.”


Contents

At least six major areas of cryobiology can be identified: 1) study of cold-adaptation of microorganisms, plants (cold hardiness), and animals, both invertebrates and vertebrates (including hibernation), 2) cryopreservation of cells, tissues, gametes, and embryos of animal and human origin for (medical) purposes of long-term storage by cooling to temperatures below the freezing point of water. This usually requires the addition of substances which protect the cells during freezing and thawing (cryoprotectants), 3) preservation of organs under hypothermic conditions for transplantation, 4) lyophilization (freeze-drying) of pharmaceuticals, 5) cryosurgery, a (minimally) invasive approach for the destruction of unhealthy tissue using cryogenic gases/fluids, and 6) physics of supercooling, ice nucleation/growth and mechanical engineering aspects of heat transfer during cooling and warming, as applied to biological systems. Cryobiology would include cryonics, the low temperature preservation of humans and mammals with the intention of future revival, although this is not part of mainstream cryobiology, depending heavily on speculative technology yet to be invented. Several of these areas of study rely on cryogenics, the branch of physics and engineering that studies the production and use of very low temperatures.

Many living organisms are able to tolerate prolonged periods of time at temperatures below the freezing point of water. Most living organisms accumulate cryoprotectants such as antinucleating proteins, polyols, and glucose to protect themselves against frost damage by sharp ice crystals. Most plants, in particular, can safely reach temperatures of −4 °C to −12 °C.

Bacteria Edit

Three species of bacteria, Carnobacterium pleistocenium, Chryseobacterium greenlandensis, and Herminiimonas glaciei, have reportedly been revived after surviving for thousands of years frozen in ice. Certain bacteria, notably Pseudomonas syringae, produce specialized proteins that serve as potent ice nucleators, which they use to force ice formation on the surface of various fruits and plants at about −2 °C. [1] The freezing causes injuries in the epithelia and makes the nutrients in the underlying plant tissues available to the bacteria. [2] Listeria grows slowly in temperatures as low as -1.5 °C and persists for some time in frozen foods. [3]

Plants Edit

Many plants undergo a process called hardening which allows them to survive temperatures below 0 °C for weeks to months.

Animals Edit

Invertebrates Edit

Nematodes that survive below 0 °C include Trichostrongylus colubriformis and Panagrolaimus davidi. Cockroach nymphs (Periplaneta japonica) survive short periods of freezing at -6 to -8 °C. The red flat bark beetle (Cucujus clavipes) can survive after being frozen to -150 °C. [4] The fungus gnat Exechia nugatoria can survive after being frozen to -50 °C, by a unique mechanism whereby ice crystals form in the body but not the head. Another freeze-tolerant beetle is Upis ceramboides. [5] See insect winter ecology and antifreeze protein. Another invertebrate that is briefly tolerant to temperatures down to -273 °C is the tardigrade.

The larvae of Haemonchus contortus, a nematode, can survive 44 weeks frozen at -196 °C.

Vertebrates Edit

For the wood frog (Rana sylvatica), in the winter, as much as 45% of its body may freeze and turn to ice. "Ice crystals form beneath the skin and become interspersed among the body's skeletal muscles. During the freeze, the frog's breathing, blood flow, and heartbeat cease. Freezing is made possible by specialized proteins and glucose, which prevent intracellular freezing and dehydration." [6] [7] The wood frog can survive up to 11 days frozen at -4 °C.

Other vertebrates that survive at body temperatures below 0 °C include painted turtles (Chrysemys picta), gray tree frogs (Hyla versicolor), box turtles (Terrapene carolina - 48 hours at -2 °C), spring peeper (Pseudacris crucifer), garter snakes (Thamnophis sirtalis- 24 hours at -1.5 °C), the chorus frog (Pseudacris triseriata), Siberian salamander (Salamandrella keyserlingii - 24 hours at -15.3 °C), [8] European common lizard (Lacerta vivipara) and Antarctic fish such as Pagothenia borchgrevinki. [9] [10] Antifreeze proteins cloned from such fish have been used to confer frost-resistance on transgenic plants. [ citation needed ]

Hibernating Arctic ground squirrels may have abdominal temperatures as low as −2.9 °C (26.8 °F), maintaining subzero abdominal temperatures for more than three weeks at a time, although the temperatures at the head and neck remain at 0 °C or above. [11]

Historical background Edit

Cryobiology history can be traced back to antiquity. As early as in 2500 BC, low temperatures were used in Egypt in medicine. The use of cold was recommended by Hippocrates to stop bleeding and swelling. With the emergence of modern science, Robert Boyle studied the effects of low temperatures on animals.

In 1949, bull semen was cryopreserved for the first time by a team of scientists led by Christopher Polge. [12] This led to a much wider use of cryopreservation today, with many organs, tissues and cells routinely stored at low temperatures. Large organs such as hearts are usually stored and transported, for short times only, at cool but not freezing temperatures for transplantation. Cell suspensions (like blood and semen) and thin tissue sections can sometimes be stored almost indefinitely in liquid nitrogen temperature (cryopreservation). Human sperm, eggs, and embryos are routinely stored in fertility research and treatments. Controlled-rate and slow freezing are well established techniques pioneered in the early 1970s which enabled the first human embryo frozen birth (Zoe Leyland) in 1984. Since then, machines that freeze biological samples using programmable steps, or controlled rates, have been used all over the world for human, animal, and cell biology – 'freezing down' a sample to better preserve it for eventual thawing, before it is deep frozen, or cryopreserved, in liquid nitrogen. Such machines are used for freezing oocytes, skin, blood products, embryo, sperm, stem cells, and general tissue preservation in hospitals, veterinary practices, and research labs. The number of live births from 'slow frozen' embryos is some 300,000 to 400,000 or 20% of the estimated 3 million in vitro fertilized births. Dr Christopher Chen, Australia, reported the world’s first pregnancy using slow-frozen oocytes from a British controlled-rate freezer in 1986.

Cryosurgery (intended and controlled tissue destruction by ice formation) was carried out by James Arnott in 1845 in an operation on a patient with cancer. Cryosurgery is not common. [ citation needed ]

Preservation techniques Edit

Cryobiology as an applied science is primarily concerned with low-temperature preservation. Hypothermic storage is typically above 0 °C but below normothermic (32 °C to 37 °C) mammalian temperatures. Storage by cryopreservation, on the other hand, will be in the −80 to −196 °C temperature range. Organs, and tissues are more frequently the objects of hypothermic storage, whereas single cells have been the most common objects cryopreserved.

A rule of thumb in hypothermic storage is that every 10 °C reduction in temperature is accompanied by a 50% decrease in oxygen consumption. [13] Although hibernating animals have adapted mechanisms to avoid metabolic imbalances associated with hypothermia, hypothermic organs, and tissues being maintained for transplantation require special preservation solutions to counter acidosis, depressed sodium pump activity. and increased intracellular calcium. Special organ preservation solutions such as Viaspan (University of Wisconsin solution), HTK, and Celsior have been designed for this purpose. [14] These solutions also contain ingredients to minimize damage by free radicals, prevent edema, compensate for ATP loss, etc.

Cryopreservation of cells is guided by the "two-factor hypothesis" of American cryobiologist Peter Mazur, which states that excessively rapid cooling kills cells by intracellular ice formation and excessively slow cooling kills cells by either electrolyte toxicity or mechanical crushing. [15] During slow cooling, ice forms extracellularly, causing water to osmotically leave cells, thereby dehydrating them. Intracellular ice can be much more damaging than extracellular ice.

For red blood cells, the optimum cooling rate is very rapid (nearly 100 °C per second), whereas for stem cells the optimum cooling rate is very slow (1 °C per minute). Cryoprotectants, such as dimethyl sulfoxide and glycerol, are used to protect cells from freezing. A variety of cell types are protected by 10% dimethyl sulfoxide. [16] Cryobiologists attempt to optimize cryoprotectant concentration (minimizing both ice formation and toxicity) and cooling rate. Cells may be cooled at an optimum rate to a temperature between −30 and −40 °C before being plunged into liquid nitrogen.

Slow cooling methods rely on the fact that cells contain few nucleating agents, but contain naturally occurring vitrifying substances that can prevent ice formation in cells that have been moderately dehydrated. Some cryobiologists are seeking mixtures of cryoprotectants for full vitrification (zero ice formation) in preservation of cells, tissues, and organs. Vitrification methods pose a challenge in the requirement to search for cryoprotectant mixtures that can minimize toxicity.

In humans Edit

Human gametes and two-, four- and eight-cell embryos can survive cryopreservation at -196 °C for 10 years under well-controlled laboratory conditions. [17]

Cryopreservation in humans with regards to infertility involves preservation of embryos, sperm, or oocytes via freezing. Conception, in vitro, is attempted when the sperm is thawed and introduced to the 'fresh' eggs, the frozen eggs are thawed and sperm is placed with the eggs and together they are placed back into the uterus or a frozen embryo is introduced to the uterus. Vitrification has flaws and is not as reliable or proven as freezing fertilized sperm, eggs, or embryos as traditional slow freezing methods because eggs alone are extremely sensitive to temperature. Many researchers are also freezing ovarian tissue in conjunction with the eggs in hopes that the ovarian tissue can be transplanted back into the uterus, stimulating normal ovulation cycles. In 2004, Donnez of Louvain in Belgium reported the first successful ovarian birth from frozen ovarian tissue. In 1997, samples of ovarian cortex were taken from a woman with Hodgkin's lymphoma and cryopreserved in a (Planer, UK) controlled-rate freezer and then stored in liquid nitrogen. Chemotherapy was initiated after the patient had premature ovarian failure. In 2003, after freeze-thawing, orthotopic autotransplantation of ovarian cortical tissue was done by laparoscopy and after five months, reimplantation signs indicated recovery of regular ovulatory cycles. Eleven months after reimplantation, a viable intrauterine pregnancy was confirmed, which resulted in the first such live birth – a girl named Tamara. [18]

Therapeutic hypothermia, e.g. during heart surgery on a "cold" heart (generated by cold perfusion without any ice formation) allows for much longer operations and improves recovery rates for patients.

The Society for Cryobiology was founded in 1964 to bring together those from the biological, medical, and physical sciences who have a common interest in the effects of low temperatures on biological systems. As of 2007, the Society for Cryobiology had about 280 members from around the world, and one-half of them are US-based. The purpose of the Society is to promote scientific research in low temperature biology, to improve scientific understanding in this field, and to disseminate and apply this knowledge to the benefit of mankind. The Society requires of all its members the highest ethical and scientific standards in the performance of their professional activities. According to the Society's bylaws, membership may be refused to applicants whose conduct is deemed detrimental to the Society in 1982, the bylaws were amended explicitly to exclude "any practice or application of freezing deceased persons in the anticipation of their reanimation", over the objections of some members who were cryonicists, such as Jerry Leaf. [19] The Society organizes an annual scientific meeting dedicated to all aspects of low-temperature biology. This international meeting offers opportunities for presentation and discussion of the most up-to-date research in cryobiology, as well as reviewing specific aspects through symposia and workshops. Members are also kept informed of news and forthcoming meetings through the Society newsletter, News Notes. The 2011–2012 president of the Society for Cryobiology was John H. Crowe. [20]

The Society for Low Temperature Biology was founded in 1964 and became a registered charity in 2003 [21] with the purpose of promoting research into the effects of low temperatures on all types of organisms and their constituent cells, tissues, and organs. As of 2006, the society had around 130 (mostly British and European) members and holds at least one annual general meeting. The program usually includes both a symposium on a topical subject and a session of free communications on any aspect of low-temperature biology. Recent symposia have included long-term stability, preservation of aquatic organisms, cryopreservation of embryos and gametes, preservation of plants, low-temperature microscopy, vitrification (glass formation of aqueous systems during cooling), freeze drying and tissue banking. Members are informed through the Society Newsletter, which is presently published three times a year.

Cryobiology (publisher: Elsevier) is the foremost scientific publication in this area, with about 60 refereed contributions published each year. Articles concern any aspect of low-temperature biology and medicine (e.g. freezing, freeze-drying, hibernation, cold tolerance and adaptation, cryoprotective compounds, medical applications of reduced temperature, cryosurgery, hypothermia, and perfusion of organs).

Cryo Letters is an independent UK-based rapid communication journal which publishes papers on the effects produced by low temperatures on a wide variety of biophysical and biological processes, or studies involving low-temperature techniques in the investigation of biological and ecological topics.

Biopreservation and Biobanking (formerly Cell Preservation Technology) is a peer-reviewed quarterly scientific journal published by Mary Ann Liebert, Inc. dedicated to the diverse spectrum of preservation technologies including cryopreservation, dry-state (anhydrobiosis), and glassy-state and hypothermic maintenance. Cell Preservation Technology has been renamed Biopreservation and Biobanking and is the official journal of International Society for Biological and Environmental Repositories.


Contents

A biomolecular target (most commonly a protein or a nucleic acid) is a key molecule involved in a particular metabolic or signaling pathway that is associated with a specific disease condition or pathology or to the infectivity or survival of a microbial pathogen. Potential drug targets are not necessarily disease causing but must by definition be disease modifying. [9] In some cases, small molecules will be designed to enhance or inhibit the target function in the specific disease modifying pathway. Small molecules (for example receptor agonists, antagonists, inverse agonists, or modulators enzyme activators or inhibitors or ion channel openers or blockers) [10] will be designed that are complementary to the binding site of target. [11] Small molecules (drugs) can be designed so as not to affect any other important "off-target" molecules (often referred to as antitargets) since drug interactions with off-target molecules may lead to undesirable side effects. [12] Due to similarities in binding sites, closely related targets identified through sequence homology have the highest chance of cross reactivity and hence highest side effect potential.

Most commonly, drugs are organic small molecules produced through chemical synthesis, but biopolymer-based drugs (also known as biopharmaceuticals) produced through biological processes are becoming increasingly more common. [13] In addition, mRNA-based gene silencing technologies may have therapeutic applications. [14]

In contrast to traditional methods of drug discovery (known as forward pharmacology), which rely on trial-and-error testing of chemical substances on cultured cells or animals, and matching the apparent effects to treatments, rational drug design (also called reverse pharmacology) begins with a hypothesis that modulation of a specific biological target may have therapeutic value. In order for a biomolecule to be selected as a drug target, two essential pieces of information are required. The first is evidence that modulation of the target will be disease modifying. This knowledge may come from, for example, disease linkage studies that show an association between mutations in the biological target and certain disease states. [15] The second is that the target is "druggable". This means that it is capable of binding to a small molecule and that its activity can be modulated by the small molecule. [16]

Once a suitable target has been identified, the target is normally cloned and produced and purified. The purified protein is then used to establish a screening assay. In addition, the three-dimensional structure of the target may be determined.

The search for small molecules that bind to the target is begun by screening libraries of potential drug compounds. This may be done by using the screening assay (a "wet screen"). In addition, if the structure of the target is available, a virtual screen may be performed of candidate drugs. Ideally the candidate drug compounds should be "drug-like", that is they should possess properties that are predicted to lead to oral bioavailability, adequate chemical and metabolic stability, and minimal toxic effects. [17] Several methods are available to estimate druglikeness such as Lipinski's Rule of Five and a range of scoring methods such as lipophilic efficiency. [18] Several methods for predicting drug metabolism have also been proposed in the scientific literature. [19]

Due to the large number of drug properties that must be simultaneously optimized during the design process, multi-objective optimization techniques are sometimes employed. [20] Finally because of the limitations in the current methods for prediction of activity, drug design is still very much reliant on serendipity [21] and bounded rationality. [22]

The most fundamental goal in drug design is to predict whether a given molecule will bind to a target and if so how strongly. Molecular mechanics or molecular dynamics is most often used to estimate the strength of the intermolecular interaction between the small molecule and its biological target. These methods are also used to predict the conformation of the small molecule and to model conformational changes in the target that may occur when the small molecule binds to it. [3] [4] Semi-empirical, ab initio quantum chemistry methods, or density functional theory are often used to provide optimized parameters for the molecular mechanics calculations and also provide an estimate of the electronic properties (electrostatic potential, polarizability, etc.) of the drug candidate that will influence binding affinity. [23]

Molecular mechanics methods may also be used to provide semi-quantitative prediction of the binding affinity. Also, knowledge-based scoring function may be used to provide binding affinity estimates. These methods use linear regression, machine learning, neural nets or other statistical techniques to derive predictive binding affinity equations by fitting experimental affinities to computationally derived interaction energies between the small molecule and the target. [24] [25]

Ideally, the computational method will be able to predict affinity before a compound is synthesized and hence in theory only one compound needs to be synthesized, saving enormous time and cost. The reality is that present computational methods are imperfect and provide, at best, only qualitatively accurate estimates of affinity. In practice it still takes several iterations of design, synthesis, and testing before an optimal drug is discovered. Computational methods have accelerated discovery by reducing the number of iterations required and have often provided novel structures. [26] [27]

Drug design with the help of computers may be used at any of the following stages of drug discovery:

  1. hit identification using virtual screening (structure- or ligand-based design) optimization of affinity and selectivity (structure-based design, QSAR, etc.) of other pharmaceutical properties while maintaining affinity

In order to overcome the insufficient prediction of binding affinity calculated by recent scoring functions, the protein-ligand interaction and compound 3D structure information are used for analysis. For structure-based drug design, several post-screening analyses focusing on protein-ligand interaction have been developed for improving enrichment and effectively mining potential candidates:

  • Consensus scoring [28][29]
    • Selecting candidates by voting of multiple scoring functions
    • May lose the relationship between protein-ligand structural information and scoring criterion
    • Represent and cluster candidates according to protein-ligand 3D information
    • Needs meaningful representation of protein-ligand interactions.

    There are two major types of drug design. The first is referred to as ligand-based drug design and the second, structure-based drug design. [2]

    Ligand-based Edit

    Ligand-based drug design (or indirect drug design) relies on knowledge of other molecules that bind to the biological target of interest. These other molecules may be used to derive a pharmacophore model that defines the minimum necessary structural characteristics a molecule must possess in order to bind to the target. [32] In other words, a model of the biological target may be built based on the knowledge of what binds to it, and this model in turn may be used to design new molecular entities that interact with the target. Alternatively, a quantitative structure-activity relationship (QSAR), in which a correlation between calculated properties of molecules and their experimentally determined biological activity, may be derived. These QSAR relationships in turn may be used to predict the activity of new analogs. [33]

    Structure-based Edit

    Structure-based drug design (or direct drug design) relies on knowledge of the three dimensional structure of the biological target obtained through methods such as x-ray crystallography or NMR spectroscopy. [34] If an experimental structure of a target is not available, it may be possible to create a homology model of the target based on the experimental structure of a related protein. Using the structure of the biological target, candidate drugs that are predicted to bind with high affinity and selectivity to the target may be designed using interactive graphics and the intuition of a medicinal chemist. Alternatively various automated computational procedures may be used to suggest new drug candidates. [35]

    Current methods for structure-based drug design can be divided roughly into three main categories. [36] The first method is identification of new ligands for a given receptor by searching large databases of 3D structures of small molecules to find those fitting the binding pocket of the receptor using fast approximate docking programs. This method is known as virtual screening. A second category is de novo design of new ligands. In this method, ligand molecules are built up within the constraints of the binding pocket by assembling small pieces in a stepwise manner. These pieces can be either individual atoms or molecular fragments. The key advantage of such a method is that novel structures, not contained in any database, can be suggested. [37] [38] [39] A third method is the optimization of known ligands by evaluating proposed analogs within the binding cavity. [36]

    Binding site identification Edit

    Binding site identification is the first step in structure based design. [16] [40] If the structure of the target or a sufficiently similar homolog is determined in the presence of a bound ligand, then the ligand should be observable in the structure in which case location of the binding site is trivial. However, there may be unoccupied allosteric binding sites that may be of interest. Furthermore, it may be that only apoprotein (protein without ligand) structures are available and the reliable identification of unoccupied sites that have the potential to bind ligands with high affinity is non-trivial. In brief, binding site identification usually relies on identification of concave surfaces on the protein that can accommodate drug sized molecules that also possess appropriate "hot spots" (hydrophobic surfaces, hydrogen bonding sites, etc.) that drive ligand binding. [16] [40]

    Scoring functions Edit

    Structure-based drug design attempts to use the structure of proteins as a basis for designing new ligands by applying the principles of molecular recognition. Selective high affinity binding to the target is generally desirable since it leads to more efficacious drugs with fewer side effects. Thus, one of the most important principles for designing or obtaining potential new ligands is to predict the binding affinity of a certain ligand to its target (and known antitargets) and use the predicted affinity as a criterion for selection. [41]

    One early general-purposed empirical scoring function to describe the binding energy of ligands to receptors was developed by Böhm. [42] [43] This empirical scoring function took the form:

    Δ G bind = Δ G 0 + Δ G hb Σ h − b o n d s + Δ G ionic Σ i o n i c − i n t + Δ G lipophilic | A | + Δ G rot N R O T >=Delta G_< ext<0>>+Delta G_< ext>Sigma _+Delta G_< ext>Sigma _+Delta G_< ext>leftvert A ightvert +Delta G_< ext>>>

    • ΔG0 – empirically derived offset that in part corresponds to the overall loss of translational and rotational entropy of the ligand upon binding.
    • ΔGhb – contribution from hydrogen bonding
    • ΔGionic – contribution from ionic interactions
    • ΔGlip – contribution from lipophilic interactions where |Alipo| is surface area of lipophilic contact between the ligand and receptor
    • ΔGrot – entropy penalty due to freezing a rotatable in the ligand bond upon binding

    A more general thermodynamic "master" equation is as follows: [44]

    • desolvation – enthalpic penalty for removing the ligand from solvent
    • motion – entropic penalty for reducing the degrees of freedom when a ligand binds to its receptor
    • configuration – conformational strain energy required to put the ligand in its "active" conformation
    • interaction – enthalpic gain for "resolvating" the ligand with its receptor

    The basic idea is that the overall binding free energy can be decomposed into independent components that are known to be important for the binding process. Each component reflects a certain kind of free energy alteration during the binding process between a ligand and its target receptor. The Master Equation is the linear combination of these components. According to Gibbs free energy equation, the relation between dissociation equilibrium constant, Kd, and the components of free energy was built.

    Various computational methods are used to estimate each of the components of the master equation. For example, the change in polar surface area upon ligand binding can be used to estimate the desolvation energy. The number of rotatable bonds frozen upon ligand binding is proportional to the motion term. The configurational or strain energy can be estimated using molecular mechanics calculations. Finally the interaction energy can be estimated using methods such as the change in non polar surface, statistically derived potentials of mean force, the number of hydrogen bonds formed, etc. In practice, the components of the master equation are fit to experimental data using multiple linear regression. This can be done with a diverse training set including many types of ligands and receptors to produce a less accurate but more general "global" model or a more restricted set of ligands and receptors to produce a more accurate but less general "local" model. [45]

    A particular example of rational drug design involves the use of three-dimensional information about biomolecules obtained from such techniques as X-ray crystallography and NMR spectroscopy. Computer-aided drug design in particular becomes much more tractable when there is a high-resolution structure of a target protein bound to a potent ligand. This approach to drug discovery is sometimes referred to as structure-based drug design. The first unequivocal example of the application of structure-based drug design leading to an approved drug is the carbonic anhydrase inhibitor dorzolamide, which was approved in 1995. [46] [47]

    Another important case study in rational drug design is imatinib, a tyrosine kinase inhibitor designed specifically for the bcr-abl fusion protein that is characteristic for Philadelphia chromosome-positive leukemias (chronic myelogenous leukemia and occasionally acute lymphocytic leukemia). Imatinib is substantially different from previous drugs for cancer, as most agents of chemotherapy simply target rapidly dividing cells, not differentiating between cancer cells and other tissues. [48]

    Additional examples include:

    • Many of the atypical antipsychotics , the prototypical H2-receptor antagonist from which the later members of the class were developed
    • Selective COX-2 inhibitor NSAIDs , a peptide HIV entry inhibitor like zolpidem and zopiclone , an HIV integrase inhibitor [49] (selective serotonin reuptake inhibitors), a class of antidepressants , an antiviral drug

    It has been argued that the highly rigid and focused nature of rational drug design suppresses serendipity in drug discovery. [50]


    Controversies About Cloning of Domestic Animals

    Ethical concerns about cloning may be broadly divided into two categories: concern about the effect of cloning on animal and human welfare, and objection to the principle of cloning, ie, to producing an animal by a means other than fertilization.

    Currently, cloning is associated with an increase in animal suffering when compared with production of animals by standard breeding methods. This is due to surgeries performed to obtain oocytes or transfer embryos, pregnancy losses, sickness and death of neonates, low-level abnormalities in surviving young, and possible distress from disease in animals produced as disease models. These concerns are somewhat mitigated by the fact that most of these findings are not unique to cloning they are also associated with other procedures that have been generally accepted as worthwhile, such as in vitro fertilization and embryo production, oocyte transfer, and embryo transfer. In addition, the accepted normal fate of many species being cloned is to be housed for maximal production and then be slaughtered and eaten. A compelling argument for cloning is that the potential benefits of the procedure to the understanding of life processes and animal disease, to human health, and to food production outweigh the cost of the procedure in terms of animal welfare.

    Additional concerns rest with the effect of cloning on the entire animal population, most commonly related to the genetic variation of the species. This is a legitimate concern in some species and for some uses, such as in dairy cattle, in which one bull may sire thousands of offspring. However, this is more related to the technology of semen freezing and distribution than to the fact that a bull itself was cloned. In companion animals, it is improbable that the few pets likely to be cloned will have an effect on the population in general. In horses, cloning may in fact increase genetic variation, because a major proposed use is to clone geldings that have been found to be superior competitors, thus rescuing genetic types that would otherwise have been lost.

    Concerns about human health focus mainly on consumption of food produced from cloned animals. After years of study, the FDA and the European Food Safety Authority concluded that consumption of meat or milk from cloned animals poses no public health risk. Therefore, remaining concerns about consumption of food from cloned animals is likely based more on principle than on actual potential for harm. Because cloning is used to produce transgenic or gene-edited animals, many perceived concerns regarding cloning are actually concerns about these genetically altered animals, which present a completely different set of potential hazards to animal and human health and the environment. In the EU, although the lack of evidence of a human health risk is recognized, marketing food from clones requires authorization. There are calls for EU rules to prohibit cloning for farming purposes and to ban the marketing of food from clones.

    A key ethical question regarding the principle of producing animals by cloning is whether this technique is violating some moral prohibition, ie, that people are “playing God” by producing embryos without using fertilization. Similar questions have arisen with each new reproductive technique that has been developed however, many people feel that cloning is a special case. This general moral aversion of the public to the concept of cloning is enhanced by the portrayal of cloning as a malevolent force in science fiction books and films.

    Counter-arguments to these moral concerns are that cloning occurs in nature in the form of identical twins that people have been producing plants and animals by “unnatural” means from the first time they planted a seed in a new area or bred a cow to a selected bull, and that this is simply a new development in the same line. Embryonic cloning was being performed for more than 10 years before the birth of Dolly with essentially no public attention, and even the birth of two lambs cloned from cultured cells of embryonic origin, announced a year before Dolly, had no public impact. Thus, it appears that the main moral issue of public concern is not the production of embryos without fertilization, but the production of embryos from cells of an existing, known animal.

    Arguments against cloning of companion animals have focused on the cost of producing a clone—tens to hundreds of thousands of dollars—when millions of unwanted dogs and cats are killed each year. However, people currently buy purebred dogs and cats for thousands of dollars when they could get animals for no or low cost. American culture supports the concept that people can spend their own money on whatever they wish.

    A related argument is that cloning turns animals into a commodity or an object, rather than a sentient being, and that producing an animal in this way shows a lack of respect for the animal as an individual. However, animals have been bought and sold since they were domesticated currently semen and embryos are frozen, shipped across the country, and used to produce desired young. Cloning does not seem to offer any unique distinction in this area.

    Commercialization of cloning brings with it the possibility of fraud and of preying on the emotions of bereaved pet owners. Cloning companies should state clearly that the technique will produce another individual with the same genetics as the original animal it does not “resurrect” an animal or create an animal identical to the donor (eg, with the same coat pattern or personality). The best simile to draw is to that of an identical twin born later in time just as with naturally occurring identical twins, they will be very similar but also different in many ways.


    De-Extinction Debate: Should Extinct Species Be Revived?

    Woolly mammoths thrived during the last ice age until rapid planetary warming--which the most recent evidence suggests followed a meteor strike--killed them off thousands of years ago. (Image: Mauricio Anton, PLOS Biology)

    Last month, hundreds of experts who study human-environment interactions called on policymakers to take immediate action to curb humanity&rsquos ecologically destructive ways. Accelerating trends of human-driven extinction, ecosystem loss, climate change, pollution, and consumption, the scientists wrote in a consensus statement, &ldquoare threatening the life-support systems upon which we all depend.&rdquo

    If current rates of extinction continue, the statement warns, we could see the loss of 75 percent of vertebrate species within three centuries.

    As conservation scientists struggle to stem the catastrophic loss of biodiversity, some synthetic biologists are working to bring extinct species back to life. You might think the two groups would be working together. But until recently, most conservation biologists knew little of the so-called "Revive and Restore" movement, which until the TEDxDeExtinction conference in March, had been meeting largely behind closed doors.

    Following a private meeting of &ldquode-extinction&rdquo pioneers at Harvard Medical School last February, the National Geographic Society and San Francisco&rsquos Long Now Foundation brought molecular biologists and conservation biologists together in October to discuss strategies for resurrecting extinct species. The organizers admitted just one journalist to the October meeting, and orchestrated media coverage of de-extinction with the TEDx conference and a National Geographic cover story a few weeks later.

    The Revive and Restore Project is the brainchild of Long Now Foundation co-founder Stewart Brand and his wife Ryan Phelan, a serial entrepreneur who most recently sold her consumer genetic testing business DNA Direct to Fortune 500 company Medco. Their top candidates for de-extinction include the passenger pigeon and the woolly mammoth. Brand and Phelan promote the project as a way to restore lost genetic diversity with its mission of ensuring &ldquodeep ecological enrichment through extinct species revival.&rdquo

    But some working on the front lines of biodiversity conservation are skeptical. In a commentary published in April, leading conservation scientists noted that few of their colleagues had considered synthetic biology&rsquos potential effects on conservation, even though it might &ldquotransform. the prospects for maintaining biodiversity.&rdquo The authors outlined several ways that recreated extinct organisms could potentially affect strategic biodiversity goals&mdashsome positive, many negative. Their point was that no one knows, but conservation biologists better start paying attention (see chart, below).

    Examples of how synthetic biology, promised or developed at even modest scales, could significantly affect the Aichi Biodiversity TargetsExamples of how synthetic biology, promised or developed at even modest scales, could significantly affect the Aichi Biodiversity Targets, adopted by the 2010 Convention on Biodiversity's Conference of the Parties. Click the image to see a larger version. (PLOS Biology, Redford et al.)

    Hank Greely, director of Stanford University&rsquos Center for Law and the Biosciences, admits a longstanding fascination with the prospect of reviving extinct species, but couldn't decide whether it was really a good idea. So he organized a conference at Stanford last week and invited philosophers, lawyers, biologists, and wildlife professionals to think through the complex ethical, legal and political issues de-extinction raises.

    &ldquoI think one of the reasons this issue has bubbled to the surface so quickly is that the technology is converging with the coolness of the idea of bringing things back, mixing with a sense of guilt we feel with driving things extinct,&rdquo University of Kansas law professor Andrew Torrance told me. But de-extinction raises several &ldquodefinitional conundrums,&rdquo he said in this talk. Are de-extinct organisms GMOs? Invasive species?

    And where would a resurrected species fit into environmental law? Conference co-organizer Alex Camacho, director of UC Irvine&rsquos Law Center for Land, Environment and Natural Resources, said the Endangered Species Act has no framework for de-extinction, since its creators couldn&rsquot possibly have imagined the prospect. Shortly after revival of an organism, a species could potentially be listed as endangered, but is it the same species? A new species? An endangered species? The ESA defines endangered as &ldquoin danger of extinction throughout all or a significant portion of its range.&rdquo But what is its range? Does it have a range? Presumably not, if you have one organism sitting in a lab, Camacho said.

    Chuck Bonham, who directs the state&rsquos Department of Fish and Wildlife agency but was not representing the agency at the conference, wrestled with the management implications of de-extinction. &ldquoHow can you be extinct if you&rsquore always available for revival?&rdquo

    De-extinction technology

    Technologies for recreating extinct species include back-breeding, cloning and genetic engineering. Though all have the potential to accomplish the task, said Beth Shapiro, an evolutionary biologist and ancient-DNA expert at the University of California at Santa Cruz, they also have drawbacks.

    With back-breeding, scientists identify traits in the closest living relative and selectively breed offspring expressing desired traits until the animals resemble their extinct cousins. Sequencing bone and tooth fragments from extinct species speeds up the work of homing in on similar genome sequences in closely related descendants. Scientists in the Netherlands are using this approach to recreate the auroch, giant wild European cattle that went extinct in 1627, from domestic cattle. Cattle have a generation time of three to six years. Trying to revive mammoths from increasingly bigger and hairier elephants, which start reproducing on average at 20 to 25 years, could take centuries.

    More problematic is cloning, where scientists remove the nucleus of an egg cell, replace it with the nucleus from a donor cell, tweak it to grow as an embryo and implant it in a surrogate mother. The process is highly fraught. Dolly, the famous cloned sheep, was the only lamb born out of 277 attempts&mdashall the other clones died in utero or shortly after birth. In what&rsquos considered the first successful de-extinction using this method, a Pyrenean ibex (a large wild goat that went extinct less than 15 years ago) carried by a hybrid ibex-goat, lived all of 12 minutes, and all in acute respiratory distress.

    If researchers attempt this with elephants as surrogates, it&rsquos likely that the much smaller elephant mother would not fare well carrying a mammoth to term.* That doesn&rsquot account for the ethics of turning such highly intelligent social animals, who appear to grieve the death of their kin, into mammoth-resurrection machines.

    A specimen of the passenger pigeon, (Ectopistes migratorius), at Cincinnati Zoo and Botanical Garden. Passenger pigeons once numbered in the billions. Overhunting and habitat loss led to a catastrophic decline within 20 years, and extinction by 1914.

    Both methods, however, require intact genomes, which means you&rsquod have to freeze cell lines taken from species before they went extinct. &ldquoIf we&rsquore going to de-extinct something that&rsquos any older than something that we recently killed, we&rsquore stuck with ancient DNA,&rdquo Shapiro said. And that means dealing with tiny fragments of DNA that are often tainted with bacteria and other contaminants.

    That leaves genome editing, finding the sequences that code for traits of interest and pasting them into an existing genome. But researchers are still refining methods to find the right place in the genome and deliver the DNA without creating problems like cancer. An even bigger problem is figuring out which parts of the genome make a mammoth woolly, the sea cow so big or passenger pigeons flock together, Shapiro said. Even if you could reconstruct the genome of an extinct species, the jump to assigning function to sequences is enormous.

    Given all these issues, Shapiro said, &ldquoI think we should consider deeply why do we want to de-extinct things.&rdquo

    And that, for many working to conserve biodiversity, is the primary question. &ldquoConservation biologists worry that if people think we can revive species they won&rsquot care about protecting what&rsquos left,&rdquo said Kate Jones, joint chair of ecology and biodiversity at University of College London and the Zoological Society of London.

    Jones, who spent most of her career thinking about what makes species go extinct, told me she understands the appeal of de-extinction. &ldquoWho wouldn&rsquot be excited about the prospect of seeing a mammoth?&rdquo she allowed. &ldquoBut the practicalities of doing it are actually quite terrifying.&rdquo And as a conservation strategy, &ldquoit&rsquos a bit useless. It&rsquos dressed up as conservation, but it&rsquos not.&rdquo

    For Jones, this isn&rsquot about de-extinction. &ldquoIt&rsquos about creating new species. They&rsquore just flashy GMOs.&rdquo

    Some senior conservation biologists refuse to engage with the topic because they think it&rsquos not a legitimate debate, Jones told me. &ldquoBut I think it&rsquos kind of inevitable that this is going to happen whether it&rsquos Stewart Brand or someone in their back garden.&rdquo

    Then there&rsquos the question of what you do with a species you&rsquove revived. Jamie Rappaport Clark, who served as head of US Fish and Wildlife under the Clinton Administration and now leads Defenders of Wildlife, urged de-extinction proponents to consider the politics of reviving species. De-extinction could justify stalling action on restoring habitat or saving species, for example. That would have doomed the Florida panther, which received an influx of genes from airlifted Texas cougars under her watch in a desperate move to save the big cat.

    She&rsquos also worried that de-extinction will provide political cover for defunding conservation. &ldquoThey&rsquoll say, &lsquoWe shouldn&rsquot be funding recovery and preventing the extinction of species because we have a way out.&rsquo It will undermine the entire integrity of the ESA, which is already under serious distress now.&rdquo

    Pile of American bison skulls waiting to be ground for fertilizer, circa 1870. (Burton Historical Collection/Detroit Public Library/Public domain)

    During a roundtable discussion, Brand raised the prospect of bringing back the saber-toothed cat to California to replace lost ecological roles of predators, at which point Bonham leapt up from his chair, joking, &ldquoI&rsquom out of here!&rdquo

    He retrieved a piece a paper from his brief case and returned to tell Brand a story about the public&rsquos uneasy relationship with predators. &ldquoWe shot the last wolf in California about 100 years ago,&rdquo Bonham said. &ldquoOne month after I came on the job, we got our first wolf back in California in 100 years.&rdquo Half the state wants him to create a wolf preserve. The other half wants to see history repeated. &ldquoWe&rsquore not ready,&rdquo he said.

    Bonham read a passage from the 1982 Fish and Wildlife grizzly bear recovery plan. &ldquoThis is an animal that cannot compromise or adjust its way of life to ours. Could not by its very nature, could not even if we allowed it the opportunity, which we did not.&rdquo The only place for the grizzly bear in California remains on the state flag. &ldquoHow in the world do you expect a saber tooth to fare any better than. the grizzly bear?&rdquo he asked Brand.

    Jones asked Brand if any of the concerns conference participants raised about de-extinctinon had altered his vision. "Not yet," he answered. "It makes me more determined. that we make completely sure that everybody understands that de-extinction and conservation are in no way competitive." He said there's now a generation of kids who now want to see woolly mammoths in a zoo. "When they do I think they'll adopt a non-tragic relationship to nature and conservation with a sense that humans can. undo even serious damage like extinction."

    Elizabeth Hadly, a Stanford paleontologist and Paul S. and Billie Achilles Chair of Environmental Biology, helped craft the recent call to action to policymakers. She thinks laboratory innovation rather than on-the-ground research is behind the de-extinction push. Funding for ecology and conservation pales compared to the big grants funding genomics and synthetic biology. Although she&rsquos at Stanford, Hadly did not attend the conference. It pains her to think about what that money could do to protect the species already here&mdashsome hanging on by a thread.

    The way we&rsquore killing elephants now, we won&rsquot have any more left in 10 or 20 years, she said. &ldquoAnd people are talking about mammoths? First of all, they were alive in an ice age. This is the completely wrong environment to bring them back to.&rdquo

    She calls de-extinction &ldquogee-whiz science at its worst&rdquo and thinks justifying it in terms of genetic diversity and ecosystem services makes no sense.

    "Spending money to reintroduce recently lost existing species&mdasheven California&rsquos grizzly bear&mdashand restore habitat is a much better use of our time and energy", she said. "Without habitat restoration", she added, "the 750 mountain gorillas left on the planet won&rsquot make it. I&rsquod much rather combine the tiger subspecies together to create a better genetic reservoir than bring back some extinct organism.&rdquo

    Clark, who spent her career working with species on the brink of extinction, offered a similar view. &ldquoThe real question,&rdquo she told me, &ldquois why would we spend all this energy and effort to bring back ancient animals but let so many others just disappear?&rdquo

    She&rsquos been spending a lot of time thinking about our moral obligation to future generations. &ldquoIs it to create a couple of sad woolly mammoths that live in a zoo? Or is it to save the wolves and the panther and the Delhi sands flower-loving flies and the fisheries?&rdquo

    For Clark, there&rsquos no question. &ldquoWe need to do a better job of stewarding what we have," she said, "before we go rushing off after cool science experiments.&rdquo


    What's the rationale behind freezing one's eggs if cells can be cloned? - Biology

    Copyright ©1995 Lee M. Silver

    6. Mutagenesis and Transgenesis

    6.1 Classical mutagenesis

    6.1.1 The specific locus test

    6.1.3 Mouse Mutant Resources

    6.2 Embryo manipulation: genetic considerations

    6.2.1 Experimental possibilities

    6.2.2 Choice of strains for egg production

    6.2.3 Optimizing embryo production by superovulation

    6.2.4 The fertile stud male

    6.2.5 Embryo transfer into foster mothers

    6.3 Transgenic mice formed by nuclear injection

    6.3.2 Tracking the transgene and detecting homozygotes

    6.4 Targeted mutagenesis and gene replacement

    6.4.2 Creating ‘gene knockouts’

    6.4.3 Creating subtle changes

    6.5 Further uses of transgenic technologies

    6.5.1 Insertional mutagenesis and gene trapping

    6.5.2 A database and a repository of genetically engineered mice

    6.1 Classical mutagenesis

    6.1.1 The specific locus test

    Genetic variation — the existence of at least two forms — is the essential ingredient present in all genetic experiments. Phenotypic variation, in particular, is used as a means for uncovering the normal function of a wild-type allele at many loci. As discussed in the first chapter of this book, it was the availability of many variant phenotypes within the fancy mouse trade that made the house mouse such an ideal organism for studies by early geneticists. In a sense though, the house mouse won by default because in the absence of domestication and artificial selection, variation in traits visible to the eye is extremely rare, and thus, other small mammals were genetically intractable. Although the fancy mouse variants provided material for a host of early genetic studies, the number of different variants was still limited, and the rate at which new ones arose spontaneously in experimental colonies was exceedingly low: it is now known that, on average, only one gamete in 100,000 is likely to carry a detectable mutation at a particular locus.

    During the 1920s, several investigators began investigating the effects of X-rays on reproduction and development. In two laboratories, at least, new mutant alleles were recovered in the offspring of irradiated parents, but the investigators failed to make any connection between irradiation and the induction of these mutations (Little and Bagg, 1924 Dobrovolskaia-Zavadskaia, 1927). The connection was finally made by Muller who, in 1927, published his classic paper explaining the induction of heritable mutations by X-rays (Muller, 1927). Since that time, geneticists who study all of the major experimental organisms — from bacteria to mice — have used both ionizing irradiation and various chemicals as agents of mutagenesis to uncover novel alleles as tools for understanding gene function.

    Large-scale mouse mutagenesis experiments were first begun at two government-based "atomic energy" laboratories: the Oak Ridge National Laboratory in Oak Ridge, Tennessee, in the U.S. and the MRC Radiobiological Research Unit first at Edinburgh, Scotland, and then at Harwell, England, in the U.K. Both of these experimental programs were begun initially after World War II as a means for quantifying the effects of various forms of radiation on mice and, by extrapolation, humans, to better understand the consequences of detonating nuclear weapons. The U.S. effort was directed by W. L. Russell and the British effort was directed by T. C. Carter (Green and Roderick, 1966). Scientists at both laboratories quickly realized the potential of their newly created resource of mutant animals, and both laboratories have since gone on to generate mutations by chemical agents as well. The very large-scale studies conducted at Oak Ridge and Harwell — where 10,000 to 60,000 first generation animals were typically analyzed in an experimental protocol — have provided most of the empirical data currently available on the mechanisms and rates at which mutations are caused by all well-characterized mutagenic agents in the mouse.

    The experiments performed by Russell and Carter, and other colleagues who followed in their footsteps, were designed to obtain discrete values for the mutagenic potential of different radiation protocols. Rather than attempt to examine all animals for all effects of a particular irradiation protocol (as was common in earlier experiments), these mouse geneticists chose instead to look only at the small fraction of animals that were mutated at a small set of well-defined "specific" loci. The rationale for the "specific locus test" was that effects on individual loci could be more easily quantitated and that the limited results obtained could still be extrapolated for an estimate of whole genome effects. Russell decided that mutation rates should be followed simultaneously at a sufficient number of loci to distinguish and avoid problems that might be caused by locus-to-locus variations in sensitivity to particular mutagens. He decided further that the same set of loci should be examined in each experiment performed. The seven loci chosen to be followed in the specific locus test were defined by recessive mutations with visible homozygous phenotypes that were easily distinguished in isolation from each other, and had no effect on viability or fertility. The seven loci are agouti (a is the recessive non-agouti allele), brown (b), albino (c), dilute (d), short-ear (se), pink-eyed dilution (p), and piebald (s). A special "marker strain" was constructed that was homozygous for all seven loci.

    In its simplest form, the specific locus test is carried out by mating females from the special marker strain to completely wild-type males that have been previously exposed to a potential mutagen. In the absence of any mutations, offspring from this cross will not express any of the seven phenotypes visible in the marker strain mother. However, if the mutagen has induced a mutation at one of the specific loci, the associated mutant phenotype will be uncovered. This test is very efficient because it only requires a single generation of breeding and visual examination is all that is required to score each animal.

    Although recessive mutations at all loci other than the specific seven will go undetected in the first generation offspring from this cross, it is possible to detect a dominant mutation at any locus so long as it is viable and produces a gross alteration in heterozygous phenotype such as a skeletal or coat color change. One should realize that the most common effect of any undirected mutagen will be to "knock-out" a gene and, in the vast majority of cases, the resulting null allele will be recessive to the wild-type. There is, however, a very small class of loci at which null alleles will act in a dominant or semidominant fashion to wild-type. These "haplo-insufficient" phenotypes are presumably caused by a developmental sensitivity to gene product dosage. Among the best characterized of the dominant-null mutations are the numerous ones uncovered at the T locus — which result in a short tail — and the W locus — which result in white spotting on the coat.

    Mutations can be induced by both physical and chemical means. The physical means is through the exposure of the whole animal to ionizing radiation of one of three classes — X-rays, gamma rays, or neutrons (Green and Roderick, 1966). The chemical means is to inject a mutagenic reagent into the animal such that it passes directly into the gonads and into differentiating germ cells. The specific locus test has provided an estimate of the relative efficiency with which each reagent induces mutations. Under different protocols of exposure, X-irradiation was found to induce mutations at a rate of 13㬮 x 10 -5 per locus, which is a 20 to 100 fold increase over the spontaneous frequency, but still not high enough to be used by any but the largest facilities as a routine means for creating mutations (Rinchik, 1991). The mutations created by irradiation are often large deletions or other gross lesions such as translocations or complex rearrangements.

    The class of known chemical agents that can induce mutations (known as mutagens) is very large and expanding all the time. However, two chemicals in particular — ethylnitrosourea (ENU) and chlorambucil (CHL) — have been found to be extremely mutagenic in mouse spermatogenic cells (Russell et al., 1979 Russell et al., 1989). Both of these chemicals produce much higher yields of mutations than any form of radiation treatment tested to date (Russell et al., 1989). Optimal doses of either ENU or CHL can induce mutations at an average per locus frequency which is greater than one in a thousand — 150 x 10 -5 with ENU and 127 x 10 -5 per locus with CHL (Russell et al., 1982 Russell et al., 1989). Although the rates at which ENU and CHL induce mutations are very similar, the types of mutations that are induced are quite different. In general, ENU causes discrete lesions which are often point mutations (Popp et al., 1983), whereas CHL causes large lesions which are often multi-locus deletions (Rinchik et al., 1990a). Originally, it was thought that the basis for mutational differences of this type was the chemical nature of the mutagen itself (Green and Roderick, 1966), but, this no longer appears to be the case. Rather, it now appears that the germ cell stage in which the mutation arises is the major determinant of the lesion type (Russell, 1990). The correlation observed between chemical and lesion type is a result of the fact that different mutagens are active at different stages of spermatogenesis. Thus, ENU acts upon pre-meiotic spermatogonia where mutations are likely to be of the discrete type, and CHL acts upon post-meiotic round spermatids where mutations are likely to be of the large lesion type (Russell et al., 1990).

    ENU was the first chemical to be identified that was sufficiently mutagenic to be used by smaller laboratories in screens for mutations at particular loci or chromosomal regions of interest (Bode, 1984 Shedlovsky et al., 1988). ENU has also been used in screens for non-locus-specific phenotypic variants that could serve as models for various human diseases (McDonald et al., 1990). Several laboratories are beginning to use ENU for saturation mutagenesis of small chromosomal regions defined by deletions as one tool (among several complementary ones) for obtaining a complete physical and genetic description of such a region (Shedlovsky et al., 1988 Rinchik et al., 1990b Rinchik, 1991). The rationale for these studies is the belief that many (although not all) genes can be uncovered phenotypically by knock-out mutations which are bred to be doubly heterozygous with a deletion. The major limitation to the global use of this approach is the very small number of genomic regions in the mouse at which large deletions have been characterized.

    The availability to Drosophila geneticists of deletions (or deficiencies as the fly people call them) that span nearly every segment of the fly genome has played a critical role in the identification and characterization of large numbers of genes and the production of both gross functional maps and fine-structure point mutation maps by the very approach just described above. Clearly, a method to accumulate a similar library of deletions for the mouse would be well-received. The mutations induced by X-rays are often large-scale genomic alterations including translocations, inversions, and deletions. Indeed, most mouse deletion mutations maintained in contemporary stocks were derived in this manner. However, the overall yield of X-ray-induced deletions is quite low, and because of other problems inherent in this approach, it is not ideal for global use.

    In 1989, chlorambucil was reported to be an attractive alternative to X-rays as an agent for the high-yield induction of deletion mutations in the mouse (Russell et al., 1989). The per locus mutation rate was found to be on the order of one in 700 in germ cells of the early spermatid class, and of the eight mutations induced at this stage that were analyzed, all were deleted for DNA sequences around the specific locus marker (Rinchik et al., 1990a). This study also showed that CHL-induced mutations were often associated with reciprocal translocations. This last finding is unfortunate because translocations can reduce fertility with consequent negative effects on strain propagation.

    There is hope that CHL can be used as a means for generating sets of overlapping deletions that span entire chromosomes (Rinchik and Russell, 1990). Projects of this type will require very large animal facilities and support resources and will consequently be confined to only a handful of labs. However, once mouse strains with deletions have been created and characterized, they can serve as a resource for the entire community.

    6.1.3 Mouse Mutant Resources

    An advantage to using the mouse as a genetic system is the strong sense of community that envelops most of the workers in the field, and it is in the context of this community that strains carrying many different mutations — both spontaneous and mutagen-induced — have been catalogued and preserved and are made available to all investigators. A catalog containing detailed descriptions of all mouse mutations characterized as of 1989 has been compiled by Margaret Green and is included as the centerpiece chapter in the Genetic Variants and Strains of the Laboratory Mouse edited by Mary Lyon and Tony Searle (Green, 1989). This catalog is now available in an electronic form that is updated regularly (see Appendix B). Of course, many more mutant animals are found and characterized with the passing of each year, and an updated list is published annually in the journal Mouse Genome. This list contains information on the individual investigators that one should contact to actually obtain the mutant mice.

    The largest collection of mutant mouse strains is maintained at the Jackson Laboratory under the auspices of the "Mouse Mutant Resource" (MMR) which is currently maintained under the direction of Dr. Muriel Davisson (Davisson, 1990). In 1990, over 250 mutant genes were maintained in this resource, accounting for two-thirds of all known mouse mutants alive at the time (Davisson, 1990). Each year, animal caretakers identify an additional 75 to 80 "deviant" animals among the two million mice that are produced by the Jackson Laboratory’s Animal Resources colonies (Davisson, 1993). Approximately 75% of the deviant phenotypes are found to have a genetic basis and breeding studies are conducted on these to determine whether or not they represent mutations at previously characterized loci. If they do, DNA samples are recovered and the lines are discarded or placed into the Frozen Embryo Repository. If a mutation is novel, its mode of transmission (autosomal/X-linked, dominant/recessive) is determined, the phenotypic effect of the mutation is characterized, and it is mapped to a specific chromosomal location with the use of breeding protocols to be described in section 9.4 (Davisson, 1990 Davisson, 1993). Descriptions of all newly characterized mutations are publicized, and mutant mouse strains are made available for purchase through the standard Jackson Laboratory catalog. In 1992, over 35,000 mice from the MMR were distributed to investigators throughout the world (Davisson, 1993).

    Space limitations make it impossible for the MMR to maintain breeding stocks of mice that contain every known mutant gene, with the total number expanding each year. Fortunately, mutant stocks that are not currently in demand by investigators can be maintained (at minimal cost) in the form of frozen embryos. The importance of embryo freezing as a storage protocol cannot be over-emphasized. Time and time again, modern-day molecular researchers have reached back to use mutations described long-ago as critical tools in the analysis of newly cloned human and mouse loci.

    6.2 Embryo manipulation: genetic considerations

    6.2.1 Experimental possibilities

    The basic technology required to obtain preimplantation embryos from the female reproductive tract, to culture them for short periods of time in petri dishes, and then to place them back into foster mothers where they can grow and develop into viable mice has been available since the 1950s (Hogan et al., 1994). Over the ensuing years, this basic technology has been used in a host of different types of experiments aimed at manipulating the process of development or the embryonic genome itself. Embryos can be dissolved into individual cells that can be recombined in new combinations to initiate the development of chimeric mice. Pronuclei and nuclei can be switched from one early embryo to another to examine the relative contributions of the cytoplasm and the genome to particular phenotypes, as well as to investigate aspects of genomic imprinting and parthenogenesis. Foreign DNA can be injected directly into pronuclei for stable integration into chromosomes which can lead to the formation of transgenic animals. Finally, embryonic cells can be converted into tissue culture cells (called embryonic stem [ES] cells) where targeted gene replacement can be accomplished. Selected ES cells can be combined with normal embryos to form chimeric animals that can pass the targeted locus through their germline. These experimental possibilities are discussed more fully later in this chapter. This section is concerned simply with genetic considerations involved in the choice of mice to be used for the generation and gestation of embryos for various experimental purposes.

    6.2.2 Choice of strains for egg production

    6.2.2.1 General considerations

    A number of factors will play a role in the selection of an appropriate strain of females who will contribute the eggs to be used as experimental material. First, in all cases, it is important that the eggs are hardy enough to resist damage from the manipulations that they will undergo. Second, the particular experimental protocol may impose a need for eggs that have special genetically-determined qualities. Third, in those case where very large numbers of eggs are required, it will be important that the strain is one that responds well to superovulation, as discussed in the next section. Finally, there is a question of genetic restrictions on the offspring that will emerge from the manipulation.

    As concerns this last criterion, for some experiments it will be important to maintain strict control over the genetic background of embryos to be used for genomic manipulation. In these cases, inbred embryos should be derived from matings between two members of the same inbred strain. If these embryos are used for germline introduction of foreign genetic material, the resulting transgenic animals will be truly coisogenic to the original inbred strain.

    For other experiments, strict genetic homogeneity will not be required. In these cases, it is possible to use F 2 embryos from superovulated F 1 females who have been mated to F 1 males of the same autosomal genotype. This breeding protocol is often preferable to the use of either a strictly inbred approach or a random-bred approach. First, in contrast to the random-bred approach, one still maintains a certain degree of control over the genetic input since only alleles derived from one or both of the inbred strains used to generate the F 1 parents will be present at any locus in each embryo. Second, in contrast to the inbred approach, the use of both females and embryos with heterozygous genotypes allows the expression of hybrid vigor at all levels of the reproductive process. In particular, heterozygous embryos are less likely to be injured by in vitro manipulations.

    6.2.2.2 The FVB/N strain is ideal for the production of transgenic mice

    One inbred strain that has been developed relatively recently from a non-inbred colony of mice with a long history of laboratory breeding at NIH has special characteristics of particular interest to investigators interested in producing transgenic mice: this strain is called FVB/N. The FVB/N strain is unique in several important ways (Taketo et al., 1991). First, its average litter size of 9.5 (with a range up to 13) is significantly higher than that found with any other well-known inbred strain (see Table 4.1). Second, fertilized eggs derived from FVB/N mothers have very large and visually prominent pronuclei this characteristic is unique among the known inbred strains and greatly facilitates the injection of DNA. Finally, the fraction of injected embryos that survive into live born animals is also much greater than that observed with all other inbred strains. For these reasons, FVB/N has quickly become the strain of choice for use in the production of transgenic animals.

    6.2.3 Optimizing embryo production by superovulation

    Although one can recover on the order of 6 to 10 eggs directly from individual naturally-mated inbred or F 1 females, it is possible to obtain much larger numbers — up to 60 eggs per animal — by inducing a state of superovulation. For many experiments, it is important to begin with a large number of embryos with superovulation, one can drastically reduce the number of females required to produce this large number. Superovulation is induced by administering two precisely-timed intraperitoneal injections of commercially-available gonadotropin reagents which mimic natural mouse hormones and initiate the maturation of an aberrantly large number of egg follicles. Superovulation, like normal ovulation, causes both a stimulation of male interest in mating as well as female receptivity to interested males. The protocol is described in detail in the mouse embryology manual by Hogan and colleagues (1994).

    Not unexpectedly, the average number of eggs induced by superovulation is highly strain-dependent. Appropriately aged females of the strains B6, BALB/cByJ, 129/SvJ, CBA/CaJ, SJL/J and C58/J can be induced to ovulate 40 to 60 eggs (Hogan et al., 1994). At the other extreme, females of the strains A/J, C3H/HeJ, BALB/cJ, 129/J 129/ReJ, DBA/2J, and C57L/J do not respond well to the superovulation protocol, producing only 15 or fewer eggs per mouse. The response of the FVB/N strain to superovulation is in-between with the production of 25 embryos or fewer per female (Taketo et al., 1991). For generating transgenic mice, however, this single negative feature of FVB/N is outweighed by the other characteristics of this strain discussed above.

    An interesting aspect of the high versus low response to superovulation is that in two cases, substrains derived from the same original inbred strain (BALB/cByJ versus BALB/cJ and 129/SvJ versus 129/J) express such clearly distinct phenotypes. This finding suggests that subtle changes in genotype can have dramatic consequences on the expression of this particular reproductive trait.

    One critical finding of both practical and theoretical importance is that F 1 hybrid females do not always express a better response to superovulation then both of their inbred parents. For example, the commonly used F 1 hybrid B6D2F 1 , which is formed by a cross between a high ovulator (B6) and a low ovulator (DBA/2J), expresses the low ovulator phenotype (Hogan et al., 1994). This observation goes against the grain of hybrid vigor and it suggests that the genetic basis for this phenotype may be much more specific and limited than it is for other general viability and fertility phenotypes. In addition, this observation suggests that for the major genes involved, the "high ovulatory" alleles are recessive.

    Two F 1 hybrids have been determined empirically to express a high level superovulation — [BALB/cByJ x B6] and [B6 x CBA/CaJ] (Hogan et al., 1994). It is also very likely that F 1 hybrids derived from matings between any of the high responders listed above will themselves be high responders as well. Many of these F 1 hybrids can be purchased directly from animal suppliers however, in most cases, suppliers cannot provide an exact day of birth which is necessary to determine the optimal time of use.

    6.2.4 The fertile stud male

    Females that have undergone ovulation — either naturally or induced — must be mated with a "fertile stud male" to produce zygotes that can be used for nuclear injection or other purposes. As discussed earlier, it is always preferable to use a fertile stud male with the same genotype as the female, whether it is inbred or an F 1 hybrid. Obviously, it is important to use visibly healthy animals in the prime of their life, between 2 and 8 months of age. In addition, past experience is often a good indicator of future performance. Males that have mated successfully on demand in the past (as indicated by a vaginal plug) are likely to do the same in the future for this reason, records should be maintained on the performance of each male used for this purpose. For optimal results, one should place only one male and one female in each cage, and after a successful mating, the male should be given a rest of two to three days.

    6.2.5 Embryo transfer into foster mothers

    6.2.3.1 Choice of strain

    Once embryos have been manipulated in culture, they must be placed back into the reproductive tract of a foster mother where they can continue their development into fully-formed live-born animals. Since the foster mother contributes only a womb, and not genomic material, to the engineered offspring, her genetic constituency should not be chosen according to the same criteria used for animals in most other experimental protocols. Only two considerations are important in the choice of a foster mother. First, and most important, she should have optimal reproductive fitness and "mothering" characteristics. This can be accomplished with either an F 1 hybrid between two standard inbred strains [B6 x CBA is recommended (Hogan et al., 1994), but others will do as well] or with outbred strains available from various commercial breeders. The best foster mothers are those that have already borne and raised at least one litter successfully thus, it is often useful to try out potential candidates by putting them through one pregnancy/mothering/weaning cycle.

    A second consideration is whether the investigator will be able to distinguish natural-born pups from those that have been fostered. This is only a factor when the foster mother has been mated to a sterile male in order to induce the required state of pseudopregnancy, and there is some question as to whether the male has been properly sterilized. The simplest method for distinguishing the two types of potential offspring is by a coat color difference for example, albino versus pigmented. If the experimental embryos are derived from non-albino-allele-carrying parents, then both the foster mother as well as her sterile stud partner (discussed below) can be chosen from commercially available outbred albino strains such as CD-1 (Charles River Breeding Laboratories) or Swiss Webster mice (from Taconic Farms). When one is certain that the sterile stud male is really sterile, coat color differences are less critical, so long as well-defined DNA differences exist if the unexpected need does arise to distinguish the genotypes of potential natural-born offspring from experimentally transferred offspring.

    6.2.3.2 Induction of pseudopregnancy and the sterile stud male

    In human females, the uterine environment becomes receptive to the implantation of fertilized eggs as a direct consequence of the hormonal induction of ovulation. In mice and most other non-primate mammals, the uterine environment becomes receptive to implantation only in response to a sufficient degree of sexual stimulation. In addition, this stimulation also causes hormonal changes which alter the normal estrus cycle under the assumption that a pregnancy will ensue. When a successful stimulatory response has occurred in the absence of fertilization, the female is said to be in a state of "pseudopregnancy." Only pseudopregnant females will allow the successful implantation and development of fostered embryos. Pseudopregnancy can be achieved in one of two ways: (1) by mating to a sterile male or (2) through the use of female masturbation tools such as vibrating rods inserted into the vagina (West et al., 1977). Most investigators have found that natural matings produce a higher percentage of pseudopregnancies than human surrogates.

    Sterile males can be derived genetically or surgically. Genetic derivation requires a breeding colony of mice that are doubly heterozygous for pseudo-allelic mutations on chromosome 17 in a region known as the t complex (Silver, 1985). Animals with the genotype T/t w2 (available from the Jackson Laboratory) are intercrossed for the purposes of both maintaining the strain as well as for the production of sterile males as diagrammed in figure 6.1.

    For those without the resources or personnel required to breed genetically sterile males, the only other choice is surgical vasectomy, which involves the severing of the vas deferens on both sides of the body (Hogan et al., 1994). The choice of mouse strain to use is based on criteria analogous to those set out for the choice of a foster mother, except that mating ability should be considered in place of mothering ability. The standard F 1 hybrids as well as "random-bred" animals can all be used with success. When it comes to choosing between individual males within a particular strain, one should use the same criteria described in section 6.2.2.2 for the choice of fertile stud males. In addition, pre-mating "sterile" males with fertile females serves to confirm the success of the vasectomy.

    6.3 Transgenic mice formed by nuclear injection

    There are two problems inherent in all methods of classical mutagenesis. The first problem is that the process is entirely random. Thus, one must start out by designing a screening assay to allow the detection of mutations at the locus of interest, and then one must hope for the appearance of mutant animals at a frequency which is at, or above, the usual per-locus rate. If a mutant allele fails to produce a phenotype that can be picked up by the screen, it will go undetected. Finally, even when mutant alleles are detected, the underlying lesion can usually not be ascertained without cloning and further molecular characterization.

    The second problem with classical mutagenesis is that induced mutations are not tagged in any way to provide a molecular entry into a locus that has not yet been cloned. Thus, if a novel locus is uncovered by an induced mutation that causes an interesting phenotype, it can only be approached through candidate gene and positional cloning approaches in the same way as any other phenotypically-defined locus. Furthermore, in the case of ENU-induced mutations, the mutant and wild-type alleles are likely to be molecularly indistinguishable with the exception of a single nucleotide that may or may not affect a restriction site.

    One can imagine two types of mutagenic approaches that would be most ideal for the two different types of situations in which mutations can provide tools for molecular analysis of development and other aspects of mammalian biology. On the one hand, a random mutagenesis approach is fine for the elucidation of novel loci so long as the mutant allele is tagged to allow direct molecular access. On the other hand, to further analyze a locus which is already cloned and characterized, one would like to generate animals that miss-express the locus in some defined manner. The technologies of transgene insertion and gene targeting have provided geneticists with the tools needed to accomplish both of these goals.

    In 1981, five independent laboratories reported the insertion of foreign DNA into the mouse germ line through the microinjection of one-cell eggs (Costantini and Lacy, 1981 Gordon and Ruddle, 1981 Harbers et al., 1981 Wagner et al., 1981a Wagner et al., 1981b). Although the incorporation of exogenous DNA into the germ line through viral infection of embryos had been reported earlier (Jaenisch, 1976), the 1981 reports implied for the first time that DNA from any source could be used to transform the mouse genome. The complexion of mouse genetics was changed forever with the development of this powerful tool. A strictly observational science was suddenly thrust into the realm of genetic engineering with all of its vast implications. The insertion of genetic material into the mouse germ line has now become sufficiently routine that the methodology is detailed in various "cookbooks" (Wassarman and DePamphilis, 1993 Hogan et al., 1994) and designer animals are even provided as a commercial service by a number of companies.

    The term transgenic has been coined to describe animals that have foreign sequences inserted stably into their genome through human intermediaries. Transgenic animals can be created by microinjection or viral infection of embryos, or through the manipulation in culture of embryonic-like "ES cells" that are subsequently incorporated back into the embryo proper for shepherding into the germ line. The latter technology will be discussed in a following section. Here I will focus on transgenic animals created by direct injection of DNA into embryos.

    The initial animal that develops from each micro-manipulated egg is called a founder. Even when multiple embryos have all been injected or infected with the same foreign DNA, the integration site — or transgene locus — in each founder will be different. However, all transgenic animals that descend from a single founder will share the same transgene locus. Protocols for the creation of transgenic mice, and extensive reviews of the technology and its uses have been described elsewhere (Palmiter and Brinster, 1986 Wassarman and DePamphilis, 1993 Hogan et al., 1994). Rules for naming transgene loci and transgenic animals are presented in section 3.3.5.

    With current protocols for the creation of transgenic mice by embryo microinjection, the site of integration is not pre-determined, and, for all practical purposes, should be considered random. Microinjection allows one to add, but not subtract genetic material in a directed manner if a particular experiment leads to the insertion of an novel version of a mouse gene into the genome, this novel allele will be present in addition to the normal diploid pair. Consequently, only dominant, or co-dominant, forms of phenotypic expression will be detectable from the transgene.

    The embryo microinjection technology can be used to explore many different aspects of mouse biology and gene regulation. One class of experiments encompasses those aimed at determining the effects of expressing a natural gene product in an unnatural manner. By combining the gene of interest with regulatory regions chosen from other genes, one can cause transgenic mice to express the product at a higher than normal level, or in alternative tissues or developmental stages. The mutant phenotypes that result from such aberrant forms of expression can be used to elucidate the normal function of the wild-type gene. Experiments of this type can be used, for example, to demonstrate the capacity of some genes to induce specific developmental changes and the oncogenic nature of others when they’re aberrantly expressed. Many other types of questions can be answered with this approach.

    In another class of experiments, one can dissect out the function of a regulatory region by forming constructs between it and a reporter gene whose expression can be easily assayed in the appropriate tissue(s). With a series of transgenic lines that have partially-deleted or mutated forms of a regulatory region, one can pinpoint which DNA sequences are involved in the turning-on and turning-off of genes in different tissues or developmental stages.

    A third of class of experiments is aimed at correcting a genetic defect in a mutant mouse through the genomic insertion of a wild-type transgene. This use of the transgenic technology provides the most powerful means available to prove that a cloned candidate gene is indeed identical to the locus responsible for a particular mutant phenotype. Furthermore, the correction of genetic defects in model mammals is a necessary prelude to any attempt to perform similar studies in humans.

    An important consideration in all transgenic experiments follows from the observation that the actual chromosomal location at which a transgene inserts can play a determining role in its expression. This will be readily apparent in cases where different founder lines with the same transgene show different patterns of transgene expression. The reason for such strain-specific differences is that some chromosomal regions are normally maintained in chromatin configurations that can act to suppress gene activity. Different transgene constructs will show different levels of sensitivity to suppression of activity when they land in such regions.

    Another potential problem can result from the insertion of the transgene into a normally-functioning endogenous locus with unanticipated consequences. In approximately 5 to 10% of all cases studied to date, homozygosity for a particular transgene locus has been found to cause lethality or some other phenotypic anomaly (Palmiter and Brinster, 1986). These recessive phenotypes are most likely due to the disruption of some normal vital gene. In less frequent cases, a transgene may land at a site that is flanked by an endogenous enhancer which can stimulate gene activity at inappropriate stages or tissues. This can lead to the expression of dominant phenotypes that are not strictly a result of the transgene itself. For all of these reasons, it is critical to analyze data from three or more founder lines with the same transgene construct before reaching conclusions concerning the effect, or lack thereof, on the mouse phenotype.

    In the vast majority of cases analyzed to date, the disruption of endogenous sequences caused by transgene integration has had no apparent effect on phenotype. However, the absence of a detectable phenotype does not necessarily mean that the transgene has integrated into a non-functional region of the genome. As discussed in chapter 5, only a small subset of all mammalian genes are actually vital, and subtle effects on phenotype are likely to go unnoticed if one performs only a cursory examination of transgenic animals. Thus, the actual frequency of insertional mutagenesis resulting from embryo microinjection is likely to be significantly higher than the numbers imply.

    6.3.2 Tracking the transgene and detecting homozygotes

    Unless a particular transgenic insertion causes an easily detectable, dominant phenotype, the presence of the transgene in an animal is most readily determined through DNA analysis. For testing large numbers of mice, the best source of DNA is from tail clippings or ear punch-outs (Gendron-Maguire and Gridley, 1993). The presence or absence of the transgene can be demonstrated most efficiently with a method of PCR analysis that is based on transgene-specific target sequences.

    The founder animal for a transgenic line will be heterozygous for the transgene insertion locus. The second homolog will be associated with a non-disrupted "wild-type" (+) allele at this locus, whereas the disrupted chromosome will carry a transgene (Tg) allele. As long as the transgene is transmitted to offspring from a heterozygous (Tg/+) parent, it will be necessary to test each individual animal of each new generation for the presence of the transgene. For this reason alone, it would be useful to generate animals homozygous for the transgene allele since all offspring from matings between homozygous Tg/Tg animals would also be homozygous and there would be no need for DNA testing.

    In rare cases, homozygous Tg/Tg animals will be phenotypically distinct from their Tg/+ cohorts. This observation is usually a good indication that the transgene has disrupted the function of an endogenous locus through the process of integration. If the homozygous recessive phenotype is lethal, it will obviously be impossible to generate a homozygous line of animals. Otherwise, the phenotype may eliminate the need for DNA analysis. In the vast majority of cases, however, homozygous Tg/Tg animals will be indistinguishable in phenotype from heterozygous Tg/+ animals, and without a recessive phenotype, the identification of homozygous animals will not be straightforward.

    One approach to confirming the genotype of a presumptive Tg/Tg animal is based on statistical genetics. In this case, confirmation is accomplished by setting up a mating between the presumptive homozygote and a non-transgenic +/+ partner. If the animal in question is only a Tg/+ heterozygote, one would expect equal numbers of Tg/+ and +/+ offspring. Through the method of chi square analysis described in section 9.1.3, one can calculate that if at least 13 offspring are born and all carry the transgene, the probability of a heterozygous genotype is less than one in a thousand. If even a single animal is obtained without the transgene, the parent’s genotype will almost certainly be Tg/+. Statistical testing of this kind must be performed independently for each presumptive Tg/Tg animal. Once homozygous Tg/Tg males and females have been confirmed, they can be re-mated to each other as the founders for a homozygous transgenic strain.

    A second approach to demonstrating transgene homozygosity requires the cloning of a endogenous sequence from the mouse genome that flanks the transgene insertion site. This task is often not straightforward because transgenic material can be present in multiple copies that are intermingled with locally rearranged endogenous sequences. Nevertheless, with the cloning of any nearby endogenous sequence, one obtains a mapping tool that, in theory, can be used to distinguish both the disrputed and nondisrupted alleles at the transgene locus through the use of one of the various techniques described in chapter 8 for detecting "codominant" DNA polymorphisms. With such a tool, and an associated assay, homozygosity for the transgene allele would be demonstrated by the absence of the wild-type nondisrupted allele. Unfortunately, this approach would require the generation of a separate endogenous clone for each and every transgenic line to be studied. Protocols for locating the transgene insertion site within the mouse linkage map are discussed in section 7.3.2.

    6.4 Targeted mutagenesis and gene replacement

    Although the transgene insertion technology described in the previous section provides a powerful tool for the analysis of gene action in the whole organism, it has one serious limitation in that it does not provide a mechanism for the directed generation of recessive alleles. This limitation can be overcome with a technology known variously as gene targeting, targeted mutagenesis, or gene replacement — the subject of this section. This powerful technology allows investigators to generate directed mutations at any cloned locus. These new mutant alleles can be passed through the germ line to produce an unlimited number of mutant offspring, and different mutations can be combined with variants at other loci to study gene interactions.

    This ultimate tool of genetic engineering was born through the combination of several technologies that had developed independently over the preceding 10 to 20 years including embryonic stem cell culture and homologous recombination, with mouse embryo manipulation and chimera formation (Sedivy and Joyner, 1992 provide an excellent review of all aspects of this field). Two independent laboratories, headed by Oliver Smithies and Mario Cappechi, finally succeeded in bringing all of these various technologies together during the mid-1980s (Capecchi, 1989 Smithies, 1993).

    One, although not the only, appeal of the gene targeting technology is the ability to create mouse models for particular human diseases (Smithies, 1993). But, in essence, gene targeting can provide investigators with powerful tools to study any cloned gene. While patterns of RNA and protein expression provide clues to the stages and tissues in which genes are active, it is only with mutations that a true understanding of function can be obtained (Chisaka and Capecchi, 1991).

    After heaping such praises on gene targeting, it is important to forewarn potential users of this technology that its application is not problem-free. First, an investigator must achieve a high level of competence and experience with several distinct, technically demanding protocols this requires a significant investment of time and energy. Second, there is the fickle nature of the technology itself as discussed below. Nevertheless, the handful of laboratories initially able to target genes successfully has expanded quickly with the training of new young investigators, and this expansion is likely to continue much further with the recent publication of several excellent volumes containing detailed chapters on experimental protocols (Joyner, 1993 Wassarman and DePamphilis, 1993 Hogan et al., 1994).

    6.4.2 Creating ‘gene knockouts’

    Once a particular gene has been cloned and characterized, the steps involved in obtaining a mouse with a null mutation in the corresponding locus can be outlined briefly as follows. First, one must design and construct an appropriate targeting vector in which the gene of interest has been disrupted with a positive selectable marker in the most commonly used protocol, a negative selectable marker is also added at a position that flanks the gene sequence. The most commonly used positive selectable marker is the neomycin resistance (neo) gene, and the most commonly used negative selectable marker is the thymidine kinase (tk) gene.

    The second step involves the introduction of the targeting vector into a culture of embryonic stem (ES) cells (usually derived from the 129 strain) followed by selection for those cells in which the internal positive selectable marker has become integrated into the genome without the flanking negative selectable marker. The third step involves screening for clones that have integrated the vector by homologous recombination rather than by the more common non-homologous recombination in random genomic sites. Once "targeted clones" have been identified, the fourth step involves the production of chimeric embryos through the injection of the mutated ES cells into the inner cavity of a blastocyst (usually of the B6 strain), and the placement of these chimeric embryos back into foster mothers who bring them to term. A recently developed alternative approach to chimera formation through the aggregation and spontaneous incorpation of ES cells into cleavage stage embryos has the advantage of not requiring sophisticated microinjection equipment (Wood et al., 1993).

    The experiment is deemed a success if the ES cells successfully enter the germline of the chimeric animals as demonstrated by breeding. If the disrupted gene is indeed transmitted through the germline, the first generation of offspring from the chimeric founder will include heterozygous animals that can be intercrossed to produce a second generation with individuals homozygous for the mutated gene. The nomenclature rules that are used to name all newly created mutations are described in section 3.4.4.

    6.4.3 Creating subtle changes

    A second generation of homologous recombination strategies have been developed to allow the placement of specific small mutations into a locus without the concomitant presence of disrupting intragenic selectable markers. The ability to create subtle changes in a gene could provide an investigator with the tools required to dissect apart the function of a gene product one amino acid at a time. A number of different approaches toward this goal have been described. The most promising of these, called "hit and run," is based on the generation of ES cell lines that have undergone homologous recombination with a targeting vector, followed by selection for an intrachromosomal recombination event that eliminates the selectable markers and leaves behind just the mutated form of the gene (Joyner et al., 1989 Hasty et al., 1991 Valancius and Smithies, 1991 Fiering et al., 1993).

    Unfortunately, at the time of this writing, the hit and run protocols are still extremely demanding and with each experiment, an investigator will only obtain a single mutant allele at the locus of interest. An alternative strategy is to break the problem into two separate tasks: (1) knocking-out the gene completely in one strain by standard homologous recombination, and (2) the independent production of one or more transgenic lines that contain subtly-altered mutant versions of the gene. By breeding the knock-out line with one of the transgenic lines, it becomes possible to generate a new line of animals in which the original wild-type allele has been replaced (although not at the same site) with a specially-designed transgene allele. There are several advantages to this approach. First, the methodology required for simply knocking-out a gene is more straightforward and better developed at the time of this writing than the hit-and-run methodology. Second, gene targeting in ES cells requires much more time and effort than the production of transgenic mice by nuclear injection. Thus, when an investigator wishes to study a variety of alleles at a particular locus, it will be much easier to create a single line of mice by gene targeting and then breed it to different transgenic lines. The one potential disadvantage to this approach is that the transgene construct may not be regulated properly and accurate patterns of expression may not occur in the animal, even when the transgene is linked to its own promoter/enhancer.

    Even when a laboratory has mastered all of the protocols required to perform gene targeting, the difference between success and failure can still be a matter of luck. Some DNA sites appear highly impervious to homologous recombination, whereas sites a few kilobases away may be much more open to integration. But even at the same site, the frequency of homologous versus non-homologous recombination events can vary by a factor of ten from one day to the next (Snouwaert et al., 1992).

    At the time of this writing, many of the factors responsible for success remain unknown. However, one critical factor that has recently become evident is the need to use source DNA for the targeting construct that has been cloned from the same strain of mice used for the derivation of the ES cell line into which the construct will be placed (van Deursen and Wieringa, 1992). In other words, the highest levels of gene replacement are obtained when the incoming DNA is isogenic with the target DNA. Apparently, the homologous recombination process is very sensitive to the infrequent nucleotide polymorphisms that are likely to distinguish different inbred strains from each other. In most cases today, ES cell lines have been derived from the 129/SvJ mouse strain (but see the subsection 6.4.5 below), and thus it is usually wise to build DNA constructs with clones obtained from 129 genomic libraries.

    The original 129/SvJ mouse, and the one still available from the Jackson Laboratory, has an off-white coat color caused by homozygosity for the pink-eye dilution (p) mutation, and forced heterozygosity for the chinchila (c ch ) and albino (c) alleles at the linked albino locus (cch p/c p). In contrast, the "129 mouse" that serves as the source of most ES cell lines used for homologous recombination has a wild-type agouti coat color. What is the basis for this difference?

    The answer is a historical one that centers on the work of Leroy Stevens, a cancer geneticist, now retired, who worked at the Jackson Laboratory. Stevens had observed that the 129/SvJ strain was unique in the occurrence of spontaneous testicular teratomas at an unusually high rate of 3 to 5% in male animals. As means to better understand the genetic parameters responsible for tumor incidence, Stevens set out to determine whether any of a variety of well-characterized single locus mutations that affect either tumor incidence or germ cell differentiation would interact with the 129 genome in a manner to increase or decrease the natural frequency of tumor formation in this strain. One of the mutations that he tested was Steel (Sl), which plays an important role in the differentiation of germ cells as well as melanocytes and hematopoetic cells. The Sl mutation expresses a dominant visible phenotype — the lightening of the normal wild-type black agouti coat color so that it has a "steely" appearance, and a reduction of pigment in the distal half of the tail. Unfortunately, it is impossible to see this phenotypic alteration on the cch p/c p coat of 129/SvJ mice which already have a nearly complete loss of pigment production. Thus, to follow the backcrossing of Sl onto 129/SvJ, it was necessary to replace the mutant alleles at the c and p loci with wild-type alleles. It is the triple congenic 129/Sv-Sl/+ , + c + p line produced by Stevens that acted as the founder for all "129 mice" that have been used in ES cell work.

    6.5 Further uses of transgenic technologies

    6.5.1 Insertional mutagenesis and gene trapping

    As indicated earlier in this chapter, one side product of many transgenic experiments is the generation of mice in which a transgene insertion has disrupted an endogenous gene with a consequent effect on phenotype. Unlike spontaneous or mutagen-induced mutations, "insertional mutations" of this type are directly amenable to molecular analysis because the disrupted locus is tagged with the transgene construct. Unexpected insertional mutations have provided instant molecular handles not only for interesting new loci but for classical loci, as well, that had not been cloned previously (Meisler, 1992).

    When insertional mutagenesis, rather than the analysis of a particular transgene construct, is the goal of an experiment, one can use alternative experimental protocols that are geared directly toward gene disruption. The main strategies currently in use are based on the introduction into ES cells of beta-galactosidase reporter constructs that either lack a promoter (Gossler et al., 1989 Friedrich and Soriano, 1991) or are disrupted by an intron (Kramer and Erickson, 1981). The constructs can be introduced by DNA transfection or within the context of a retrovirus (Robertson, 1991). It is only when a construct integrates into a gene undergoing transcriptional activity that functional beta-galactosidase is produced, and producing cells can be easily recognized by a color assay. Of course, the production of "beta-gal" will usually mean that the normal product of the disrupted gene can not be made and thus, this protocol provides a means for the direct isolation of ES cells with tagged mutations in genes that function in embryonic cells. Mutant cells can be incorporated into chimeric embryos for the ultimate production of homozygous mutant animals that will display the phenotype caused by the absence of the disrupted locus. This entire technology, referred to as "gene trapping" (Joyner et al., 1992), is clearly superior to traditional methods for the production of mutations at novel loci that use chemical mutagens or irradiation.

    6.5.2 A database and a repository of genetically engineered mice

    A computerized database (called TBASE) has been developed to help investigators catalog the strains that they produce and find potentially useful strains produced by others (Woychik et al., 1993). The database is available over the Internet through the Johns Hopkins Computational Biology Gopher Server and is linked to the on-line mouse databases maintained by the Jackson Laboratory Informatics Group.

    Although the gene replacement technology has been employed with success by more and more laboratories, it is still the case, and likely to remain so, that an enormous amount of time and effort goes into the production of each newly engineered mouse strain. Clearly, it does not make sense to derive strains with the same gene replacement more than once. However, with the high costs of animal care and maintenance, it is often difficult for researchers to maintain strains that they are no longer actively using. Furthermore, many individual research colonies are contaminated by various viruses, and as such, virus-free facilities are reluctant to import mice from anywhere other than reputable dealers. The Jackson Laboratory has recently come to the rescue by setting up a clearing house to preserve what are likely to be the most useful of these strains for other members of the worldwide research community. For the first time, JAX will be importing mice from large numbers of individual researchers. Each strain will be re-derived by cesarean section into a germ-free barrier facility, and will be made available for a nominal cost, without experimental restriction, to all members of the research community.

    With the various technologies that have now been developed to manipulate the genomes of embryonic cells combined with ever-more sophisticated molecular tools, it can be stated without exaggeration that the sky is the limit for what can be accomplished with the mouse as a model genetic system. It is always impossible to predict what the future holds, but one can imagine the use of both gene addition and gene replacement technologies as routine tools for assessing the functions of sequential segments of DNA obtained by walking along each mouse and human chromosome. With recent reports of success in the insertion of intact YAC-sized DNA molecules of 250 kb or more in length into the germline of transgenic animals, it becomes feasible to analyze even larger chunks of DNA for the presence of interesting genetic elements (Jakobovits et al., 1993 Schedl et al., 1993). In fact, it is only with experiments of this type that it will be possible to completely uncover all of the pathways through which a gene is regulated, and all of the pathways through which a gene product may function. Just as the study of neurons in isolation can not possibly provide a clue to human consciousness, the study of individual genes outside of the whole animal can not possibly provide a clue to the network of interactions required for the growth and development of a whole mouse or person.


    A new lease of life

    Some say no, cloning is playing God and will end badly. Natural selection means the strong survive, and the weak die out. Cloning interferes with this process and will not work. Even if we successfully clone extinct or endangered animals, they would not survive in the wild. And if they do, they will disrupt existing ecosystems and endanger other species.

    Others say yes, we don’t just have a duty to do it: our planet depends on reversing the rate of extinction. Humans have degraded habitats and killed off thousands of species. As biodiversity declines, the remaining species become even more vulnerable to disease, environmental disaster and climate change. By diversifying the gene pool, cloning may safeguard the future of life on Earth.


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