cloning may make possible some extraordinary advances in the science of genetic engineering

8/2/2019 Cloning may make possible some extraordinary advances in the science of genetic engineering

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Cloning may make possible some extraordinary advances in the science of genetic

engineering. It may speed up mammalian research, and make possible the introduction of

desired traits into higher mammals. There are many different levels at which geneticengineering may occur, some of which have been going on for a long time. Read my

summary of one way in which cloning may make possible new advances in genetic

engineering. This page provided courtesy of:

The BioFact Report

Frequently Asked Questions

Tell me about thecloning of sheep, cows, and now mice!

How can I clone humans? Cloning of humans might be illegal in the United States,

so do so at your own risk. The information provided here is an actual recipe based

on current technology, but there might be problems, or better methods.

Forget serious cloning information, I want to read some Cloning Jokes!

Types of Genetic Engineering

Natural Selection, nature's own genetic engineering.

Selective Breeding, our success in altering the course of natural selection.

Genetic Manipulation, the current state of the art in genetic engineering.

True Genetic Engineering, the next step.

Playing God! If you're offended by genetic engineering, read what's theoreticallypossible. Beyond the best genetic engineering we can currently devise is much,

much more: the re-engineering of life itself. Other Bio-technology, Human Cloning and Genetic Engineering Links.

Natural Selection

Natural Selection is nature's own form of genetic engineering. The most fit organisms

survive through natural selection. The rate of evolution of new species through natural

selection is incredibly slow, but methods have been discovered by which nature has

optimized the process.

The entire genome (all the genes) of higher animals and plants are broken up intofunctional components known as exons and separated by regions called introns. Special

genes known as transposable elements serve to mix and match functional components of

genes in an effort to maximize the likelyhood of creating better genes and organisms. Thereis some evidence that bacteria, one of the simplest organisms, had introns and exons in

some past era, but lost them in favor of efficiency and other means of acquiring new DNA.

Selective Breeding
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Selective Breeding or "Unnatural Selection", is man's most basic effort at genetic

engineering by creating our own selective pressures. Many conventional farm animals,

domesticated dogs and cats were likely created ages ago by selectively breeding animalstogether with desired traits. Gregor Mendel helped to establish the rules of genetics through

his work selectively breeding plants in the 1800's. Selective Breeding has worked well for

engineering animals and plants, but it can take whole human lifetimes to bring about smallchanges in a species.

Through unnatural selection certain attributes and characteristics can be enhanced by

selectively killing all organisms that do not have the desired traits. This has been suggested

by some as a viable option for genetically engineering humans. Parents could produce alarge number of fertilized eggs through in vitro fertilization. Each could be grown for a

while in vitro and then be tested for desired traits. Only an egg with all the traits desired by

the parents would then be implanted in the mother. There are obvious drawbacks, not theleast of which is the large number of fertilized eggs that are not selected. This option is not

a viable alternative for many couples for religious reasons.

Another drawback is that selecting for a very large number of traits is close to impossible.

Each gene desired at least doubles the number of fertilized eggs required. Certain traits arethe result of many genes acting in concert, which could inflate egg requirements very

quickly. Last of all, fertilized eggs must have one copy of each gene from each parent.

Even with an infinite number of eggs a bad gene cannot be totally eliminated if one parenthas two copies of that gene.

Genetic Manipulations

Genetic Manipulations are becoming common as a means of genetic engineering. There are

many methods of introducing new genetic material into a cell or organism, or altering theexisting material. Radiation and mutagenic compounds are able to reek havoc on DNA.

Special viruses have been altered and put to use which can introduce new genetic material

to an organism. Transposable elements, natures own gene shuffling tools, have been put to

use moving genes around in cells and organisms. Gene Targeting is a way of replacing aspecific gene with another within a cell.

These kinds of genetic manipulations are great for research with animals. Gene targeting

seems to be the most precise way of altering known genes. Gene therapies often try toreplace or repair defective genes in tissues where the genes are in use. Gene therapy does

not usually alter the "germ line", that is the reproductive cells, so even if gene therapy

corrects a problem, the problem can still be inherited by children.

Gene Therapy on the reproductive cells, or better yet, on a fertilized egg could be used tointroduce whatever genes are desired into an organism, even a human, when they are still a

single cell. With cloning technology, not even a fertilized egg is needed, just a cell that will

grow in cell culture. This is where genetic engineering stands right now. It is technicallypossible to repair and/or replace any known gene, but it is not very efficient and requires a


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large number of cells, of which only a few will be properly repaired. The other limitation is

the number of known genes.

The functions of all the genes are not known, only those of a very small percentage of thetotal genes in organisms such as humans. Research in animals is uncovering the functions

of the precursors of human genes, and that research helps in determining the precisefunction of human genes, but research is proceeding slowly. There may come a time when

we have the option of children who are Albert Einstien, Micheal Jordon and Bill Gates (ortheir female equivalents) roled into one, but not yet.

What's preventing genetic manipulation of all the known genes in human eggs? Cloning

has not been demonstrated to work with human cells for one thing, but Doctor RichardSeed may be working on that right now. There may be public opposition to human cloning

that is slowing research. The cost of genetic manipulations is relatively high and takes quite

a while. Supply and demand may be the key. Demand for children guaranteed not to have

any of the known genetic diseases is outweighed by the costs, but they will eventually meet

somewhere in the middle as the number of correctable diseases rises and the costs fall.

I can envision special gene constructions just for the purpose of cleanly replacing disease

genes with the functional versions. Libraries of functional and optimal versions of genes

-within DNA constructs necessary to introduce them into cells- will likely be created soonby genetic engineering companies, if they have not been started already. I have my own

ideas about what those constructions must include. Current gene therapies are very sloppy

when it comes to altering genes, but I have some ideas on making it cleaner and perhapsmore precise. I intend to share those ideas with the world from right here on this page some

time soon.

True Genetic Engineering

What I would call true genetic engineering is the creation of whole new genes and proteins,or even new organisms. We understand the genetic code and can create random or specific

proteins quite readily, but creating new proteins precisely for a given purpose -for example,

to strongly catalyze a particular chemical reaction- is still beyond us. Research into thestructure and folding of proteins may yield some answers. Mixing and matching of the

components of known proteins and organisms may yet be mastered, but that is a large step

beyond even the manipulation of known genes, but there is still much more beyond that.

Playing God!Is substituting one gene for another or introducing functional genes, as in gene therapy,

playing God? Is creating new genes, proteins and organisms playing God? Perhaps, but it is

all possible within a certain set of rules that have been handed down to the human race by 4billion years of evolution, or perhaps by the creator himself. The genetic code was probably

decided quite early in evolution, being defined almost entirely before the first eukaryotic

cells (cells with a nucleus) over 1 billion years ago.


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It may be possible to one day change the genetic code, so that every sequence of three

nucleic acids codes for a different amino acid. This would be possible through a relatively

simple change in the sequence of DNA coding transfer RNA. Changing the sequence of allthe other DNA so that it would recognize the new genetic code and produce functional

proteins would be the hard part, but why stop there!

While we're at it we could replace the 20 amino acids that are included in the genetic code

with all new amino acids. The current coding system of three nucleic acids per amino acidwould allow for as many as 64 different amino acids. Think of the diversity of protein

function possible with so much more variety! DNA itself could have more variety, how

about 6 nucleic acids instead of 4. A three nucleic acid sequence could then code for 216amino acids, or use just two nucleic acids to code for 36 different amino acids. Proteins

could be right handed instead of left, DNA and RNA could spiral in the other direction, etc.

In fact life may be possible using entirely different chemicals than life here on earth, no

DNA, RNA or protein as we recognize them. I actually think that's quite unlikely. The

components of life are in and of themselves quite simple molecules, most of which wouldprobably be used again if life evolved somewhere else, or was created artificially. Some of

the elements of the process are most likely entirely random, such as the handedness ofprotein, and the direction in which the DNA helix rotates. The genetic code itself is

probably random, and there are rare occurences of non-standard genetic codes still found

on earth, such as in the DNA of some cellular organelles and certain bacteria.

That's as far as I can go on the subject of Genetic Engineering, but there are many ideashere that could be more fully fleshed out. As I come across more information and read

about more of the research that's being done, I will update this page. If you come across

more detailed information on the Internet covering any of the topics listed here I may be

interested in linking to those pages. You may send e-mail to me, Arthur Kerschen

Genetic Inequality: Human Genetic

Engineering

By: Danielle Simmons, Ph.D. (Write Science Right) 2008 Nature Education

Citation: Simmons, D. (2008) Genetic inequality: Human genetic engineering. Nature

Education 1(1)

As genetics allows us to turn the tide on human disease, it's also granting the power to

engineer desirable traits into humans. What limits should we create as this technologydevelops?
mailto:[email protected]://www.nature.com/scitable/topicpage/genetic-inequality-human-genetic-engineering-768http://printreadingpage%28%29/http://www.nature.com/scitable/topicpage/genetic-inequality-human-genetic-engineering-768http://www.facebook.com/share.php?u=http://twitter.com/share?url=http://www.nature.com/scitable/topicpage/genetic-inequality-human-genetic-engineering-768mailto:[email protected]


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Genes influence health and disease, as well as human traits and behavior. Researchers are

just beginning to use genetic technology to unravel the genomic contributions to these

different phenotypes, and as they do so, they are also discovering a variety of otherpotential applications for this technology. For instance, ongoing advances make it

increasingly likely that scientists will someday be able to genetically engineer humans to

possess certain desired traits. Of course, the possibility of human genetic engineering raisesnumerous ethical and legal questions. Although such questions rarely have clear and

definite answers, the expertise and research of bioethicists, sociologists, anthropologists,

and other social scientists can inform us about how different individuals, cultures, andreligions view the ethical boundaries for the uses of genomics. Moreover, such insights can

assist in the development of guidelines and policies.

Testing for Traits Unrelated to Disease

Much of what we currently know about the ramifications of genetic self-knowledge comesfrom testing for diseases. Once disease genes were identified, it became much easier to

make a molecular or cytogenetic diagnosis for many genetic conditions. Diagnostic testingsupplies the technical ability to test presymptomatic, at-risk individuals and/or carriers to

determine whether they will develop a specific condition. This sort of testing is aparticularly attractive choice for individuals who are at risk for diseases that have available

preventative measures or treatments, as well as people who might carry genes that have

significant reproductive recurrence risks. Indeed, thanks to advances in single-celldiagnostics and fertilization technology, embryos can now be created in vitro; then, only

those embryos that are not affected by a specific genetic illness can be selected and

implanted in a woman's uterus. This process is referred to aspreimplantation geneticdiagnosis.

Foradult-onset conditions, ethical concerns have been raised regarding whether genetictesting should be performed if there is no cure for the disease in question. Many people

wonder whether positive diagnosis of an impending untreatable disease will harm the at-risk individual by creating undue stress and anxiety. Interestingly, social science research

has demonstrated that the answer to this question is both yes and no. It seems that if genetic

testing shows that an individual is a carrier for a recessive disease, such as Tay-Sachsdisease or sickle-cell anemia, this knowledge may have a negative impact on the

individual's well-being, at least in the short term (Marteau et al., 1992; Woolridge &

Murray, 1988). On the other hand, if predictive testing for an adult-onset genetic disorder

such as Huntington's disease reveals that an at-risk individual will develop the disorderlater in life, most patients report less preoccupation with the disease and a relief from the

anxiety of the unknown (Taylor & Myers, 1997). For many people who choose to havepredictive testing, gaining a locus of control by having a definitive answer is helpful. Somepeople are grateful for the opportunity to make life changesfor instance, traveling more,

changing jobs, or retiring earlyin anticipation of developing a debilitating condition later

in their lives.

Of course, as genetic research advances, tests are continually being developed for traits andbehaviors that are not related to disease. Most of these traits and behaviors are inherited as
http://www.nature.com/scitable/topicpage/Prenatal-Screen-Detects-Fetal-Abnormalities-PGD-and-306http://www.nature.com/scitable/topicpage/Prenatal-Screen-Detects-Fetal-Abnormalities-PGD-and-306http://www.nature.com/scitable/topicpage/Genetic-Causes-of-Adult-Onset-Disorders-34609http://www.nature.com/scitable/topicpage/Genetic-Causes-of-Adult-Onset-Disorders-34609http://www.nature.com/scitable/topicpage/Mendelian-Genetics-Patterns-of-Inheritance-and-Single-966http://www.nature.com/scitable/topicpage/Mendelian-Genetics-Patterns-of-Inheritance-and-Single-966http://www.nature.com/scitable/topicpage/Genetic-Causes-of-Adult-Onset-Disorders-34609http://www.nature.com/scitable/topicpage/Prenatal-Screen-Detects-Fetal-Abnormalities-PGD-and-306http://www.nature.com/scitable/topicpage/Prenatal-Screen-Detects-Fetal-Abnormalities-PGD-and-306http://www.nature.com/scitable/topicpage/Genetic-Causes-of-Adult-Onset-Disorders-34609http://www.nature.com/scitable/topicpage/Mendelian-Genetics-Patterns-of-Inheritance-and-Single-966http://www.nature.com/scitable/topicpage/Mendelian-Genetics-Patterns-of-Inheritance-and-Single-966http://www.nature.com/scitable/topicpage/Genetic-Causes-of-Adult-Onset-Disorders-34609


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complex conditions, meaning that multiple genes and environmental, behavioral, or

nutritional factors may contribute to the phenotype. Currently, available tests include those

for eye color, handedness, addictive behavior, "nutritional" background, and athleticism.But does knowing whether one has the genetic background for these nondisease traits

negatively affect one's self-concept or health perception? Studies are now beginning to

address this question. For example, one group of scientists performed genetic testing formuscle traits on a group of volunteers enrolled in a resistance-training program (Gordon et

al., 2005). These tests looked forsingle-nucleotide polymorphisms that would tell whether

an individual had a genetic predisposition for muscle strength, size, and performance. Theinvestigators found that if the individuals did not receive affirmative genetic information

regarding muscle traits, they credited the positive effects of the exercise program to their

own abilities. However, those study participants who did receive positive test results were

more likely to view the beneficial changes as out of their control, attributing any suchchanges to their genetic makeup. Thus, a lack of genetic predisposition for muscle traits

actually gave subjects a sense of empowerment.

The results of the aforementioned study may be surprising to many people, as one majorconcern associated with testing for nondisease traits is the fear that those people who do

not possess the genes for a positive trait may develop a negative self-image and/or

inferiority complex. Another matter bioethicists often consider is that people may discover

that they carry some genes associated with physiological or behavioral traits that arefrequently perceived as negative. Moreover, many critics fear that the prevalence of these

traits in certain ethnic populations could lead to prejudice and other societal problems.

Thus, rigorous social science research by individuals from diverse cultural backgrounds iscrucial to understanding people's perceptions and establishing appropriate boundaries.

Building Better Athletes with Gene Doping
http://www.nature.com/scitable/topicpage/Using-SNP-Data-to-Examine-Human-Phenotypic-706http://disporigimg%28%22/scitable/content/ne0000/ne0000/ne0000/ne0000/54787/10.1038_ng0997-71_full.jpg%22,%20%22The%20double-muscled%20Belgian%20blue%20cow%20breed%22,%20%22true%22,%20%22Figure%201%22,%20%22Double%20muscled%20animals%20have%20an%20increase%20in%20muscle%20mass%20of%20up%20to%2020%25%20greater%20than%20normal%20animals.%20%20The%20increased%20muscle%20is%20due%20to%20the%20fact%20that%20these%20animals%20have%20a%20mutation%20in%20a%20specific%20gene%20that%20normally%20is%20involved%20in%20muscular%20hypertrophy.%22,%20%22true%22,%20%22Used%20with%20permission.%20All%20rights%20reserved.%22,%20'472',%20'337',%20'http://www.nature.com');http://www.nature.com/scitable/topicpage/Using-SNP-Data-to-Examine-Human-Phenotypic-706


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Figure 1: The double-muscled Belgian blue cow breed

Double muscled animals have an increase in muscle mass of up to 20% greater than normalanimals. The increased muscle is due to the fact that these animals have a mutation in a

specific gene that normally is involved in muscular hypertrophy.

1997Nature Publishing Group Grobet, L. et al. A deletion in the bovine myostatin gene

cuases the double-mustard phenotype in cattle.Nature Genetics17, 71. Used withpermission. All rights reserved.

Over the years, the desire for better sports performance has driven many trainers and

athletes to abuse scientific research in an attempt to gain an unjust advantage over theircompetitors. Historically, such efforts have involved the use of performance-enhancing

drugs that were originally meant to treat people with disease. This practice is called doping,

and it frequently involved such substances as erythropoietin, steroids, and growthhormones (Filipp, 2007). To control this drive for an unfair competitive edge, in 1999, the

International Olympic Committee created the World Anti-Doping Agency (WADA), which

prohibits the use of performance-enhancing drugs by athletes. WADA also conducts

various testing programs in an attempt to catch those athletes who violate the anti-dopingrules.

Today, WADA has a new hurdle to overcomethat ofgene doping. This practice is

defined as the nontherapeutic use of cells, genes, or genetic elements to enhance athletic

performance. Gene doping takes advantage of cutting-edge research in gene therapy thatinvolves the transfer of genetic material to human cells to treat or prevent disease (Well,

2008). Because gene doping increases the amount of proteins and hormones that cells

normally make, testing for genetic performance enhancers will be very difficult, and a newrace is on to develop ways to detect this form of doping (Baoutina et al., 2008).

The potential to alter genes to build better athletes was immediately realized with theinvention of so-called "Schwarzenegger mice" in the late 1990s. These mice were given

this nickname because they were genetically engineered to have increased muscle growthand strength (McPherron et al., 1997; Barton-Davis et al., 1998). The goal in developing

these mice was to study muscle disease and reverse the decreased muscle mass that occurs

with aging. Interestingly, the Schwarzenegger mice were not the first animals of their kind;that title belongs to Belgian Blue cattle (Figure 1), an exceptional breed known for its

enormous muscle mass. These animals, which arose via selective breeding, have a mutated

and nonfunctional copy of the myostatin gene, which normally controls musculardevelopment. Without this control, the cows' muscles never stop growing (Grobet et al.,

1997). In fact, Belgian Blue cattle get so large that most females of the breed cannot give

natural birth, so their offspring have to be delivered by cesarean section. Schwarzeneggermice differ from these cattle in that they highlight scientists' newfound ability to inducemuscle development through genetic engineering, which brings up the evident advantages

for athletes. But does conferring one desirable trait create other, more harmful

consequences? Are gene doping and other forms of genetic engineering something worthexploring, or should we, as a society, decide that manipulation of genes for nondisease

purposes is unethical?
http://disporigimg%28%22/scitable/content/ne0000/ne0000/ne0000/ne0000/54787/10.1038_ng0997-71_full.jpg%22,%20%22The%20double-muscled%20Belgian%20blue%20cow%20breed%22,%20%22true%22,%20%22Figure%201%22,%20%22Double%20muscled%20animals%20have%20an%20increase%20in%20muscle%20mass%20of%20up%20to%2020%25%20greater%20than%20normal%20animals.%20%20The%20increased%20muscle%20is%20due%20to%20the%20fact%20that%20these%20animals%20have%20a%20mutation%20in%20a%20specific%20gene%20that%20normally%20is%20involved%20in%20muscular%20hypertrophy.%22,%20%22true%22,%20%22Used%20with%20permission.%20All%20rights%20reserved.%22,%20'472',%20'337',%20'http://www.nature.com');http://disporigimg%28%22/scitable/content/ne0000/ne0000/ne0000/ne0000/54787/10.1038_ng0997-71_full.jpg%22,%20%22The%20double-muscled%20Belgian%20blue%20cow%20breed%22,%20%22true%22,%20%22Figure%201%22,%20%22Double%20muscled%20animals%20have%20an%20increase%20in%20muscle%20mass%20of%20up%20to%2020%25%20greater%20than%20normal%20animals.%20%20The%20increased%20muscle%20is%20due%20to%20the%20fact%20that%20these%20animals%20have%20a%20mutation%20in%20a%20specific%20gene%20that%20normally%20is%20involved%20in%20muscular%20hypertrophy.%22,%20%22true%22,%20%22Used%20with%20permission.%20All%20rights%20reserved.%22,%20'472',%20'337',%20'http://www.nature.com');http://disporigimg%28%22/scitable/content/ne0000/ne0000/ne0000/ne0000/54787/10.1038_ng0997-71_full.jpg%22,%20%22The%20double-muscled%20Belgian%20blue%20cow%20breed%22,%20%22true%22,%20%22Figure%201%22,%20%22Double%20muscled%20animals%20have%20an%20increase%20in%20muscle%20mass%20of%20up%20to%2020%25%20greater%20than%20normal%20animals.%20%20The%20increased%20muscle%20is%20due%20to%20the%20fact%20that%20these%20animals%20have%20a%20mutation%20in%20a%20specific%20gene%20that%20normally%20is%20involved%20in%20muscular%20hypertrophy.%22,%20%22true%22,%20%22Used%20with%20permission.%20All%20rights%20reserved.%22,%20'472',%20'337',%20'http://www.nature.com');http://disporigimg%28%22/scitable/content/ne0000/ne0000/ne0000/ne0000/54787/10.1038_ng0997-71_full.jpg%22,%20%22The%20double-muscled%20Belgian%20blue%20cow%20breed%22,%20%22true%22,%20%22Figure%201%22,%20%22Double%20muscled%20animals%20have%20an%20increase%20in%20muscle%20mass%20of%20up%20to%2020%25%20greater%20than%20normal%20animals.%20%20The%20increased%20muscle%20is%20due%20to%20the%20fact%20that%20these%20animals%20have%20a%20mutation%20in%20a%20specific%20gene%20that%20normally%20is%20involved%20in%20muscular%20hypertrophy.%22,%20%22true%22,%20%22Used%20with%20permission.%20All%20rights%20reserved.%22,%20'472',%20'337',%20'http://www.nature.com');http://www.nature.com/http://www.nature.com/http://www.nature.com/scitable/topicpage/Sports-Gene-Doping-and-WADA-764http://www.nature.com/scitable/topicpage/Sports-Gene-Doping-and-WADA-764http://www.nature.com/scitable/topicpage/Sports-Gene-Doping-and-WADA-764http://www.nature.com/scitable/topicpage/Gene-Therapy-760http://disporigimg%28%22/scitable/content/ne0000/ne0000/ne0000/ne0000/54787/10.1038_ng0997-71_full.jpg%22,%20%22The%20double-muscled%20Belgian%20blue%20cow%20breed%22,%20%22true%22,%20%22Figure%201%22,%20%22Double%20muscled%20animals%20have%20an%20increase%20in%20muscle%20mass%20of%20up%20to%2020%25%20greater%20than%20normal%20animals.%20%20The%20increased%20muscle%20is%20due%20to%20the%20fact%20that%20these%20animals%20have%20a%20mutation%20in%20a%20specific%20gene%20that%20normally%20is%20involved%20in%20muscular%20hypertrophy.%22,%20%22true%22,%20%22Used%20with%20permission.%20All%20rights%20reserved.%22,%20'472',%20'337',%20'http://www.nature.com');http://disporigimg%28%22/scitable/content/ne0000/ne0000/ne0000/ne0000/54787/10.1038_ng0997-71_full.jpg%22,%20%22The%20double-muscled%20Belgian%20blue%20cow%20breed%22,%20%22true%22,%20%22Figure%201%22,%20%22Double%20muscled%20animals%20have%20an%20increase%20in%20muscle%20mass%20of%20up%20to%2020%25%20greater%20than%20normal%20animals.%20%20The%20increased%20muscle%20is%20due%20to%20the%20fact%20that%20these%20animals%20have%20a%20mutation%20in%20a%20specific%20gene%20that%20normally%20is%20involved%20in%20muscular%20hypertrophy.%22,%20%22true%22,%20%22Used%20with%20permission.%20All%20rights%20reserved.%22,%20'472',%20'337',%20'http://www.nature.com');http://disporigimg%28%22/scitable/content/ne0000/ne0000/ne0000/ne0000/54787/10.1038_ng0997-71_full.jpg%22,%20%22The%20double-muscled%20Belgian%20blue%20cow%20breed%22,%20%22true%22,%20%22Figure%201%22,%20%22Double%20muscled%20animals%20have%20an%20increase%20in%20muscle%20mass%20of%20up%20to%2020%25%20greater%20than%20normal%20animals.%20%20The%20increased%20muscle%20is%20due%20to%20the%20fact%20that%20these%20animals%20have%20a%20mutation%20in%20a%20specific%20gene%20that%20normally%20is%20involved%20in%20muscular%20hypertrophy.%22,%20%22true%22,%20%22Used%20with%20permission.%20All%20rights%20reserved.%22,%20'472',%20'337',%20'http://www.nature.com');http://disporigimg%28%22/scitable/content/ne0000/ne0000/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Creating Designer Babies

Genetic testing also harbors the potential for yet another scientific strategy to be applied in

the area ofeugenics, or the social philosophy of promoting the improvement of inheritedhuman traits through intervention. In the past, eugenics was used to justify practices

including involuntary sterilization and euthanasia. Today, many people fear thatpreimplantation genetic diagnosis may be perfected and could technically be applied toselect specific nondisease traits (rather than eliminate severe disease, as it is currently used)

in implanted embryos, thus amounting to a form of eugenics. In the media, this possibility

has been sensationalized and is frequently referred to as creation of so-called "designerbabies," an expression that has even been included in the Oxford English Dictionary.

Although possible, this genetic technology has not yet been implemented; nonetheless, it

continues to bring up many heated ethical issues.

Trait selection and enhancement in embryos raises moral issues involving both individualsand society. First, does selecting for particular traits pose health risks that would not have

existed otherwise? The safety of the procedures used for preimplantation genetic diagnosisis currently under investigation, and because this is a relatively new form of reproductive

technology, there is by nature a lack of long-term data and adequate numbers of researchsubjects. Still, one safety concern often raised involves the fact that most genes have more

than one effect. For example, in the late 1990s, scientists discovered a gene that is linked to

memory (Tang et al., 1999). Modifying this gene in mice greatly improved learning andmemory, but it also caused increased sensitivity to pain (Wei et al., 2001), which is

obviously not a desirable trait. Beyond questions of safety, issues of individual liberties

also arise. For instance, should parents be allowed to manipulate the genes of their childrento select for certain traits when the children themselves cannot give consent? Suppose a

mother and father select an embryo based on its supposed genetic predisposition to

musicality, but the child grows up to dislike music. Will this alter the way the child feelsabout its parents, and vice versa? Finally, in terms of society, it is not feasible for everyone

to have access to this type of expensive technology. Thus, perhaps only the most privileged

members of society will be able to have "designer children" that possess greater

intelligence or physical attractiveness. This may create a genetic aristocracy and lead tonew forms of inequality.

At present, these questions and conjectures are purely hypothetical, because the technology

needed for trait selection is not yet available. In fact, such technology may be impossible,

considering that most traits are complex and involve numerous genes. Still, contemplationof these and other issues related to genetic engineering is important should the ability to

create genetically enhanced humans ever arise.

Chapter 21Genetic manipulation of

animals
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21.1An overview of genetic manipulation of animals

Go to:

Experimental animals have been used in biomedical research for decades. In many cases,

aspects of physiology and biochemistry have been investigated, and artificial manipulationshave often been confined to examining the effect of altering the animal's environment or

some aspect of itsphenotype. Some animals, notablyDrosophila and mice, have been

particularly amenable to genetic analyses and traditional genetic manipulation of animalshas involved carefully selected breeding experiments or exposure of animals to powerful

chemical or radioisotopic mutagens. A new era in animal research was ushered in during

the early 1980s when successful experiments designed to genetically modify animals byinserting foreign DNA were first reported. These new methodologies were expected to

have many advantages for research but two major areas have benefited:

Gene function. While the use of cultured cells and cell extracts can be extremely

valuable in studying gene expression and function, the ability to insert genes intowhole animals or to selectively delete or alter single predetermined genes in an

animal provides enormous power in studying gene function.

Animal models of disease. Nature has provided some animal models of disease andsome have been generated by random mutagenesis programmes, but not in a

predetermined way. The new technologies held the promise of altering at will even

single genes within a living animal in such a way as to mimic mutations faithfullyin an analogous gene in humans, thereby providing a higher chance of resembling

human disease phenotypes.

In order to create genetically modified animals, it is necessary to modify the DNA of

germline cells so that the modified DNA is heritable. As a result, certain cells that have thecapacity to differentiate into the different cells of an adult animal (or at least to give rise to

germ line cells) were considered to be the optimal targets for introducing foreign DNA.

The fertilized oocyte is one such cell, being totipotent. Other target cells are cells of very

early stage embryos, including embryonic stem (ES) cells. Although such cells arepostzygotic they represent a stage in development where there has been incomplete

separation of thesoma and thegermline. Such cells are therefore capable of giving rise to

both somatic and germline cells.

When a foreign DNA molecule is artifically introduced into the cells of an animal, a

transgenic animalis produced. The foreign DNA molecule is called a transgene and may

contain one or many genes. By inserting a transgene into a fertilized oocyte or cells fromthe early embryo, the resulting transgenic animalmay be able to transmit the foreign DNAstably in its germline. Many different types of transgenic animals have been created

including transgenicDrosophila, transgenic frogs, transgenic fish and a variety of

transgenic mammals including mice, rats and various livestock animals. The technology oftransgenesis and its applications are considered inSection 21.2.
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Although transgenes often integrate into the host chromosomes without affecting the

expression of any endogenous genes, occasionally the integration event alters endogenous

gene expression (insertional mutation), producing a recognizablephenotype. Thisconstitutes a form ofin vivo mutagenesis, albeit at an unselectedtarget gene.Gene

targeting was developed as a method ofin vivo mutagenesis in which the mutation is

introduced into apreselectedendogenous gene. This can be achieved in somatic cells, butgene targeting in culturedES cells is particularly powerful because it can lead to the

construction of an animal in which all nucleated cells contain a mutation at the desired

locus (see Section 21.3 below). In mammals, gene targeting has been possible only in micebut research on ES cells from other species may extend the capacity for gene targeting in

the near future. Note that in some cases gene targeting is used to produce a subtle mutation

and as a result the ES cells used for blastocyst implantation do not contain any foreign

sequences. The resulting mice may be described as genetically modified but not astransgenic.

The ability to produce transgenic mice and particularly the ability to perform specific

changes in a predetermined gene by gene targeting has permitted the design of many newanimal models of human disease (Section 21.4). Another experimental approach involving

genetic manipulation of animals has had a major impact recently. In 1997 a new era in

mammalian genetics was heralded when the procedure ofsomatic cell nuclear transfer

permitted cloning of an adult mammal for the first time. This involved transfer of thenucleus from an adult cell into an enucleated oocyte and the technology has subsequently

been applied as an alternative route to generating transgenic animals (Section 21.5).

21.2The creation and applications of transgenic animals

Go to:

Of the different transgenic animals that have been made thus far, transgenic Drosophila,

transgenic frogs, and transgenicfishhave been very important for understanding aspects of

gene function and development in these species. Transgenic sheep and other transgenic

livestock animals have been produced largely to serve as bioreactors, whole-animalexpression cloning systems in which introduced genes are expressed to give large amounts

of therapeutic or commercially valuable gene products (seeSection 22.1.2). But it has been

transgenic mice that have been the most useful to biomedical research, both in providinganimal models of disease and in permitting the most useful analyses of mammalian gene

function.

21.2.1Transgenic animals can be produced following transfer of clonedDNA into fertilized oocytes and cells from very early stageembryos

Transgenesis involves transfer of foreign DNA into totipotent or pluripotent embryo cells(either fertilized oocytes, cells of the very early embryo or cultured embryonic stem cells)

followed by insertion of the transferred DNA into host chromosomes. If the foreign DNA

integrates into the chromosomes of a fertilized oocyte, the developing animal will be fully
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transgenic since all nucleated cells in the animal should contain the transgene. If

chromosomal integration occurs later, at a postzygotic stage, the animal will be a mosaic,

with some cells containing the transgene and some others lacking it. If the transgene ispresent in germline cells it can be passed through sperm or egg cells into some of the

animal's progeny, and PCR-based tests can be used to quickly screen for the presence of the

transgene. Progeny that are transgene positive can be expected to be fully transgenic; alltheir cells should have developed from a fertilized oocyte containing the transgene.

Pronuclear microinjection

To obtain transgenic mice by this route, females are superovulated, mated to fertile males

and sacrificed the next day. Fertilized oocytes are recovered from excised oviducts. TheDNA of interest is then microinjected using a micromanipulator into the male pronucleus

of individual oocytes (Figure 21.1). Surviving oocytes are reimplanted into the oviducts of

foster females and allowed to develop into mature animals (see Gordon, 1992).

Figure 21.1

Construction of transgenic mice by pronuclear microinjection. Very fine glass pipettes areconstructed using specialized equipment: one, a holding pipette, has a bore which can

accommodate (more...)

During this procedure, the microinjected DNA (transgene) randomly integrates into

chromosomal DNA, usually at a single site, although rarely two sites of integration arefound in a single animal. Individual insertion sites typically contain multiple copies of the

transgenes integrated into chromosomal DNA as head-to-tail concatemers (it is not unusual

to find 50 or more copies at a single insertion site). As a result of chromosomal integration,

the transgenes can be passed on to subsequent generations in mendelian fashion: if theforeign DNA has integrated at the one-cell stage, it should be transmitted to 50% of the

offspring.

Transfer into pre- or postimplantation embryos

Cells from very early stage embryos may be totipotent or at least pluripotent and canprovide a route for foreign DNA to enter the germline. DNA can be transferred into

unselected cells of very early embryos, as described in this section or into cell lines derived

from embryonic stem cells, as described inSection 21.2.2.

One method that allows foreign DNA to integrate into the chromosomes of the target cellsuses retroviruses, RNA viruses which naturally undergo an intermediate DNA form prior

to integrating into cellular genomes (see Figure 18.2). Infection of preimplantation mouse
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embryos with aretrovirussuch as Moloney murine leukemia virus or injection of the

retrovirus into early postimplantation mouse embryos results in mosaic offspring.

Retroviruses should integrate rarely and at random into accessible cells, and the use ofreplication- defective retroviruses provides heritable markers for clonal descendants of the

target cell (unlike wild-type viruses which spread from cell to cell). This approach has been

used, therefore, for studying cell lineage using reporter genes.

InDrosophila, efficient chromosomal integration is possible by using sequences from aDrosophila transposable element known as the P element to permit insertion of single

copies of a gene at random in the genome. The gene or DNA fragment to be inserted is first

manipulated so as to be flanked by the two terminal sequences of the P element. Themodified DNA is then microinjected into a very youngDrosophila embryo along with a

separate plasmid containing a gene encoding a transposase. In the presence of the

transposase the terminal P element sequences allow the intervening DNA fragment totranspose and as a result of the ensuing transposition events, the injected DNA often enters

the germline in a single copy.

21.2.2Cultured embryonic stem (ES) cells provide a powerful route togenetic modification of the germline

The microinjection of foreign DNA into fertilized oocytes is technically difficult and not

suited to large-scale production of transgenic animals or to sophisticated genetic

manipulation. A popular alternative, but one which has so far been restricted to theconstruction of genetically modified mice, involves transferring the foreign DNA initially

into cultured embryonic stem (ES) cells. Mouse ES cells are derived from 3.54.5 day

postcoitum embryos and arise from the inner cell mass of the blastocyst (see Figure 21.2).The ES cells can be cultured in vitro and retain the potential to contribute extensively to all

of the tissues of a mouse, including the germline, when injected back into a host blastocystand reimplanted in a pseudopregnant mouse.

Figure 21.2

Genetically modified ES cells as a route for transferring foreign DNA or specific mutationsinto the mouse germline. Cells from the inner cell mass were cultured following excision

of oviducts and(more...)

The developing embryo is a chimera: it contains two populations of cells derived fromdifferent zygotes, those of the blastocyst and the implanted ES cells. If the two strains of

cells are derived from mice with different coat colors, chimeric offspring can easily be

identified (see Figure 21.2). Use of genetically modified ES cells results in a partially
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transgenic mouse. Because the injected ES cells can form all or part of the functional germ

cells of thechimera, it is possible to derive fully transgenic mice. This is usually

accomplished by screening the offspring of matings between chimeras (usually males) andmice with a coat colorrecessive to that of the strain from which the ES cells were derived

(see Figure 21.2).

The big advantage ofES cells is that they can be grown readily in culture. This means that

a variety of genetic manipulations can be conducted in cultured ES cells. Importantly, the

desired genetic modification can be verified in tissue culture, before injecting the

genetically modified cells into a blastocyst prior to implantation. For example, the desired

gene can be ligated to amarkergene, such as the neo gene, enabling a positive selection forcells that have been successfully transfected (see Box 10.1). The presence of the desired

gene can also be verified quickly by a PCR-based assay. ES cells also offer the huge

advantage ofgene targetingby homologous recombination, a method which can permit aprogramed selective alteration of a singlepredeterminedgene and also highly specific

ways of chromosome engineering. Such approaches are extremely powerful for

understanding gene function (Section 21.3) and also for creating animal models of disease(Section 21.4).

The ES cell approach to constructing transgenic mice was made possible by the successful

establishment in the early 1980s of stable cell lines from isolated mouseES cells. ES cells

were not so readily identifed in other mammals, although there have been some importantrecent successes (seeBox 21.1).

Box 21.1

Isolation and manipulation of mammalian embryonic stem cells. The embryo properderives from the inner cell mass (ICM) of a blastocyst (Box 2.2). Mouse embryonic stem

(ES) cells were first isolated (more...)

21.2.3Transgenic animals have been used for a variety of studiesinvestigating mammalian gene expression and function

Transgenic animals have been extremely important for analyzing human genes (Hanahan,1989; Camperet al., 1995; Theuring, 1995), and have helped greatly in our understanding

of a variety of fundamental biological processes, notably in immunology, neurobiology,cancer and developmental studies. The following list is far from exhaustive but is intended

to illustrate some major types of application:

Investigating gene expression and its regulation. Although evidence forcis-actingregulatory elements is often inferred initially from studies using cultured cells, they
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need to be validated in whole animal studies. Transgenes consisting of the

presumptive regulatory sequence(s) coupled to a reporter gene, such as lacZ,

provide a sensitive method of detecting gene expression and a powerful way ofinvestigating regulation of gene expression. Long-range control of gene expression

is often investigated using YAC transgenes, seeSection 21.2.4.

Investigating gene function by targeted gene inactivation. Specific genes can beinactivated by agene targetingprocedure to introduce a transgene into the target

gene (insertional inactivation). The effect on thephenotype of creating a null

mutation in the gene of interest can provide powerful clues to gene function.

Investigating dosage effects and ectopic expression. In some cases, valuable

information can be gained by over-expressing a transgene (e.g. Schedl et al., 1996)

or by expressing it ectopically (the transgene is coupled to a tissue-specific

promoterwhich causes expression in cells; the phenotypic consequence mayprovide valuable clues to function).

Cell lineage ablation. Transgenes can be designed consisting of a tissue-specific

promotercoupled to a sequence encoding a toxin, for example diphtheria toxin

subunit A or ricin. When the promoter becomes active at the appropriate stage oftissue differentiation, the toxin is produced and kills the cells. Thus, certain cell

lineages in the animal can be eliminated (cell ablation) and the phenotypicconsequences monitored.

Investigating gain of function. In principle, any mammalian gene that produces a

dominant negative effect or gain of function can be investigated by introduction ofan appropriate transgene. In some cases, this can provide proof of a suspected

biological function. A classical example concerns the Sry gene. A variety of

different genetic analyses had implicated this gene as a major male-determining

gene but convincing proof was obtained using a transgenic mouse approach. Theexperiment consisted of transferring a cloned Sry gene into a fertilized 46,XX

mouse oocyte. As a result of this artificial intervention, the resulting mouse, which

nature had intended to be female, turned out to be male (Koopman et al., 1991).

Modeling human disease.Insertional inactivationis often used to model loss of

function mutations whilst gain of function mutations can often be modeled by

inserting a mutant transgene (see Section 21.4).

21.2.4The use of YAC transgenes and inducible promoters has greatlyextended the applications of transgenic animals

Inducible promoters

For many applications it is desirable to have a transgene expressed under the control of atissue-specificpromoteror an inducible promoter (see Section 8.2.4). In the former case,genetic engineering can be used to splice known tissue-specific promoters from cloned

genes to the gene of interest. In some cases, coupled regulatory elements can confer both

position-independent and tissue-specific expression as with sequence elements from the -globin locus control region (Grosveld et al., 1987).
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Various attempts have also been made to create inducible transgenic mice (e.g. by using

heavy metal ions to induce expression of an integrated gene which has a coupled

metallothioneinpromoter, etc.). Generally, the use of inducible promoters has beenhampered by leakiness in gene expression and by relatively low levels of induction, and

they have often been applicable to a limited range of tissues. More recently, however, more

promising systems have been developed. For example, methods employing tetracycline-regulated inducible expression have permitted construction of both highly inducible

transfected cells (with much greater efficiency than the constitutive system) and transgenic

mice (Shockett et al., 1995). In the latter case, the expression of a reporter gene, such as theluciferase gene, can be controlled by altering the concentration of tetracycline in the

drinking water of the animals.

YAC transgenics

Early studies of gene expression and regulation in transgenic animals involved transfer of

small genes. However, expression of small transgenes often fails to follow the normal

temporal and spatial patterns of expression or match the expression level of the endogenoushomolog. An increasing number of human genes are known to be very large (see Figure

7.7). Even in the case of small genes, important regulatory elements that are required forcorrect expression may be located many kilobases upstream of the coding sequence (see

Figure 8.23). In order to be able to study the expression and regulation of a human gene

under the control of its own cis-acting regulatory elements, it was therefore necessary toestablish transfection conditions which would allow the transfer of large DNA clones.

A major breakthrough in transgenic studies was the development of so-called YAC

transgenics (Lamb and Gearhart, 1995). The first report to be published described transfer

of a 670 kb YAC containing the humanHPRT(hypoxanthine phosphoribosyltransferase)

gene into mouse ES cells(Jakobovits et al., 1993). This was accomplished by spheroplastfusion (i.e. fusion of ES cells with YAC-containing yeast cells that have been stripped of

the hard cell wall; seeSection 4.3.4). Fragments from the yeast genome can integrate at thesame time, however, and so alternative methods have sought to purify an individual YAC

by size-fractionation on a preparative gel using pulsed-field gel electrophoresis (assuming

that the YAC migrates at a position in the gel that is different from any yeast chromosome).

The purified YAC can be inserted into a fertilized oocyte by pronuclear microinjection (seeabove). This method is, however, limited to small YACs: the DNA of large YACs is more

likely to fragment following microinjection with very fine micropipettes. Alternatively,

purified YACs have been transferred into ES cells by using liposomes, artificial lipidvesicles that are used to transport molecules into a cell following fusion of the lipid coat

with the plasma membrane of the recipient cell (see Figure 22.6).

YAC transgenics have permitted study of large genes such as the 400 kb humanAPPgene,

a gene known to contribute to Alzheimer's disease, encoding the amyloid protein precursor.A YAC transgene containingAPPshowed tissue- and cell type-specific expression patterns

closely mirroring that of the endogenous mouse gene (for this and other examples, see

Lamb and Gearhart, 1995). Long-range gene regulation mechanisms (locus control regions,imprinting and other chromatin domain effects; see Chapter 8) can be modeled. An
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interesting application has been in the production of fully human antibodies in the mouse

by transfer of human YACs containing large segments of the human heavy and kappa light

chain immunoglobulin loci into mouse ES cells, and thence the creation of transgenic miceable to produce human antibodies (seeMendez et al., 1997 and Figure 21.3). Finally, YAC

transgenics may also find a role in modeling disease caused by large-scale gene dosage

imbalance (see Section 21.4.4).

Figure 21.3

Use of YAC transgenesis to construct a mouse with a human antibody repertoire. YACscontaining Ig sequences are obtained by screening YAC libraries with suitable Ig probes.

The recovery of(more...)

Transchromosomic animals

Ultimately, even YACs have upper limits for the size of foreign inserts that can betransferred. Mammalian artificial chromosomes have also been generated, including first

generation human artificial chromosomes (Harrington et al., 1997; Ikeno et al., 1998).

Such systems will have the capacity of transferring hundreds and possibly thousands ofgenes into transgenic animals, although a preferred route may be by using nuclear transfer

technology (Section 21.5) rather thanES cells. Recently, however, transfer of whole

chromosomes or chromosome fragments into ES cells has been possible by microcell-

mediated chromosome transfer (seeSections 10.1.1 and10.1.3). Using this approachTomizuka et al. (1997) were able to transfer human chromosomes or chromosome

fragments derived from normal fibroblasts into mouse ES cells. The resulting chimeric

transchromosomic mice were viable, and the chromosome fragments appeared to showfunctional expression and could be transmitted through the germline.

21.3Use of mouse embryonic stem cells in gene targeting

and gene trapping

Go to:

21.3.1Gene targeting by homologous recombination in ES cells can beused to produce mice with a mutation in a predetermined gene

Gene targeting involves engineering a mutation in apreselectedgene within an intact cell.It can therefore be viewed as a form of artificial site-directed in vivo mutagenesis (as

opposed to the various methods of site-directed in vitro mutagenesis described in Section

6.4). The mutation may result in inactivation of gene expression (a knock-out mutation),
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or altered gene expression, and so can be useful for studying gene function (see below). In

addition, the same method can be used to correct a pathogenic mutation by restoring the

normalphenotype, and so has therapeutic potential (see Section 22.3).

Gene targeting typically involves introducing a mutation by homologous recombination. A

cloned gene (or gene segment) closely related in sequence to endogenous target gene istransfected into the appropriate cells. In some of the cells, homologous recombination

occurs between the introduced gene and its chromosomal homolog. Gene targeting byhomologous recombination has been achieved in some somatic mammalian cells, such as

myoblasts. However, the most important application involves mouse ES cells: once a

mutation has been engineered into a specific mouse gene within the ES cells, the modifiedES cells can then be injected into the blastocyst of a foster mother and eventually a mouse

can be produced with the mutation in the desired gene in all nucleated cells (Capecchi,

1989; Melton, 1994).

Homologous recombination in mammalian cells is a very rare occurrence (unlike in yeast

cells, for example, where it occurs naturally at high frequencies, enabling sophisticatedgenetic manipulation). The frequency of homologous recombination is increased, however,

when the degree ofsequence homology between the introduced DNA and the target gene isvery high. As a result, the introduced DNA clone is a mouse sequence which should

preferably be isogenic (derived from the same mouse strain as the strain of mouse from

which the ES cellswere derived). Even then, the frequency of genuine homologousrecombination events is very low and may be difficult to identify against a sizeable

background of random integration events.

To assist identification of the desired homologous recombination events, the targeting

vector(often a plasmid vector) contains a markergene, such as the neo gene (see Box

10.1), which permits selection for cells that have taken up the introduced DNA. PCRassays are used to screen for evidence of a homologous recombination event (by using a

marker-specificprimerplus a primer derived from a sequence present in the target gene butabsent from the introduced homologous gene segment). The targeting construct is

transferred into cultured mouse ES cells by electroporation, a method in which pulses of

high voltage are delivered to cells, causing temporary relaxation of the selective

permeability properties of the plasma membranes. Two basic approaches have been used:

Insertion vectors target thelocus of interest by a single reciprocal recombination,

causing insertion of the entire introduced DNA including the vectorsequence

(Figure 21.4A). This is the most reliable way of causing a knock-out mutation.

Replacement vectors are designed to replace some of the sequence in thechromosomal gene by a homologous sequence from the introduced DNA (Figure

21.4B). This can occur as a result of a double reciprocal recombination or bygene

conversion. The replacement method can inactivate a gene when the introducedsequence contains one or more premature termination codons or lacks critical

coding sequences. It can also be used to correct a pathogenic mutation.
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Figure 21.4

Gene targeting by homologous recombination can inactivate a predetermined chromosomal

gene within an intact cell. (A) Insertion vector method. The introduced vector DNA (blue)is cut at a unique(more...)

The replacementvectorapproach, as well as the insertion vector method, often leaves

foreign sequences at the target locus. In some cases, however, a more subtle mutation is

required. For example, it may be desirable to investigate the effect of changing a singlecodon. Various two-step recombination techniques can be used to accomplish this method,

and the resulting mouse, although genetically modified lacks any foreign sequences and so

can no longer be described as transgenic (see Figure 21.5 and Melton, 1994).

Figure 21.5

Double replacement gene targeting can be used to introduce subtle mutations. Both the

methods in Figure 21.4 result in introduction of a substantial amount of exogenoussequence (more...)

Gene targeting in mice is popularly used for producing artificial mouse models of human

disease (Section 21.4). In addition, it provides a powerful general method of studying gene

function. The gene in question is selectively inactivated, producing a knock-out mouse,and the effect of the mutation on the development of the mouse is monitored carefully.

Sometimes there is little or no phenotypic consequence after inactivating a gene that would

be expected to be crucially important, such as some genes which encode atranscription

factor known to be expressed in early embryonic development. The lack of aphenotype insuch cases is often thought to be due to genetic redundancy (another gene is able to carry

out the function of the gene that has been knocked out). As a result, in some cases double

or even triple gene knock-outs have been carried out to analyze gene function, as in thecase of some of theHox genes (see, for example, Manley and Capecchi, 1997).

A useful example of investigating functional redundancy concerns studies of the mouseEngrailedgenes,En-1 andEn-2. Both of these genes are homeobox genes which had been

considered to play crucial roles in brain formation.En-1 knock-outs have seriousabnormalities but surprisinglyEn-2 knock-outs have only minor problems. Expression of

theEn-1 gene is switched on 810 hours before that of theEn-2 product, suggesting that

perhaps theEn-1 product can compensate for the lack ofEn-2 product inEn-2 knock-outs.To test for the possibility of functional redundancy,Hanks et al. 1995 used a variant of the

knock-out procedure known as the knock-in technique. In this case the transgene used to
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knock-out the target endogenous gene is itself designed to be expressed under the control

of the cis-acting elements of the knocked-out gene. A transgene containing anEn-2 coding

sequence was used to knock-out the endogenousEn-1 gene. In so doing, the introducedEn-2 sequence came under control of theEn-1 regulatory sequences and was expressed

before the endogenousEn-2 gene was switched on (see Figure 21.6). The resultingEn-1

knock-out mouse had a normalphenotype, demonstrating that the knocked-inEn-2 genewas functionally equivalent toEn-1 (Hanks et al., 1995).

Figure 21.6

The knock-in method replaces the activity of one chromosomal gene by that of anintroduced gene. TheEn-1 gene shown at top has two exons and coding sequences are

shown by filled boxes. Its promoter(more...)

21.3.2Site-specific recombination systems, notably the Cre-loxPsystem, extend the power of gene targeting

Several site-specific recombination systems from bacteriophages and yeasts have been

characterized and are promising tools forg

cloning may make possible some extraordinary advances in the science of genetic engineering

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