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 genetic engineering 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 the cloning 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 theoretically possible. 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 into functional 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. There is 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

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 animals together 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 small changes 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 a large 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 the least 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 are the 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 parent has 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 the existing 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 a specific 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 to replace 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 to introduce 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 technically possible to repair and/or replace any known gene, but it is not very efficient and requires a

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 the total 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 precise function 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 (or their 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 Richard Seed 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 soon by 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 perhaps more 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 the structure 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 4 billion 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.

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 all the 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 acid would 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 216 amino 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 would probably 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 of protein, 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 ideas here 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 technology develops?

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 other potential 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 raises numerous 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, and religions 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 comes from testing for diseases. Once disease genes were identified, it became much easier to make a molecular or cytogenetic diagnosis for many genetic conditions. Diagnostic testing supplies 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 a particularly 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-cell diagnostics 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 as preimplantation genetic diagnosis. For adult-onset conditions, ethical concerns have been raised regarding whether genetic testing 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 atrisk 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-Sachs disease 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 disorder later 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 have predictive testing, gaining a locus of control by having a definitive answer is helpful. Some people 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 and behaviors that are not related to disease. Most of these traits and behaviors are inherited as

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 for muscle traits on a group of volunteers enrolled in a resistance-training program (Gordon et al., 2005). These tests looked for single-nucleotide polymorphisms that would tell whether an individual had a genetic predisposition for muscle strength, size, and performance. The investigators 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 such changes 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 major concern 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 are frequently 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 is crucial to understanding people's perceptions and establishing appropriate boundaries.

Building Better Athletes with Gene Doping

Figure 1: The double-muscled Belgian blue cow breed Double muscled animals have an increase in muscle mass of up to 20% greater than normal animals. 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. 1997 Nature Publishing Group Grobet, L. et al. A deletion in the bovine myostatin gene cuases the double-mustard phenotype in cattle. Nature Genetics 17, 71. Used with permission. 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 their competitors. 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 growth hormones (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-doping rules. Today, WADA has a new hurdle to overcomethat of gene 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 that involves 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 new race 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 the invention of so-called "Schwarzenegger mice" in the late 1990s. These mice were given this nickname because they were genetically engineered to have increased muscle growth and 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 muscular development. 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. Schwarzenegger mice differ from these cattle in that they highlight scientists' newfound ability to induce muscle 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 worth exploring, or should we, as a society, decide that manipulation of genes for nondisease purposes is unethical?

Creating Designer Babies

Genetic testing also harbors the potential for yet another scientific strategy to be applied in the area of eugenics, or the social philosophy of promoting the improvement of inherited human traits through intervention. In the past, eugenics was used to justify practices including involuntary sterilization and euthanasia. Today, many people fear that preimplantation genetic diagnosis may be perfected and could technically be applied to select 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 "designer babies," 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 individuals and 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 diagnosis is 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 research subjects. 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 and memory, 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 children to 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 feels about 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 to new 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, contemplation of 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

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 manipulations have often been confined to examining the effect of altering the animal's environment or some aspect of its phenotype. Some animals, notably Drosophila and mice, have been particularly amenable to genetic analyses and traditional genetic manipulation of animals has 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 by inserting 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 into whole 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 and some 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 faithfully in 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 the capacity 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 are postzygotic they represent a stage in development where there has been incomplete separation of the soma and the germline. 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 animal is 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 from the early embryo, the resulting transgenic animal may be able to transmit the foreign DNA stably in its germline. Many different types of transgenic animals have been created including transgenic Drosophila, transgenic frogs, transgenic fish and a variety of transgenic mammals including mice, rats and various livestock animals. The technology of transgenesis and its applications are considered in Section 21.2.

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 recognizable phenotype. This constitutes a form of in vivo mutagenesis, albeit at an unselected target gene. Gene targeting was developed as a method of in vivo mutagenesis in which the mutation is introduced into a preselected endogenous gene. This can be achieved in somatic cells, but gene targeting in cultured ES 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 mice but 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 as transgenic. 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 new animal 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 of somatic cell nuclear transfer permitted cloning of an adult mammal for the first time. This involved transfer of the nucleus 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 transgenic fish have 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-animal expression cloning systems in which introduced genes are expressed to give large amounts of therapeutic or commercially valuable gene products (see Section 22.1.2). But it has been transgenic mice that have been the most useful to biomedical research, both in providing animal models of disease and in permitting the most useful analyses of mammalian gene function.

21.2.1Transgenic animals can be produced following transfer of cloned DNA into fertilized oocytes and cells from very early stage embryos
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

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 is present 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; all their 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. The DNA 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 are constructed 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 are found 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 the foreign 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 can provide 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 in Section 21.2.2. One method that allows foreign DNA to integrate into the chromosomes of the target cells uses retroviruses, RNA viruses which naturally undergo an intermediate DNA form prior to integrating into cellular genomes (see Figure 18.2). Infection of preimplantation mouse

embryos with a retrovirus such 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 of replication- 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. In Drosophila, efficient chromosomal integration is possible by using sequences from a Drosophila 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. The modified DNA is then microinjected into a very young Drosophila 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 to transpose 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 to genetic 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 the construction 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 blastocyst and reimplanted in a pseudopregnant mouse.

Figure 21.2
Genetically modified ES cells as a route for transferring foreign DNA or specific mutations into 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 from different 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

transgenic mouse. Because the injected ES cells can form all or part of the functional germ cells of the chimera, it is possible to derive fully transgenic mice. This is usually accomplished by screening the offspring of matings between chimeras (usually males) and mice with a coat color recessive to that of the strain from which the ES cells were derived (see Figure 21.2). The big advantage of ES 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 a marker gene, such as the neo gene, enabling a positive selection for cells 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 of gene targeting by homologous recombination, a method which can permit a programed selective alteration of a single predetermined gene 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 mouse ES cells. ES cells were not so readily identifed in other mammals, although there have been some important recent successes (see Box 21.1).

Box 21.1
Isolation and manipulation of mammalian embryonic stem cells. The embryo proper derives 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 studies investigating mammalian gene expression and function
Transgenic animals have been extremely important for analyzing human genes (Hanahan, 1989; Camper et 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 for cis-acting regulatory elements is often inferred initially from studies using cultured cells, they

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 of investigating regulation of gene expression. Long-range control of gene expression is often investigated using YAC transgenes, see Section 21.2.4. Investigating gene function by targeted gene inactivation. Specific genes can be inactivated by a gene targeting procedure to introduce a transgene into the target gene (insertional inactivation). The effect on the phenotype 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 promoter which causes expression in cells; the phenotypic consequence may provide valuable clues to function). Cell lineage ablation. Transgenes can be designed consisting of a tissue-specific promoter coupled to a sequence encoding a toxin, for example diphtheria toxin subunit A or ricin. When the promoter becomes active at the appropriate stage of tissue differentiation, the toxin is produced and kills the cells. Thus, certain cell lineages in the animal can be eliminated (cell ablation) and the phenotypic consequences 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 of an 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. The experiment 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 inactivation is 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 greatly extended the applications of transgenic animals

Inducible promoters
For many applications it is desirable to have a transgene expressed under the control of a tissue-specific promoter or 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).

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 metallothionein promoter, etc.). Generally, the use of inducible promoters has been hampered 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 tetracyclineregulated 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 the luciferase 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 endogenous homolog. 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 for correct 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 to establish 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 human HPRT (hypoxanthine phosphoribosyltransferase) gene into mouse ES cells (Jakobovits et al., 1993). This was accomplished by spheroplast fusion (i.e. fusion of ES cells with YAC-containing yeast cells that have been stripped of the hard cell wall; see Section 4.3.4). Fragments from the yeast genome can integrate at the same 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 (see above). 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 lipid vesicles 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 human APP gene, a gene known to contribute to Alzheimer's disease, encoding the amyloid protein precursor. A YAC transgene containing APP showed 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

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 mice able to produce human antibodies (see Mendez 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. YACs containing 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 be transferred. 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 of genes into transgenic animals, although a preferred route may be by using nuclear transfer technology (Section 21.5) rather than ES cells. Recently, however, transfer of whole chromosomes or chromosome fragments into ES cells has been possible by microcellmediated chromosome transfer (see Sections 10.1.1 and 10.1.3). Using this approach Tomizuka 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 show functional 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 be used to produce mice with a mutation in a predetermined gene
Gene targeting involves engineering a mutation in a preselected gene 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),

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 normal phenotype, 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 is transfected into the appropriate cells. In some of the cells, homologous recombination occurs between the introduced gene and its chromosomal homolog. Gene targeting by homologous 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 modified ES 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 sophisticated genetic manipulation). The frequency of homologous recombination is increased, however, when the degree of sequence homology between the introduced DNA and the target gene is very 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 cells were derived). Even then, the frequency of genuine homologous recombination 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 marker gene, such as the neo gene (see Box 10.1), which permits selection for cells that have taken up the introduced DNA. PCR assays are used to screen for evidence of a homologous recombination event (by using a marker-specific primer plus a primer derived from a sequence present in the target gene but absent 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 the locus of interest by a single reciprocal recombination, causing insertion of the entire introduced DNA including the vector sequence (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 the chromosomal gene by a homologous sequence from the introduced DNA (Figure 21.4B). This can occur as a result of a double reciprocal recombination or by gene conversion. The replacement method can inactivate a gene when the introduced sequence contains one or more premature termination codons or lacks critical coding sequences. It can also be used to correct a pathogenic mutation.

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 replacement vector approach, 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 single codon. 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 exogenous sequence (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 a transcription factor known to be expressed in early embryonic development. The lack of a phenotype in such 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 the case of some of the Hox genes (see, for example, Manley and Capecchi, 1997). A useful example of investigating functional redundancy concerns studies of the mouse Engrailed genes, En-1 and En-2. Both of these genes are homeobox genes which had been considered to play crucial roles in brain formation. En-1 knock-outs have serious abnormalities but surprisingly En-2 knock-outs have only minor problems. Expression of the En-1 gene is switched on 810 hours before that of the En-2 product, suggesting that perhaps the En-1 product can compensate for the lack of En-2 product in En-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

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 an En-2 coding sequence was used to knock-out the endogenous En-1 gene. In so doing, the introduced En-2 sequence came under control of the En-1 regulatory sequences and was expressed before the endogenous En-2 gene was switched on (see Figure 21.6). The resulting En-1 knock-out mouse had a normal phenotype, demonstrating that the knocked-in En-2 gene was functionally equivalent to En-1 (Hanks et al., 1995).

Figure 21.6
The knock-in method replaces the activity of one chromosomal gene by that of an introduced gene. The En-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-loxP system, extend the power of gene targeting
Several site-specific recombination systems from bacteriophages and yeasts have been characterized and are promising tools for genome engineering (Kilby et al., 1993). Thus far, the Cre-loxP recombination system from bacteriophage P1 has been the most widely used. The natural function of the Cre (causes recombination) recombinase is to mediate recombination between two loxP sequences that are in the same orientation (the loxP sequence consists of 34 bp and comprises two inverted 13 bp repeats separated by a central asymmetric 8 bp spacer; Figure 21.7). As a result of recombination, the intervening sequence between the two loxP sites is excised (see Figure 4.15). Using gene targeting, loxP sequences can be stitched into a desired gene or chromosomal location, and the subsequent provision of a gene encoding the Cre product can result in an artificially generated site-directed recombination event (see Chambers, 1994). As described below, several applications can be envisaged (Lobe and Nagy, 1998).

Figure 21.7
Structure of the loxP recognition sequence. Note that the central 8 bp sequence which is flanked by the 13 bp inverted repeats is asymmetric and confers orientation.

Tissue- and cell type-specific knock-outs

Some genes are vital to early development and simple knock-out experiments are generally not helpful because death ensues at the early embryonic stage. To overcome this problem,

methods have been developed to inactivate expression of the target gene in only selected, predetermined cells of the animal (conditional knock-out). The animal can therefore survive and the effect of the knock-out can be studied in a tissue or cell type of interest. An early example of a conditional knock-out sought to study the role of DNA polymerase (an enzyme that is essential for embryonic development) in T lymphocytes (Gu et al., 1994). The gene targeting procedure replaced part of the endogenous gene by an introduced homologous gene segment flanked by loxP sequences. Mice carrying this targeted mutation were then mated with a strain of mice which carried a Cre transgene gene under the control of a T cell-specific promoter. Offspring with the loxP-flanked pol sequences plus the Cre transgene were identified and survived to adulthood. The Cre product was expressed only in T cells, leading to inactivation of the target gene in these cells by excision of DNA polymerase gene segment between the two loxP sequences (see Figure 21.8 for the general method).

Figure 21.8
Gene targeting using the Cre-loxP recombination system can be used to inactivate a gene in a desired cell type. (A) Illustration of a standard homologous recombination (more...)

Tissue- and cell type-specific gene activation

This approach is the opposite to that described above: it involves selective activation of a gene in certain cells of the animal to switch on a foreign gene only in predetermined cells of the animal (Barinaga, 1994).

Chromosome engineering
Another important recent development is a strategy for chromosome engineering in ES cells which relies on sequential gene targeting and Cre-loxP recombination. Gene targeting is used to integrate loxP sites at the desired chromosomal locations and, subsequently, transient expression of Cre recombinase is used to mediate a selected chromosomal rearrangement (Ramirez-Solis et al., 1995; Smith et al., 1995; Figure 21.9). Chromosome engineering strategies of this type offer the exciting possibility of creating novel mouse lines with specific chromosomal abnormalities for genetic studies.

Figure 21.9
Chromosome engineering can be accomplished using cre-loxP systems. (A) Use of targeted insertion of loxP sites to facilitate a chromosomal translocation. See Smith et al. (more...) The multiple targeting and selection steps in ES cells used in the above chromosome engineering methods can be avoided using the novel approach of Herault et al. 1998. This targeted meiotic recombination method takes advantage of the homologous chromosome pairing that occurs naturally during meiosis at the first cell division. A transgene is designed to express Cre recombinase under the control of a Sycp1 promoter (the Sycp1 gene encodes the SCYP1 protein which is part of the synaptonemal complex that facilitates crossing over). As a result Cre recombinase is produced in male spermatocytes during the zygotene to pachytene stages when chromosome pairing occurs.

21.3.3Gene trapping in mouse ES cells allows an efficient approach to functional analysis

Large-scale approaches to investigating animal gene function have principally relied on exposing animals to high doses of radiation, or to potent chemical mutagens such as ethylnitrosurea (ENU) or ethyl methylsulfonate (EMS). In mouse mutagenesis programs, for example, males are typically exposed to high levels of a suitable mutagen to induce a high frequency of mutation in sperm DNA. The progeny of irradiated mice are then screened for obvious phenotypic abnormalities. Such mutagenesis screens have been very useful in producing novel mutants, but a major problem is that the mutations occur essentially at random. Identification of the structural change(s) in the DNA of a single mutant animal may therefore require a laborious positional cloning approach. A mutation that is induced by inserting a known foreign DNA sequence (transgene) into the mouse genome has a major advantage over one induced by chemical mutagens or X rays: it leaves a sequence tag at the locus which is mutated. As a result, rapid molecular characterization of the mutated locus is possible. Mouse ES cells provide a way of introducing such mutations into the germ line, and so random insertional mutagenesis in ES cells using transgenes was considered a useful way of producing a large number of mouse mutants which could quickly be characterized at the molecular level. Unlike gene targeting, the transgene should not be related to endogenous sequences, but should integrate essentially at random by nonhomologous recombination. However, because large sections of the mouse genome are noncoding, many random insertion events may not result in gene disruption. In order to improve the efficiency of recovering mutations that are likely to have a phenotypic effect (by altering a gene or its expression) the gene trap approach was devised (see Evans et al., 1997). The underlying principle is that the transgene which is inserted into ES cells contains a defective reporter gene or marker gene which lacks some component needed for gene expression. The reporter or marker gene is designed to be expressed only after it inserts into a gene (within an intron or exon) or at a promoter. When it integrates at such positions it can acquire the expression element that it lacks.

Different gene trap strategies are possible. In some cases, the reporter lacks a functional promoter and so relies on chance integration next to an appropriate cis-acting sequence element that can activate its transcription. Other approaches have used a marker gene coupled to a suitable promoter but lacking a downstream polyadenylation signal. Here, the marker gene is designed to be expressed after integrating into a host cell gene such that a fusion RNA product is made that utilizes 3 host sequences in order to acquire a poly (A) tail (see Figure 21.10). Already, this type of approach has been applied on a large scale, with a recent report describing disruption and sequence identification of 2000 genes in mouse embryonic stem cells (Zambrowicz et al., 1998).

Figure 21.10
Gene trapping uses an expression-defective transgene to select for chromosomal integration events that occur within or close to a gene. A specimen host cell gene with 4 exons (E1 E4) is shown (more...)

21.4Creating animal models of disease using transgenic technology and gene targeting
Go to: Animal models of human disease are crucially important to medical research. They allow detailed examination of the physiological basis of disease. They also offer a front-line testing system for studying the efficacy of novel treatments before conducting clinical trials on human subjects. Although many individual human disorders do not have a good animal model, animal models exist for some representatives of all the major human disease classes: genetically determined diseases, disease due to infectious agents, sporadic cancers and autoimmune disorders (see Leiter et al., 1987; Darling and Abbott, 1992; Clarke, 1994; Bedell et al., 1997). Some animal models of human disease originated spontaneously; others have been generated artificially by a variety of different routes (Table 21.1).

Table 21.1
Classes of animal models of disease. Until recently, the great majority of available animal disease models were ones which arose spontaneously or had been artificially induced by random mutagenesis using exposure to high doses of mutagenic chemicals or X-rays (next section). More recently gene targeting and transgenic technologies have provided direct ways of obtaining animal models of disease, and targeted mutations in the mouse have been particularly valuable. Interestingly, it has become increasingly clear that disease phenotypes due to comparable mutations in human and mouse gene homologs often show considerable differences (Section 21.4.5).

21.4.1Animal models of disease occurring spontaneously or as a result of exposure to chemical mutagens or radiation may be difficult to identify

Spontaneous animal disease models

Mutant human phenotypes, especially those associated with obvious disease symptoms, are subject to intense scrutiny: most individuals who suffer from a disorder seek medical advice. If they present with a previously undescribed phenotype their case may well be referred to experts who often will document the phenotype in the medical literature. Given the motivation of both affected individuals and their families, physicians and interested medical researchers, and the large population size for screening (current total global population is ~6 billion individuals), there is a remarkably effective screening process for mutant human phenotypes. In contrast, many animal disease phenotypes will go unrecorded. Only a small percentage of the animal population is in captivity, and recording of spontaneous mutant phenotypes is largely dependent on examination of animal colonies bred for research purposes and, to a lesser extent, livestock and pet populations. Only mutants with obvious external anomalies are likely to be noticed. Despite the difficulty in identifying spontaneous animal mutants, a number of animal phenotypes have been described as likely models of human diseases (see Table 21.2 for some examples). In some cases, the animal mutant phenotype closely parallels the corresponding clinical phenotype, but in others there is considerable divergence because of species differences in biochemical and developmental pathways (see Erickson, 1989; Erickson, 1996; Wynshaw-Boris, 1996; see also Section 21.4.5). Additionally, phenotypic differences may result because of different classes of mutation at orthologous loci.

Table 21.2
Examples of spontaneous animal mutants.

Random mutagenesis using chemicals and irradiation

Classical methods of producing animal mutants have involved controlled exposure to mutagenic chemicals, notably ethyl nitrosurea (ENU) and ethyl methylsulfonate (EMS) or to high doses of X-rays. Large numbers of Drosophila and mouse mutants have been obtained by this method and, very recently, efficient large-scale mutagenesis screens have been conducted on both mice and also zebrafish (which offers some advantages as an animal model; see Box 21.2). A major problem with chemically-induced and irradiationinduced mutations, however, is that they are generated essentially at random. In order to identify a mutant phenotype of interest, a laborious screen for mutants needs to be conducted by close examination of the phenotypes following mutagenesis. The mutant phenotypes which have been described in these studies, as well as for spontaneous mutants, show a clear bias towards phenotypes with obvious external abnormalities, simply because of the ease of identifying them. Nevertheless, several important models of human disease have been created using such methods.

Box 21.2
The potential of animals for modeling human disease. Primates should provide the best animal models of human disease because they are so closely related to us (see Section 14.6.2). Humans and great (more...)

21.4.2Mice have been widely used as animal models of human disease largely because specific mutations can be created at a predetermined locus
Spontaneous and artificially produced disease phenotypes have been described in a wide range of animal species with differing potentials for modeling human disease. In some cases, the species may be too evolutionarily remote from humans to provide useful disease models. For example, numerous Drosophila mutants have been generated and studies of some developmental mutants have enabled the identification of human genes that are important in development, but they cannot serve as useful models of human disease. Other species, such as the zebrafish, which are also evolutionarily remote from us, offer some advantages as model organisms and models have been reported for some human diseases (Box 21.2). Mammals would be expected to provide better disease models but, for a variety of reasons, our closest relatives, the great apes, have not been very useful in providing

disease models. Instead, other mammals, notably mice, have been used widely to model human disease (see Box 21.2). In the case of animal disease models which are artificially induced by exposure to mutagenic chemicals or radiation, or which originate spontaneously, there is little or no artificial control over the resulting phenotype and frequently, the identification of an animal disease model is serendipitous. The great advantage of transgenic/genetargeted mouse models of disease is that specific disease models can be constructed to order. Provided that the relevant gene clones are available, including mutant genes in some cases, mice can be generated with a desired alteration in a chosen target gene. All the major classes of disease, inherited disorders, cancers, infectious diseases and autoimmune disorders can be modeled in this way (Table 21.3; Smithies, 1993; Clarke, 1994; Bedell et al., 1997). In most cases, the transgenic/gene targeting approaches have been used to model single gene disorders but, increasingly, attempts are being made to produce mouse models of complex genetic diseases, such as Alzheimer's disease, atherosclerosis and essential hypertension effects.

Table 21.3
Examples of transgenic or gene-targeted mouse models of human disease.

21.4.3Single gene disorders resulting from loss of function and gain of function mutations can be conveniently modeled by gene targeting and by integration of mutant genes respectively

Modeling loss of function mutations by gene targeting in mice

Many disease phenotypes, including those of essentially all recessively inherited disorders and many dominantly inherited disorders, are thought to result from loss of gene function. The simplest way of modeling the disease for single gene disorders of this type is to make a knock-out mouse. The first step is to isolate the orthologous mouse gene and to use a segment of it to knock out the endogenous gene in mouse ES cells using gene targeting. Following injection of the genetically modified ES cells into the blastocyst of a foster mother, and continued development, founder mice are obtained with the targeted mutation in a sizeable proportion of their germ cells. These mice can be interbred and the offspring can be screened for the presence of the desired mutation, and for the presence of the wildtype allele using PCR assays of cells collected from tail bleeds.

The gene targeting event is intended to create a null allele (where there is complete absence of gene expression), but sometimes the result may be a leaky mutation and the mutant allele retains some gene expression. This may explain why gene targeting has produced mouse models of a disease with divergent phenotypes. For example, the mouse model of cystic fibrosis described by Snouwaert et al. 1992 had a severe phenotype while that reported by Dorin et al. 1992 had a mild phenotype because of a leaky mutation. Differences in phenotype may also occur because of modifier genes using different mouse strains (see Section 21.4.5).

Modeling gain of function mutations by insertion of a mutant gene

This general experimental design has been used frequently in conjunction with the pronuclear microinjection technique of gene transfer. The disease to be modeled must be one where the presence of an introduced DNA is itself sufficient to induce pathogenesis, and can include inherited gain of function mutations, oncogenes, etc. To model such disorders, it is necessary to clone a mutant gene or, if necessary, design one by in vitro mutagenesis. The mutant gene is then simply inserted as a transgene, e.g. by microinjection into fertilized oocytes. Because there is no requirement for the introduced mutant gene to integrate at a specific location, human mutant genes will suffice although, in some cases, mouse mutant genes have been used. The two examples below illustrate this approach.

An early example was intended to assess whether a leucine substitution found at codon 102 in a prion protein gene in a patient with Gerstmann-Strussler-Scheinker (GSS) syndrome was pathogenic. An analogous mutation was artificially designed in a cloned mouse prion protein gene and the mutant gene was then injected into fertilized oocytes to produce transgenic mice. The mice went on to develop spontaneous neurodegeneration, reminiscent of that found in the human syndrome (Hsiao et al., 1990). A variety of other experiments using prion protein transgenes have been very helpful in understanding prions (Gabizon and Taraboulos, 1997). Expanded triplet repeats causing neurodegenerative disorders (see Box 16.7) constitute another type of gain of function mutation. Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited disorder which results from unstable expansion of a CAG triplet repeat in the ataxin gene. It is characterized by degeneration of cerebellar Purkinje cells, spinocerebellar tracts and some brainstem neurons. Transgenic mice were produced by introduction of one of two transgenes driven by a Purkinje cell-specific promoter: the normal human ataxin gene (SCA1), and a mutant ataxin gene containing an expanded CAG repeat. Both types of transgene were stable in parent to offspring transmissions, but only those with the expanded allele developed ataxia and Purkinje cell degeneration, confirming the gain of function hypothesis (Burright et al., 1995).

21.4.4Considerable effort is currently being devoted to constructing mouse models of cancers and other complex genetic disorders

Modeling human cancers

(See Ghebranious and Donehower, 1998; Macleod and Jacks, 1999.)

Gain of function. Disease due to inappropriate inactivation of a proto-oncogene can be modeled by constructing a transgenic mouse: the appropriate oncogene is introduced into the mouse genome by simple transgene integration. Loss of function. Disease due to inactivation of tumor suppressor genes can be modeled by constructing knock-out mice through gene targeting. For example, several models have been generated by inactivating the mouse homologs of the TP53 and RB1 genes but the phenotypes show only broad similarity to the homologous human phenotypes, respectively Li-Fraumeni syndrome and retinoblastoma (see also Section 21.4.5).

Modeling chromosomal disorders

Existing mouse models for human chromosomal disorders are sparse. In some cases this is due to insufficent conservation of synteny between the two species. Taking the example of Down syndrome (trisomy 21), human chromosome 21 shares a large region of genetic homology with mouse chromosome 16 (see Figure 14.23), but trisomy 16 (Ts16) mice are not good models because they die in utero. The Ts16 mouse could never be expected to be a good model of human trisomy 21: it is not trisomic for all human chromosome 21 genes (the genes in the distal 23 Mb of human chromosome 21 have orthologs on mouse chromosomes 17 and 10) and is trisomic for some genes which have human orthologs on chromosomes other than chromosome 21. In order to produce a better Down syndrome model in the mouse, attention has focused on the Down syndrome critical region at 21q21.3-q22.2 (deduced from observing the phenotypes of rare Down syndrome patients with partial trisomy 21). Within this region, the human minibrain gene at 21q22.2 may be an important contributory locus to the associated learning defects: transgenic mice in which a 180 kb YAC containing the 100 kb human minibrain gene but apparently no other gene, develop learning defects (Smith et al., 1997). A segmental trisomy 16 mouse, Ts65Dn, obtained by standard methods of irradiating mice was shown by Reeves et al. 1995 to have learning and behavior deficits. Other more recently produced models are discussed by Kola and Hertzog (1998). Future efforts in modeling chromosomal disorders can be expected to take advantage of gene targeting using Cre-loxP. As described in Section 21.3.2 this system offers tremendous potential for genome engineering and can be used to engineer chromosome translocations at defined positions on preselected chromosomes. YAC transgenics can also be expected to be important in investigating over-expression of genes in other chromosomal disorders and in disorders resulting from aberrant gene dosage for large

regions, such as Charcot-Marie-Tooth type 1A, which is due to overexpression as a result of a 1.5-Mb duplication in the PNP22 gene region (see Figure 16.7).

Modeling complex diseases

Increasingly, the focus in human genetics is moving towards understanding the pathogenesis of complex genetic diseases such as atherosclerosis, essential hypertension, diabetes, etc. Such disorders have a complex etiology with multiple genetic and environmental components. Some valuable animal models have been produced for some of these disorders but gene targeting approaches are expected in the future to provide additional badly needed models (Smithies and Maeda, 1995). As long as suitably promising genes can be identified as being involved in the pathogenesis, then breeding experiments can be used to bring different combinations of disease genes together, and the effect of different genetic backgrounds in different strains of mice and of different environmental factors can be assessed. This approach may not be so daunting as it sounds because, increasingly, many complex disease phenotypes are considered to be due mostly to the combination of only a very few major susceptibility genes. For example, a digenic model of spina bifida occulta was generated serendipitously in offspring obtained by crossing a mouse heterozygous for the Patch mutation (pdgfrb; platelet-derived growth factor receptor) with a mouse homozygous for the undulated mutation (pax-1) (see Helwig et al., 1995).

21.4.5Mouse models of human disease may be difficult to construct because of a variety of human/mouse differences
It is not uncommon for spontaneous or artificially generated mouse models of disease to show phenotypes that are considerably different from the homologous human disorders. For example, gene targeting to inactivate several mouse tumor suppressor genes has often produced disappointing mouse models, as in the case of TP53 and RB1 (retinoblastoma) knock-outs. There may be problems in achieving the desired type of mutation. For example, in gene targeting intended null mutations may be offset in some cases by exon skipping or some other form of leaky transcription, or the expression of a transgene may be affected by various parameters causing an unexpected phenotype. Setting aside these possibilities, there are several areas where differences between mice and humans could be expected to result in divergent disease phenotypes for mutations in orthologous genes (Erickson, 1989; Erickson, 1996; Wynshaw-Boris, 1996):

Biochemical pathways. Although biochemical pathways in mammals are generally well conserved, some differences are known between the pathways of humans and mice. The human retina appears to depend heavily on the accurate function of the Rb gene product, but other vertebrate retinas do not. As a result, spontaneous retinoblastoma mouse mutants have not been described, and retinoblastoma is not a feature of Rb1 knock-out mice. Another example may be provided by ganglioside degradation pathways and while deficiency of the hexosaminidase gene HEXA results in the severe Tay-Sachs disorder, inactivation of the mouse homolog Hexa

results in abnormal accumulation of ganglioside in neurons but without motor deficits or learning deficits (Wynshaw-Boris, 1996). Developmental pathways. The differences in human and mouse developmental pathways are not well understood but are expected to be significant for some organ systems, such as in brain development. Absolute time. Because of the huge difference in the average lifespans of mice and humans, certain human disorders in which the disease is of late onset may possibly be difficult to model in mice. Genetic background: the importance of modifier genes. Most human populations are outbred. Laboratory strains of mice, however, are very inbred. Often a particular phenotype can vary considerably in different strains of mice because of differences in their alleles at other loci (modifier genes), which can interact with the locus of interest.

A useful example of the importance of genetic background is the Min (multiple intestinal neoplasia) mouse which was generated by ENU mutagenesis and results from mutations in the mouse Apc gene. Mutations in the orthologous human gene, APC, cause adenomatous polyposis coli and related colon cancers and the Min mouse has been regarded as a good model for such disorders. The phenotype of the Min mouse is, however, dramatically modified by the genetic background. For example the number of colonic polyps in mice carrying APCMin is strikingly dependent on the strain of mouse. Similar phenotypic variability is found in human families where different members of the same family may have strikingly different tumor phenotypes although they possess identical mutations in the APC gene. Some of the variability could be due to environmental factors, but the involvement of modifier genes had been strongly suspected. The Min mouse provides a well-defined genetic system for mapping and identifying modifier genes (Dietrich et al., 1993; MacPhee et al., 1995).

21.5Manipulating animals by somatic cell nuclear transfer

Go to:

21.5.1Principles and practice of animal cloning

The term clones indicates genetic identity and so can describe genetically identical molecules (DNA clones), genetically identical cells or genetically identical organisms. Animal clones occur naturally as a result of sexual reproduction. For example, genetically identical twins are clones who happened to have received exactly the same set of genetic instructions from two donor individuals, a mother and a father. A form of animal cloning can also occur as a result of artificial manipulation to bring about a type of asexual reproduction. The genetic manipulation in this case uses nuclear transfer technology: a nucleus is removed from a donor cell then transplanted into an oocyte whose own nucleus has previously been removed. The resulting renucleated oocyte can give rise to an individual who will carry the nuclear genome of only one donor individual, unlike

genetically identical twins. The individual providing the donor nucleus and the individual that develops from the renucleated oocyte are usually described as clones, but it should be noted that they share only the same nuclear DNA; they do not share the same mitochondrial DNA, unlike genetically identical twins. Nuclear transfer technology was first employed in embryo cloning, in which the donor cell is derived from an early embryo, and has been long established in the case of amphibia. However, it was only comparatively recently when McGrath and Solter reported nuclear transplantation in the mouse embryo and paved the way for modern mammalian cloning (see Fulka et al., 1998). Subsequently, nuclear transplantation was conducted successfully in the eggs of domestic animals, including sheep and cows. Unlike embryo cloning the prospect of cloning adults had seemed remote. During their development from (ultimately) the fertilized oocyte, adult somatic cells undergo an extensive series of cell division and differentiation steps. Until recently it was thought that these processes were accompanied by irreversible modifications to the genome. Even in frogs transplantation of an adult cell nucleus had never been reported to give rise to an adult animal; instead, the renucleated embryos underwent early development but thereafter failed to develop to term. A variety of early experiments in mice were also unsuccessful before the landmark study of Wilmut et al. 1997 reported successful cloning of an adult sheep. For the first time, an adult nucleus had been reprogrammed to become totipotent once more, just like the genetic material in the fertilized oocyte from which the donor cell had ultimately developed. In the Wilmut et al. study, the donor cells were derived from a cell line established from adult mammary gland cells and were fused to an enucleated metaphase II-arrested oocyte (Figure 21.11A). The donor cells were deprived of serum before use, forcing them to exit the cell cycle into a quiescent stage, Go (Stewart, 1997). A certain degree of gene silencing is a characteristic feature of the nuclei of Go cells. As egg cells are normally fertilized by transcriptionally inactive sperm cells, Go cells may be more amenable to full genetic reprogramming. Another consideration is the degree of chromosome condensation and of access to chromatin remodeling factors such as transcription factors in the oocyte. In any event, the cloning was extremely inefficient: out of a total of 434 oocytes that were submitted to the procedure, only 29 developed to the transferable stage and of these only one developed to term, being born as the now famous Dolly (Figure 21.11B). Subsequent doubts about the exact origin of the donor cell and whether Dolly really was an adult clone (as opposed to a contaminant fetal cell) have been allayed by genetic testing of Dolly and the adult mammary gland donor cells. Importantly, successful animal cloning has also been achieved by other groups with comparatively high success in cloning of adult mice (Wakayama et al., 1998) and cows (Kato et al., 1998).

Figure 21.11
A sheep called Dolly was the outcome of the first successful attempt at animal cloning. (A) Experimental strategy used by Wilmut et al. (1997). During culture in serum-depleted medium the normal (more...)

21.5.2The successful cloning of an adult animal has ma

Technology, Genetic Manipulation, and the Future

December 1991 By Egbert Schuurman [Dr. Egbert Schuurman is a professor at the technical universities in both Delft and Eindhoven in the Netherlands. Since 1983 he has also been a member of the upper house of the Dutch Parliament representing one of his country's smaller political partiesthe Reformed Political Federation. An expert in technological issues related to the environment and human life, Dr. Schuurman has published widely and participated in numerous international conferences. [The following selected excerpts are taken from Schuurman's "The Future: Our Choice or God's Gift?"a recent English translation of a Dutch booklet. The translator and editor is Petrus Simons. The book is available from Exile Press, P.O. Box 12-294, Wellington, New Zealand. Ed.] Technology Without Limits [The parable about King Midas teaches us that too much of a good thing can lead to death.] If used wisely, gold is a delight and able to play a meaningful role in life. Too much of it causes great problems.

It is no different with science and technology. They are fine gifts which our God and Creator has entrusted to us so that we might use them wisely and to his glory, to serve him and our fellow man. In our culture, however, these wonderful gifts have been used to conquer the world without stopping at any barriers. By means of a thoroughgoing scientific analysis we have been breaking down reality into its smallest possible components. Once we have these basic bits, we try to use them to reconstruct a different whole, according to our wishes. In recent times, this has happened with the discovery and identification of the basic building blocks of living beings: genes. Biotechnology, using recombinant DNA techniques, is now able to breed new types of plants and animals. What about human beings? Should we not remake them as well? Roots of the New Faith During the 16th century, two major movements came into conflict. One was called the Renaissance, which had been developing since the 14th century, and the other the Reformation, which had started with the rediscovery of the Gospel by Martin Luther. Each had a profound impact on Western society. Yet, gradually, the Renaissance came to dominate the spirit of the ages that followed. The people of the Renaissance saw the world as a place in which man is free to work but is answerable only to himself for his actions. He should not be dictated to by the church. He should strive for self-realization and self- fulfillment. He should attempt to discover himself and explore all the possibilities of the world around him. By doing this consistently, man would become a new being, he would experience a new birth (this is what "renaissance" means). This rebirth does not come from God, but results from man's own actions in history. In this renaissance man discovers his own creativity. Man is a creator. By harmoniously developing all aspects of his life, by becoming an all-round human being, man would achieve redemption from the imperfections of his past. This new man could also sin, but only by becoming unfaithful to himself, by subjecting himself to alien authority, be it God's authority or the authority of the church. Man is free to determine his own destiny. If rules are needed, then he will determine those, using his capacity to reason. The world is not a theater in which the drama of God, man and the devil is acted out, or in which God carries out his rescue mission through his Son. It is rather a selfcontained, beautiful structure, a great work of art. In the enormous powers of nature man discovers something of himself. There are hardly any limits put to his endeavors. Man's free will propels him to ever-greater heights. Technism and Genetic Manipulation Genetic manipulation is a new scientific-technical possibility [that has arisen in the course of a long line of scientific and technological development, fired by Renaissance aspirations.] In company with a growing number of philosophers I have come to view technism as the dominating spiritual driving force behind science and technology. Although

it has been the prevailing influence, I emphasize that there have also been other factors, and this suggests that it may be possible to find another, more acceptable road to the future. "Technism" is man's pretension to control the whole of reality by means of science and technology. This control should serve to solve all problems and to secure material welfare and happiness. Man himself wants to be lord and master, creator, redeemer and renewer. The Enlightenment reinforced the contents and consequences of technism. Through this 18th-century movement the spirit of the Renaissancean unlimited trust in man's own power to renew lifebecame connected with the physical sciences. The heroic man of the Enlightenment believes that by means of the physical sciences he is capable of solving all problems so as to renew himself as well as society. [When it comes to health care, the implications of technism are that] man relates to reality in the same way as a dictator relates to people. He only knows them insofar as they can be manipulated. Technical possibilities are being overrated when the value of life, so valuable and worthy of protection, is sacrificed to it. This happens in the case of abortion and in cases where the dying process is drawn out by means of technology. In-vitro fertilization (IVF)the test-tube-baby techniquehas made it possible to use human embryos in a laboratory. Initially it was intended to implant them into mothers who were unable to conceive children in the natural way. More and more, however, experiments are being done with the embryos obtained. It has been suggested that they may be used for the production of medicines. This raises their economic value so that they might become commercial objects. There are also researchers who are in favor of complete scientifictechnical control of the procreation process through an experimental approach. IVF might also allow the selection of embryos. One might prefer boys to girls or vice versa. Moreover, one could be more interested in children with a high intelligence than in others. The next step would be to manipulate genes on the basis of a knowledge of the genetic makeup of the embryo. Someone has said that every individual letter in the book (the genome) of a human being will be able to be read. Thereafter "sick" or "damaged" genes can be replaced by new ones. Perhaps some new genes could be added. Thus the "ennobling" of the human race could become a possibility. Genetics would evolve into eugenics. New possibilities will lead to pressures on every couple. On the basis of genetic examination and information they will receive free advice, without any obligation. Yet, as a result of public opinion and pressure, many parents will have to face problematic decisions. Initially, abortion will be advised when prenatal diagnostics indicate the possible birth of a handicapped child. In official documents this is called "secondary prevention" and "optimization of the reproductive process." If knowledge of the gene composition of both parents points to an increased risk of conceiving handicapped children, then fertilization will take place more and more in test tubes. This provides the possibilities for prenatal diagnostics, experiments, elimination and selection.

Nobel-prize winner Walter Gilbert, who teaches biochemistry at Harvard University, believes that this is simply the result of the age-old exhortation "Know yourself." This has opened the door to genetic manipulation. Any principled resistance to genetic manipulation of the embryo ceases to have meaning. In the Christian viewif it is not secularizedresistance to experiments with and manipulation of embryos remains complete because every human being is believed to have been created in God's image and is unique in God's eyes. Its beginning lies in conception. God's love and care extend to the life of every human being. That life is never perfect. It is not up to man to set the norm for an existence worthy of a human person, nor to determine unilaterally the quality of life. A Different Way Am I suggesting that the latest scientific-technical possibilities should not be used at all? That would be a wrong conclusion. I would like to plead for a different view of science and technology: rather than playing a key role in human affairs they should play second fiddle. Instead of being demanding masters, they should become humble servants. When is genetic manipulation of people justified in the light of growing wisdom and when not? Let us start with the latter. If science is influenced by the will to power, then the object to be controlled will not be recognized according to its own nature and meaning. The whole object will be reduced to a knowledge of the abstractions made. This procedure will result in reductions and will lead, in practice, to technization. Such technization will also affect man if the whole of the human person is controlled by means of genetic manipulation. This happens, or could happen, with embryos in the laboratory. In such genetic manipulation it is not appreciated that the whole person is at stake even if only as a potential person. [For this reason I believe we should reject] genetic manipulation of the genome. At the same time this points to a framework within which genetic manipulation could be a good thing. In this I do not question responsible genetic manipulations of bacteria, fungi or virusesprovided appropriate safety measures are takenfor the production of new medicines for sick people which surpass existing medicines or are able to combat diseases that hitherto could not be cured. In other words, genetic manipulation of particular organs is possible. Genotherapy on somatic cells in which the whole man is respected in love may contribute to responsible health care. For instance, this is the case in the fight against certain bone marrow diseases, particularly sicklecell anemia, or diabetes through manipulation of the spleen. As with so many therapies, geno-treatment is likely to cause a permanent change in the human body, so it is all the more urgent to exclude those organs from genetic manipulation which interfere with the integrity of the human person. I mention in this connection the germ cells of the genital organs and the brain (in total). Those who advocate genetic manipulations of cells of genital organs because successful repairs would be transferable, ignore not only the structural objections just discussed but also the major uncertainties for the next generation. The consequences of the modification to be made cannot be fully appreciated because of

the complexity of the germ cells and of the mystery of the genome which cannot be scientifically fathomed. [From a Christian point of view all things exist by God's power] and have their destiny in him. If we abstract from this intimate relationship of everything with God, then the proper nature and the meaning of things cannot be appreciated. For this reason, a Christian perspective requires a strong emphasis upon the limitations of science. 866-CPJUSTICE (866-275-8784) Local: 202-220-1347 email: inquiries@cpjustice.org Mailing Address:

opyright 2012. All Rights Reserved. ivacy Policy