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Applications of Biotechnology Techniques

INTRODUCTION

The art and science of producing genetically engineered animals have advanced very rapidly in the past few years. It now is possible to generate animals with useful novel properties for dairy, meat, or fiber production, for environmental control of waste production, for biomedical purposes or other human consumption, or that are nearly identical copies of animals chosen for useful traits, such as milk or meat production, high fertility, and the like. This chapter addresses the current state of the art of these technologies and then point out specific concerns that arise as a consequence of their application. Subsequent chapters will discuss how the technical issues can directly affect human health, the food supply, animal welfare, and the environment.

INTRODUCTION OF NOVEL GENES

A number of methods are presently employed for genetic engineering of various animal species. Most of these were developed originally in mouse and Drosophila models and have only more recently been extended to other domesticated animals. Access to the germline of mammals can be obtained by: (1) direct manipulation of the fertilized egg, followed by its implantation into the uterus; (2) manipulation of the sperm used to generate the zygote; (3) manipulation of early embryonic tissue in place; (4) the use of embryonic stem



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Animal Biotechnology: Science-Based Concerns 2 Applications of Biotechnology Techniques INTRODUCTION The art and science of producing genetically engineered animals have advanced very rapidly in the past few years. It now is possible to generate animals with useful novel properties for dairy, meat, or fiber production, for environmental control of waste production, for biomedical purposes or other human consumption, or that are nearly identical copies of animals chosen for useful traits, such as milk or meat production, high fertility, and the like. This chapter addresses the current state of the art of these technologies and then point out specific concerns that arise as a consequence of their application. Subsequent chapters will discuss how the technical issues can directly affect human health, the food supply, animal welfare, and the environment. INTRODUCTION OF NOVEL GENES A number of methods are presently employed for genetic engineering of various animal species. Most of these were developed originally in mouse and Drosophila models and have only more recently been extended to other domesticated animals. Access to the germline of mammals can be obtained by: (1) direct manipulation of the fertilized egg, followed by its implantation into the uterus; (2) manipulation of the sperm used to generate the zygote; (3) manipulation of early embryonic tissue in place; (4) the use of embryonic stem

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Animal Biotechnology: Science-Based Concerns (ES) cell lines which, after manipulation and selection ex vivo, can then be introduced into early embryos, some of whose germline will develop from the ES cells (Smith, 2001); and (5) manipulation of cultured somatic cells, whose nuclei then can be transferred into enucleated oocytes and thereby provide the genetic information required to produce a whole animal. The last two methods have the advantage of allowing cells containing the modification of interest to be selected prior to undertaking the expensive and lengthy process of generating animals. Usable ES cells are not available for all species of interest, however, and generation of embryos by nuclear transfer (NT) from somatic cells is becoming the method of choice for genetic engineering and duplication of nearly genetically identical animals (Westhusin et al., 2001). Manipulation of the avian germline is difficult since ES lines are not available and the early embryo is difficult to access. Much current work focuses on the use of blastodermal cells or primordial germ cells, which can be cultured briefly and manipulated to modify the germline prior to introduction into fresh embryos to create chimeras from which modified lines can eventually be developed, albeit with low efficiency (Aritomi and Fujihara, 2000). There are two basic approaches presently in use for inserting DNA into vertebrate germline cells, transfection and infection with retrovirus vectors. A third approach, based on the use of mobile genetic elements, has been commonly used for insects and is being explored for germline modification of vertebrates (Izsvak et al., 2000). Transfection Transfection methods include: (1) direct microinjection of DNA into the cell nucleus; (2) electroporation—introduction of DNA through transient pores created by controlled electrical pulses; (3) use of polycations to neutralize charges on DNA and the cell surface that prevent efficient uptake of DNA; (4) lipofection, or enclosure of DNA in lipid vesicles that enter a cell by membrane fusion much in the manner of a virus, and (5) sperm-mediated transfection, possibly in conjunction with intracytoplasmic sperm injection (ICSI) or electroporation (see Chapter 6). The manner of introduction of DNA is a technical issue, determined empirically for each system, and makes little difference to the final outcome. In general (with the exception of homologous recombination, discussed below), the structure of DNA introduced into a cell by any of these methods is highly variable and uncertain. Often, only a fragment of the transfected DNA is integrated into the chromosome, frequently in multiple copies, that often are integrated in long tandem arrays (Gordon and Ruddle, 1985). When transfecting cultured somatic or ES cells, a selectable marker, such as the gene encoding phosphotransferase, is often included as part

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Animal Biotechnology: Science-Based Concerns of the DNA to permit selection for its presence either in eukaryotic cell lines or in the bacteria in which the DNA was mass-produced. Retrovirus Vectors Retroviruses are infectious elements that replicate by a unique process involving copying of the viral RNA genome into DNA (a process called reverse transcription) followed by its specific and stable introduction into host cell DNA (integration). The integrated DNA then can be expressed using the normal transcriptional machinery of the cell. Retroviruses commonly are used to introduce genes of interest into cells in culture or into somatic tissue in experimental animals (Miller, 1997). They also have been used for germline modification of fish (Amsterdam et al., 1997), mollusks (Lu et al., 1996) chickens (Thoraval et al., 1995), mice (Soriano et al., 1986), and, more recently, cattle (Chan et al., 1998). To make a retrovirus vector, a DNA construct containing the gene of interest is flanked by sequences necessary for replication as a virus. These sequences include transcriptional promoters in the long terminal repeats (LTR’s), which flank the integrated DNA, or provirus. Signals necessary for packaging of the transcript in virions (virus particles), for reverse transcription, and for integration of the resulting DNA also must be included. Introduction of such a DNA construct into cells that express viral proteins, but that are incapable of making infectious virus (i.e., helper, or packaging, cells), leads to the creation of infectious virions containing an RNA copy of the gene of interest. After infection of cells with such virions, the RNA is copied into DNA and integrated at random sites in the cell genome. Again, selectable markers often are included in the construct to select cells containing the desired virus construct. Transposons Transposons are DNA elements that (in the presence of the appropriate gene products, or transposases) can transfer their information from one site to another in the same cell. A variety of transposons have been found in insects (Handler, 2001) and fish (Ivics et al., 1997), and some are routinely used as vectors for the generation of transgenic insects (Braig and Yan, 2002). No active transposons of these types have been observed in mammals, although the human genome contains thousands of copies of a DNA sequence related to the mariner transposon of Drosophila (Lander et al., 2001), suggesting that there might have been active elements in our recent evolutionary history. Nevertheless, several recent reports suggest that naturally-occurring transposons found in insects, or even bacteria, might provide a useful and

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Animal Biotechnology: Science-Based Concerns efficient means of introducing genes into the germline of animals. Mariner, for example, has been shown to be active in chick zygotes, transferring its DNA from a microinjected plasmid into the germline, albeit at low efficiency (Sherman et al., 1998). A modified version of Sleeping Beauty, a related element from fish, has been developed to give a high yield of germline or somatic transformants in cultured cells (Ivics et al., 1997) and laboratory mice (Personal communication, P. Hackett, University of Minnesota). In practice, it remains to be seen whether—and how efficiently—genes of interest can be transferred in this way. Another transposon system being investigated for this purpose is the T DNA of Agrobacterium tumefaciens, a natural pathogen of plants. This bacterium can fuse with plant cells, leading to transposition of the DNA (along with whatever genes it carries) into the nuclear DNA of the host. This technique is widely used for the generation of transgenic plants (Halford and Shewry, 2000). Remarkably, it recently has been shown that Agrobacterium can fuse with human cells in culture, leading to transfer of T DNA carrying a marker gene (Kunik et al., 2001). None of the transposon-based techniques currently are used for the generation of transgenic livestock, but they might lead to more efficient methods for this purpose. DIRECTED GENETIC MANIPULATION Another goal of transgenic technology is the creation of engineered animals that lack specific genes (knockout), or have these genes replaced by one that has been engineered in a specific way (knockin; see Box 2.1). For example, transplantation of organs or tissues from non-primates (such as pigs) to humans (xenotransplantation) is currently impossible, due in part to a dramatic (“hyperacute”) immune response by human recipients to a carbohydrate on the surface of pig cells (galactose-1,3-galactose); this carbohydrate is not found in old-world primates (Galili, 2001). Inactivation of the enzyme (galactosyl transferase, GT) in donor pigs could alleviate this problem, and pigs with one allele of the gene encoding this enzyme recently have been produced (Lai et al., 2002), giving rise to expectation that completely GT-deficient animals soon will be available. Another important goal is to eliminate from cattle the gene encoding prion-related protein (PrP), the protein associated with scrapie in sheep and bovine spongiform encephalopathy (BSE, or mad cow disease). Removal of this gene from mice has, at most, subtle phenotypic consequences, yet renders them completely resistant to these diseases (Bueler et al., 1992). If the mouse model holds true in cattle, homozygous knockout of bovine PrP could lead to the elimination of BSE.

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Animal Biotechnology: Science-Based Concerns BOX 2.1 Knockout and Knockin Technology In order to study the relationship between proteins and gene function, scientists now can prevent the manufacturing of a protein by a specific gene. By disabling a gene from a test organism, and then producing descendants that contain two copies of the disabled gene, it is possible to observe the descendants’ development in the absence of a particular protein. This practice, referred to as knockout technology, is an attempt to shut down or turn off a particular gene. Thus far, the mouse has been the mammal in which knockout technology has been most generally applied (University of Guelph, 2001). In essence, a “knockout” organism (e.g., the mouse) is created when an embryo cell (an embryonic stem cell—or ESC—which is a cell that has yet to divide into different tissue cells; NRC, 2002b) is genetically engineered, and then inserted into a developing embryo. The embryo then is inserted surgically into the womb of a host (e.g., a female mouse). Once the embryo has matured, a portion of its stem cells will produce egg and sperm with the knocked-out gene. A gene also can be altered in function, in contrast to being deleted. When a gene is altered but not shut down, a “targeted mutation” effect is created. This practice is referred to as knockin technology, whereby a life form has an altered gene “knocked” into it (MGD, 2002). Gene knockout/knockin technology is well established as an experimental tool in mice due to the availability of ES cell lines. The principleis to take advantage of a rather rare event that occurs after introduction of DNA into cells—homologous recombination between identical sequences in the genome and the transfecting DNA (Bronson and Smithies, 1994). In the most common protocol, a selectable marker (such as the neomycin resistance gene) is inserted within a piece of DNA corresponding to a portion of a gene of interest. After transfection of cells by this construct and selection for the marker (by growth in a medium containing the neomycin-related antibiotic G418, in this example), the selected cells are screened to identify the small fraction that has one copy of the gene of interest disrupted by the marker. Progeny animals derived from the cells will be heterozygous for the “knocked-out” or “knocked-in” gene; breeding to obtain homozygous animals is straightforward. Because the process is so inefficient, very large numbers of transfected cells must be screened, making the use of cultured cells essential, since it would be impracticable to screen large numbers of progeny from microinjected eggs. The galactosyl transferase-knockout pigs discussed above were generated from cultured fetal fibroblasts manipulated in this way. Nuclei from these cells then were transferred into oocytes as described in the next section.

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Animal Biotechnology: Science-Based Concerns PROPAGATION BY NUCLEAR TRANSFER In February of 1997, Dolly the sheep was introduced (Wilmut et al., 1997) and the public subsequently was inundated with opinions about the power and potential of creating new animals from somatic cells. Dolly represented the most recent advance in genetic technology—the production of multiple individuals nearly genetically identical to an adult animal. In this technique, somatic cells from an appropriate tissue are grown in culture and their nuclei are injected into enucleated oocytes obtained from another individual of the same or a closely related species. After a further period of culture, the partially developed embryos are transferred into a foster mother. This technology is being developed rapidly for many species of interest (Table 2.1) and promises to become a rapid and efficient means of propagating domestic animals with desired traits, whether those are naturally derived and selected or genetically engineered (Betthauser et al., 2000; Lanza et al., 2001; Westhusin et al., 2001). The process often is referred to as “cloning” (see Chapter 1). The nuclear transfer technique was based on previous studies in frogs conducted during the previous 5 decades (Briggs et al., 1951; Prather et al., 1999), but, until Dolly, it was unclear whether nuclei from highly differentiated somatic cells could be reprogrammed to a pattern of gene expression suitable for directing normal development of a mammalian embryo. TABLE 2.1 State of the art of transgenic technology for selected organisms. Organism Transfection Viral vectors Transposon ES cells Nuclear transfer Mouse 4a 2 1 4a 2 Cow 3 1 0 0 2 Sheep 3 0 0 0 2 Goat 3 0 0 0 2 Pig 3 0 0 0 2 Rabbit 3 0 0 1 0 Chicken 1 2 1 0 0 Altlantic salmon 3 0 0 0 0 Channel catfish 2 0 0 0 0 Tilapia 3 0 0 0 0 Zebrafish 1 0 0 1 1 Crustaceans 1 1 0 0 0 Mollusks 1 1 0 0 0 Drosophila 2 2 2 2 0 Mosquito 1 0 2 0 0 NOTE: 0: No significant progress. 1: Has been accomplished experimentally (proof of concept). 2: Routine experimental use. 3: Commercialization sought. 4: Widespread production. a For experimental uses. See (Dove, 2000)

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Animal Biotechnology: Science-Based Concerns The ability to reprogram the nucleus of donor cells for successful nuclear transfer appeared initially to require the use of methods that facilitate cell cycle synchrony (Stice et al., 1998), since only after the donor cells were induced to become quiescent could offspring be obtained by transfer of the donor nucleus to enucleated oocytes (Wilmut et al., 1998). The necessity for quiescence is not as clear today, since nuclei from actively dividing cells have now also been used successfully for this purpose (Cibelli et al., 1998; Kasinathan et al., 2001; Kuhholzer et al., 2001). The ability to reprogram the donor cells also depends on the species and nuclear transfer procedure. One hypothesis is that differences in timing of embryonic genome activation contributes to differences in cloning efficiency among species (Stice et al., 1998). Currently, only oocytes can be used successfully, as they are the only recipient cells that convert differentiated nuclei into undifferentiated stages resembling pronuclei in freshly fertilized zygotes, a step which is essential for the complete development of the reconstructed embryo (Campbell, 1999; Fulka et al., 2001). How the enucleated oocyte (cytoplast) accomplishes this reprogramming is currently unknown. At present, propagation of animals by nuclear transfer is inefficient, with an average of less than 10 percent of the embryos resulting in live offspring, although the success rate appears to be increasing with experience (Cibelli et al., 2002). Most of the failures occur during development (most often in the first third of the pregnancy for cattle and sheep), and there appears to be an increased rate of perinatal death relative to normally-conceived offspring. In cattle, at least, the developmental and perinatal problems appear to be as much a function of the in vitro culture technology as of the nuclear transfer itself (see Chapter 6). However, even with this existing low efficiency, there are many potential applications for reproducing highly desired genotypes, including rare or endangered species, household pets, elite sires or dams, breeds with desirable production traits but low fertility, sterile animals such as castrates and mules, or transgenic animals that have high value and for which rapid propagation is desirable. Another important application of this technology is in the dissemination of germplasm as embryos and consequent reduction of the associated risk of disease spread (Prather et al., 1999). It also is important to note that there are significant differences between cattle and swine in terms of the utility of this technique. In cattle, the ejaculate from a single bull can be used to breed 400 to 500 females in AI programs. In contrast, the ejaculate from a single boar can be used to breed only 10 to 20 females. Thus embryos obtained from NT might be the method of choice for the dissemination of swine germplasm rather than AI (Prather et al., 1999). A number of variables influence the success rate of nuclear transfer. These include: species, source of the recipient ova, cell type of donor nuclei, treatment of donor cells prior to nuclear transfer, and the techniques employed for nuclear transfer (Westhusin et al., 2001).

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Animal Biotechnology: Science-Based Concerns TECHNICAL ISSUES WITH GERMLINE MODIFICATION Expression of Randomly-Inserted Genes A key consideration in development of transgenic animals is ensuring that the gene product of interest is expressed in the correct tissue and at the appropriate level and time. Specifics of how this goal is accomplished vary considerably from one system to another, but some general principles can be elucidated. First, the natural regulatory elements (promoters) for most genes that direct tissue-specific transcription are complex, large, and poorly understood. For this reason, well-characterized promoters for other genes are appended to DNA encoding the desired gene product. Second, the expression of transgenes, especially those derived by transfection, is strongly under the influence of control elements in the DNA around the integration site (Wolf et al., 2000). These positional effects often lead to silencing of the gene of interest (or other genes near the integration site), or, more rarely, to unregulated expression (Bonifer et al., 1996; Henikoff, 1998). They can be alleviated to some extent by the inclusion of sequences, such as insulators, or locus-control regions (Wolf et al., 2000), but it is impossible to predict whether a given construct will show the desired pattern of expression after integration. While these effects do not directly affect the safety or utility of those animals that are eventually used, they do introduce considerable inefficiency into the system A further problem with obtaining correct expression of an introduced transgene is that introduced genes are subjected to silencing by processes including methylation of C residues at CpG dinucleotides, which frequently are found in chromosomal regions important in the regulation of gene expression. Methylation is a major mechanism for turning off the expression of inappropriate genes in somatic cells. Silencing can occur in somatic tissues, but is particularly acute with introduced genes after passage through the germline, where there is widespread DNA methylation at an early stage of embryogenesis (Jaenisch, 1997). Normal genes in their proper place have signals—in most cases unknown—that reverse the methylation at the appropriate developmental stage. Such signals generally are not present on many commonly used promoter elements, such as retroviral LTR sequences, and expression directed by these elements rarely survives passage through the germline (Pannell and Ellis, 2001). As a natural example, humans carry thousands of endogenous proviruses that have resulted from retroviral infection of the germline of our distant ancestors; yet only a very few are ever expressed at any significant level (Boeke and Stoye, 1997). Proviruses based on commonly-used murine leukemia virus (MLV) vectors introduced by deliberate infection of the germline suffer a similar fate (Jahner and Jaenisch, 1985). Recently, it has been found that vectors based on human immunodeficiency

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Animal Biotechnology: Science-Based Concerns virus (HIV) can be used to efficiently insert genes into the germline of mice, and that genes inserted in this way are not subject to silencing following germline transmission (Lois et al., 2002; Pfeiffer et al., 2002). Such vectors promise to yield more efficient and reliable means of generating transgenic animals of many species. Similar vector systems based on the distantly related feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV) also are being developed (Curran et al., 2000; Berkowitz et al., 2001). Again, methylation-induced shutoff of gene expression is an issue affecting the strategy and efficiency of production of transgenic animals, much less their safety as producers of useful products. Necessity for Selection As the discussion above indicates, germline modification remains a hit-or-miss technology and, with most techniques, only a very small fraction of the progeny obtained has the desired properties of expression, copy number, and lack of genetic damage. Thus, large numbers of animals must be screened for the presence and copy number of the inserted sequence, for its properly regulated expression, for the ability of this expression to survive transmission through the germline and, finally, for the desired phenotypic characteristics and absence of unintended genetic side effects (see below and Chapter 6). Such screening could require several generations of breeding before one can be confident of the absence of recessive genetic damage, and the failure rate of the overall process is very high. As nuclear transfer technology improves, techniques requiring direct introduction of DNA into the animal germline followed by extensive screening of progeny are likely to be replaced by much simpler manipulation and selection of cells in culture, followed by recreation of animals with the desired properties directly from the nuclei of the manipulated cells (Brink et al., 2000). CONCERNS RELATED TO GERMLINE TECHNOLOGY There are a number of safety issues that arise as a consequence of manipulation of the germline. These can be divided into several levels of concern: from the animal (or group of animals); to the human handler, recipient, or user of the animal or its products; to the human population as a whole; and the environment. All of these levels are discussed here in the context of the technology used; many are presented in more detail in the following chapters.

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Animal Biotechnology: Science-Based Concerns As discussed above, introduction of DNA into a cell—whether somatic or germline—is not a well-controlled process and can lead to a number of undesired genetic consequences. Unintended Genetic Side Effects Introduction of DNA into random sites in the germline is a mutagenic event that will affect any gene that happens to be at or near the site of integration. The most obvious effect is the disruption of the integrity of a gene into which the insertion occurs. Since a large fraction of the mammalian genome is noncoding DNA derived from various kinds of silenced transposable elements, not all integration events will lead to gene inactivation; however a fraction of animals selected for the presence of a transgene has been found to carry associated genetic lesions. In mice, for example, it has been estimated that about 5 percent of MLV proviruses integrated into the germline have led to mutations o this sort (Boeke and Stoye, 1997). Direct DNA introduction can lead to numbers of integrated copies at multiple sites, leading to a risk of creating animals with a variety of genetic defects, which should be carefully screened for in the course of subsequent breeding. For example, one of the very first transgenic mouse lines generated, intended to contain an inserted active oncogene, also suffered a lesion that caused a severe recessive developmental limb defect (Woychik et al., 1985). A number of other examples of insertional inactivation by transgenes introduced into mice are known, and this approach has been proposed as a useful technique for mutagenesis (Woychik and Alagramam, 1998). Two additional points should be noted. First, the successfully transfected embryo might have inserted DNA sequences other than those that express the transgene, so the point of damage can be at a location different from the active transgene. Second, damage of this sort is often (but not always) recessive, so that it can only be detected by inbreeding to derive animals homozygous at the site(s) of the inserted DNA, adding to the complexity of the screening process. A related effect is the activation of gene expression in the vicinity of the transgene through the action of the introduced promoter elements. This sort of inappropriate activation of expression is the mechanism of cancer induction in animals infected by a variety of retroviruses, and it has been well-studied as a model for oncogenesis. There are a number of mechanisms by which the expression of genes adjacent to (or even at some distance from) the integration site can be activated, including promoter and enhancer insertion, as well as gene fusion and introduction of elements that stabilize messenger RNA (Rosenberg and Jolicoeur, 1997). Indeed, alteration of expression of genes at genome sites far removed from a transgene has been reported in cell lines, apparently due to altered methylation (Muller et al., 2001). Whether this effect

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Animal Biotechnology: Science-Based Concerns also occurs in transgenic animals is not known. Activation effects are likely to reveal themselves as dominant mutations that can have a variety of phenotypic consequences, from derailing normal development to causing a high rate of cancer later in life. Unexpected Effects of the Modification Even if expressed as desired, the genetic engineering itself can often have unexpected effects on the physiology of the engineered organism. One example of such an unwanted effect relates to the xenotransplantation model described above. The galactosyl transferase deficiency in humans, which leads to hyperacute rejection of organ from pigs, also is thought to offer a level of protection against zoonotic infection by enveloped viruses (Weiss, 1998). This effect occurs because the surface proteins of viruses produced by nonhuman cells are also engineered with the same galactosyl-galactose structure found on host cell proteins, and are therefore subject to the same potent immune response. This response would lead to the rapid elimination of viruses transmitted from animals before infection could occur. Pigs that are engineered by knockout of this gene would, therefore, have the potential to transmit viruses, such as influenza, much more readily to human handlers. A related concern is that human cell-surface proteins introduced into animal species as transgenes could render those animals susceptible to human viruses, increasing their risk of disease and providing alternative hosts for the spread of human disease. For example, the human poliovirus receptor (CD155) renders mice susceptible to poliovirus infection when introduced as a transgene (Racaniello and Ren, 1994). Also, the human complement-response modifying proteins CD46 and CD55, which are being introduced into pigs to protect xenografts from rejection, also serve as receptors for human viruses—measles and Coxsackie, respectively (Weiss, 1998). Their presence in transgenic pigs not only could render these animals susceptible to infection by the human viruses, but also could provide a new evolutionary pathway for adaptation of pig viruses to human cells. Since the receptors for many other viruses have not yet been identified, the potential for this sort of surprise exists whenever a human cell-surface protein is introduced into another animal species. Marker Genes Vector constructs used for creating transgenic organisms usually contain genes other than the desired transgene. These genes are typically drug-resistance markers obtained from bacteria, which also can confer resistance to the same or similar drugs on eukaryotic cells. The neo gene encoding

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Animal Biotechnology: Science-Based Concerns neomycin phosphotransferase, for example, has been used widely for selecting cells in culture infected with retrovirus or other gene constructs (vectors). In most cases, marker genes remain in vectors used for generation of transgenic (especially knockout animals). While many researchers in the field consider them a relatively harmless convenience, there is a potential for them to cause undesired side effects to the host species (such as aiding in the generation of novel antibiotic resistant pathogens) or the ultimate consumer (such as acting as novel allergens). While their potential for real harm is probably very small, it is difficult, maybe impossible, to prove that marker genes are harmless in consumer products. Such genes are usually unnecessary to the product itself and can usually be dispensed with by sound experimental design. Their presence raises concerns about the food and environmental safety of genetically engineered animal products. Undesired Inserts In addition to insertion of the correct element at multiple locations, the preparation of material used to generate the transgene (or knockout) might contain additional sequences unrelated (or only partially related) to the one of interest and intent. Even extensively purified DNA fragments derived from plasmids grown in E. coli might still contain large amounts of contaminating material derived from the host bacterium. Because such fragments can be heterogeneous in size and sequence, they are difficult to detect in DNA preparations by standard methods like gel electrophoresis. A particular problem in this regard arises with retroviral vectors, because host cells (especially of mouse origin) often contain large numbers of endogenous virus and virus-like sequences that can, in some cases, constitute a majority of the genomes present in vector preparations (Chakraborty et al., 1994; Scadden et al., 1990). Inadvertent introduction of such sequences into the germline of transgenic animals not only has the potential for creating unintended genetic damage, but also can contribute by recombination to the generation of novel infectious viruses. A well-known example is the inadvertent generation of replication-competent MLV’s containing multiple such recombinants during the growth of a vector containing a globin gene (Purcell et al., 1996). These viruses were highly pathogenic in rhesus monkeys, causing a fatal lymphoma, similar to the disease induced by MLV in mice.

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Animal Biotechnology: Science-Based Concerns Potential for Mobilization When viral vectors are used for the introduction of genes into the germline of animals, there exists a potential for inadvertent transmission of the gene to other individuals (not necessarily of the same species). This undesirable effect could occur if such an animal were to be infected with a virus sufficiently similar to the vector to package the vector into virions. For example, if a transgenic chicken were created using an avian retrovirus vector, then infection of the transgenic chicken with any related virus (such viruses are quite commonly found in commercial poultry operations) could lead to the production and release of a virus that could transmit the gene to other animals where its presence and expression might be highly undesirable, such as among wild bird populations. Generation of a replicating virus could occur in the absence of exogenous infection, since many species contain endogenous retroviruses in their genomes that could serve as agents of this kind of mobilization. For example, in cats carrying murine leukemia virus-based vector constructs, the introduced genes could be mobilized to other cats (or, at least theoretically, to their human hosts) by the endogenous feline leukemia viruses found in most animals. As discussed above, the use of vectors based on HIV has the potential to improve the efficiency of introduction of new genes into the germline of many animal species. Such germline vectors could, in principle, also be mobilized by HIV or a sufficiently close relative. Viruses closely related to HIV are found only in African primates; however, viruses of the same genus (Lentivirus) are fairly common in cats (feline immunodeficiency virus or FIV), cattle (bovine immunodeficiency virus or BIV), and sheep (visna-maedi virus or VMV; Rosenberg and Jolicouer, 1997). Despite the distant relationship, FIV has been shown to transfer HIV-based vector constructs from one cell to another, raising a serious concern that similar transfer of genes introduced by an HIV (or any lentivirus) vector could be mobilized among animals infected with these common viruses (Berkowitz et al., 2001; Browning et al., 2001). A related concern arises with the use of mariner and related transposons (including sleeping beauty) to introduce germline DNA. Related elements have been found in large numbers (14 thousand copies) in the human genome (Lander et al., 2001) and planaria, nematodes, centipedes, mites, insects (Robertson, 1997), and humans (Robertson and Zumpano, 1997), suggesting the possibility of horizontal gene flow via transposition among highly diverse hosts (Robertson and Lampe, 1995; Hartl et al., 1997; Hamada et al., 1997; Kordis and Gubensek, 1998; 1999; Jordan et al., 1999; Sundararajan et al., 1999). These potentially could be mobilized by the constructs used to transfer mariner-like elements into the germline, and their insertion into genes could give rise to unexpected genetic damage. Horizontal gene transfer also might be mediated by ingestion of DNA (Houck et al., 1991; Yoshiyama et al., 2001).

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Animal Biotechnology: Science-Based Concerns The possible importance of horizontal gene transfer in eukaryotes is controversial (Cummings, 1994; Capy et al., 1994); the most compelling argument for horizontal gene flow in eukaryotes is the ubiquity of transposable elements and endogenous retroviruses in genomic DNA, with no known means for their distribution other than by horizontal gene transfer. It should be noted that any groups using transposable elements for genetic engineering could express the transposase or hopase in the trans configuration and then delete the gene for these enzymes from the transgene constructs, so that once inserted into the host’s chromosome, the element is immobilized. Were this a requirement applied to transposable element vector systems for genetic engineering of animals, the hazards at issue could be minimized or eliminated, so long as active elements capable of mobilizing the introduced sequences were not already present in the host animal. Potential for Creation of New Pathogens In addition to their potential for mobilization by interaction with related viruses, transgene sequences also can contribute elements to infecting agents that might modify their ability to cause disease. The donation of drug-resistance genes to bacteria as a consequence of their widespread presence in transgenic livestock is one theoretical example, although the resistance gene would have to be one not found in the environment for the risk of such an event to be significantly enhanced over the natural background. Another example is the possible generation of new retroviruses following recombination between endogenous or exogenous viruses and ones used as vectors for transgenes. This recombination event could result in the provision of new genes or regulatory elements (such as LTR’s capable of more efficient expression) that could adversely modify the pathogenic potential of the infecting virus. A recent natural example is the generation, through recombination between an infectious avian retrovirus and a distantly related endogenous element, of a highly virulent virus, called HPRS-103, or subgroup J avian leukemia virus (ALV) (Payne et al., 1992; Benson et al., 1998). This virus apparently arose as the result of a single, very rare event, but subsequently has been spread worldwide and has become a source of considerable economic loss to poultry breeders (Venugopal, 1999). ISSUES RELATED TO SOMATIC CELL NUCLEAR TRANSFER TECHNOLOGY The generation of animals using nuclear transfer from somatic cells has received a great deal of attention recently, and it is clear that this technology is

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Animal Biotechnology: Science-Based Concerns fast becoming a practical way to rapidly propagate animals with valuable properties (Polejaeva et al., 2000). The DNA genomes of somatic cell nuclei used for this procedure differ in two important ways from those of germline cells. First, they have shortened telomeres at the ends of the chromosomes, a consequence of multiple rounds of cell division in the absence of telomerase, the enzyme responsible for maintenance of telomere length. Since loss of telomere length is the principal mechanism limiting the lifespan of cells in culture (Urquidi et al., 2000), the lack of appropriate-length telomeres might be expected to reduce the lifespan of the newly generated offspring or their progeny, but, surprisingly, telomere length (and lifespan of cultured cells) are restored to normal values following generation of cattle by somatic cell nuclear transfer (Betts et al., 2001), even when senescent cells are used to donate nuclei (Lanza et al., 2000). Second, the methylation state of the DNA of somatic cells is quite different from that of germline cells (Rideout et al., 2001). Since methylation (at CG sequences) plays a major role in the overall regulation of gene expression, it might be expected that inappropriate methylation states might lead to gross developmental abnormalities in embryos produced by somatic cell nuclear transfer. Indeed, it is possible that the inability of the embryo to properly reprogram methylation and expression is a major cause of the developmental abnormalities often seen in the generation of NT-produced embryos (Rideout et al., 2001). However, the apparently rapid increase in success rate of this procedure with experience, combined with the fact that animals who survive to adulthood are apparently normal (Betthauser et al., 2000), implies that correct methylation can be restored in NT embryos under the proper conditions. “Correct conditions” might involve having the transferred nucleus in the proper stage of the cell cycle (Gibbons et al., 2002), but this point is controversial. Furthermore, in a direct study (Kang et al., 2001), it was found that correct methylation and expression levels of several key genes were restored in pig embryos derived from adult cell nuclei. Thus, although nuclear reprogramming is a significant practical issue in the efficient application of this technology, it does not appear to present as insurmountable a barrier as once thought. Apparently the developmental process has a much more robust error-correction system than believed possible a few years ago. The committee carefully considered the possible concerns that might be raised by use of somatic cell nuclear transfer technology. A few issues regarding animal welfare could be identified (see Chapter 6), including the possibility of inappropriate gene expression during development due to altered methylation patterns, or other developmental problems, such as oversized fetuses (Young et al., 1998), as well as concerns that the widespread application of this technology might reduce genetic diversity of animal populations. However, the effects of cloning are more difficult to anticipate because competing processes are at issue. On the one hand, cloning by its nature produces identical copies of a particular individual, reducing genetic

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Animal Biotechnology: Science-Based Concerns variability relative to what would have been transmitted via conventional breeding. On the other hand, cloning makes it possible to save and utilize genetic variability that would not otherwise be available, for example, the genetic resources from a steer proven to be high performing. The tradeoff between the competing processes is hard to quantify in the absence of simulation modeling with validation from field observations. Whatever the mechanism causing it, loss of genetic diversity could limit the potential for future genetic improvement of breeds by selective breeding or biotechnologic approaches. Further, disease could spread through susceptible populations more rapidly than through more genetically diverse populations. This latter concern is well documented and several studies illustrate the susceptibility of species with low genetic diversity to infectious disease. Diversity of animal populations, particularly at major histocompatibility (MHC) loci, is a major factor preventing spread of disease, particularly viral disease (Xu et al., 1993; Schook et al., 1996; Kaufman and Lamont, 1996; Lewin et al., 1999). Different MHC types recognize different viral or bacterial epitopes encoded by pathogens for presentation to the immune system. In genetically diverse populations, pathogens can evade the immune response only if they adapt to each individual MHC type following transmission from one individual to another. The requirement for this evolutionary process provides a population of animals with significant protection against the spread of infection. Pathogens can more easily evade host immune response in genetically uniform populations (Yuhki and O’Brien, 1990). The consequences of the failure of immunorecognition is illustrated by the deadly epidemics of diseases—such as measles—spread by initial contact between Europeans and isolated New World populations that lacked adequate MHC diversity. Not only could enhanced susceptibility create significant risk for the spread of “new” infectious diseases in “monocultures” of cloned or highly inbred animal populations; it also could create new reservoirs for spread of zoonotic infections—like new strains of influenza—to humans. The seriousness of these concerns, particularly relative to current practice (see Chapter 1) obviously must vary considerably from one type of animal to another, and might be alleviated with further technologic advances. CONCLUSIONS The technology for modifying the germline of domestic animals is being advanced at a very rapid pace. Indeed, some major advances were reported during the brief period in which this report was being prepared. Although many of the detailed issues discussed in this chapter will no doubt soon become outdated to be replaced by new ones not yet considered, some general issues will remain. In particular, there will (probably) always be concerns regarding

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Animal Biotechnology: Science-Based Concerns the use of unnecessary genes in constructs used for generation of engineered animals, the use of vectors with the potential to be mobilized or to otherwise contribute sequences to related environmental organisms, and the effects of the technology on the welfare of the engineered animals themselves.