Cloning and Gene Transfer in Hannnals. N. First, J. Lohse, J. Robl, E. Critser and J. Rutledge. University of Wisconsin.

Cloning and Gene Transfer in Hannnals N. First, J. Lohse, J. Robl, E. Critser and J. Rutledge University of Wisconsin Introduction The impact of artificial insemination on increased milk production by dairy cows is well known and often cited as the technological achievement having the greatest impact on livestock production. Milk production per cow more than doubled from 1951 to 1981 largely due to use of superior bulls through A.I. This improvement has been brought about through intensive and accurate selection of superior bulls. Even greater increases in milk or meat production could be realized if we could: i) exercise similar selection intensity and accuracy in choosing the female parents or 2) phenotypically identify and multiply (clone) embryos resulting from favorable combinations of sperm and egg or 3) introduce genes responsible for enhanced milk or meat production into existing strains of cattle. Research at the University of Wisconsin supported by the American Breeders Service and its parent company, W. R. Grace, is aimed at developing methods for multiplying or cloning embryos and methods for transferring cloned genes into bovine embryos. Clonin_ of Embryos We have studied cloning by several methods and at different stages of development. The two methods most likely to be useful adjuncts to our present embryo transfer industry are: I) the production of twins by microsurgically dividing late stage multicellular embryos of the morula or blastocyst stage 136

and 2) nuclear transfer. The first is now in commercial use for production of twins in cattle and the latter offers possibilities for repeated cloning of embryos to produce large numbers of a single genotype. Developmental stages of a bovine egg from maturation in the follicle through fertilization and early embryonic development in the oviduct and uterus are shown in Figure I. The reader is encouraged to consult this figure as often as necessary to understand the stages under discussion. Splitting Embryos to Produce Twins This method of cloning embryos was developed at Cambridge, England (Willadsen et al., 1981) and has been successfully applied to produce cloned calves by Colorado State University Embryo Transfer Lab (Williams et al., 1982, 1984). Willadsen et al. (1981) examined the potential for splitting embryos into halves or quarters (Table i). The survival was high for half embryos but lower for quarter embryos. Our studies (O'Brien et al., 1983) and studies of Tsunoda and McLaren (1983) suggest that the signal for initiation of blastocyst formation is inherent in each blastomere from the first cell division. Separation of blastomeres to produce 2 or 4 blastocysts from l- embryo reduces the number of cells by one half and one fourth in the resulting blastocyst. Separation into one-quarter embryos often leaves insufficient cells in the inner cell mass to reliably develop a fetus from the embryo. This insufficiency is likely the case for quartered morula or blastocysts as well. In his original experiment Willadsen placed the halved embryos in zonae pellucidae, coated them with agar, incubated them in a sheep for i day and then transferred them to a cow. Most other studies (Williams et al., 1982, 1984; Ozil et al., 1981; Lambeth et al., 1983; Brem et al., 1984; Northey, 137

Critser and First, unpublished) have achieved good success by placing halved embryos in a surrogate zona pellucida and transferring immediately to the uterus of a recipient cow thereby avoiding the agar coating and incubation in a sheep. A question relevant to the application of splitting as a means for multiplying embryos concerns whether the clone must be placed in a surrogate zona pellucida. We have evaluated the ability of bovine morula to develop in vitro i) with a zona pellucida (control) 2) without a zona pellucida and 3) after splitting. Splitting embryos at the morula stage resulted in development to the blastocyst stage in culture. Overall 30 of 34 control morula, 29 of 34 morula without zona pellucida and 51 half embryos from 34 embryos after splitting were cultured to normal appearing blastocysts. Thus, the zona pellucida does not appear essential for development of late stage bovine embryos in culture. Data from 17 transfers performed in Dr. Bavister's laboratory at the University of Wisconsin suggest the zona is not absolutely essential for survival of bovine embryos transferred to recipients. The pregnancy rate was 38% with zona present and 33% without zona pellucida (Hoppe and Bavister, 1983). Further research is needed to determine if a surrogate zona pellucida is essential or desirable. Presently the most frequently used method of splitting embryos involves microsurgical cutting of the morula or blastocyst with a fragment of razor blade controlled by a micromanipulator and at a magnification of approximately 100x (Figure 2). When cutting a blastocyst the cut must bisect the inner cell mass (fetal portion) of the embryo. One half is left in the original zona pellucida. The other half is removed by aspiration and placed in a surrogate zona. The empty surrogate zona pellucida is obtained by removing the contents of low genetic value unfertilized or fertilized eggs. The half embryos are 138

then immediately transferred to the uteri of appropriately staged recipient COWS, Studies at Colorado suggest that survival of split embryos iafter transfer is highest when the embryo is split at the early blastocyst stage. Pregnancy rates achieved by splitting embryos at early morula, late morula, early blastocyst and late blastocyst stages were 16, 48, 60 and 48%, respectively (Williams et al., 1984). The compactness of cells and quality of the embryo are also critical for achievement of- pregnancy after transfer of half embryos. Pregnancy rates from embryos visually rated excellent, good or poor were 52, 29 and 6%, respectively (Brem et al., 1984). The signal from half embryos to the mother for establishment of pregnancy may sometimes be insufficient. The probability of pregnancy is approximately doubled by the transfer of 2 half embryos to one recipient (Ozil et al., 1982). We have been splitting embryos for American Breeders Service for the purpose of producing twin bulls from matings of highly selected high performance parents. Twin bulls reduce the cost of progeny testing each bull, provide a doubled or reserve supply-_f semen and allow evaluation of existing progeny test programs. Thus far we have transferred singly more than 80 half embryos of which 35% resulted in maintained pregnancies. When embryos were split and transferred immediately the pregnancy rate was 60 to 75%. We view this method as having limited potential for greatly increasing the supply of embryos and inappropriate for producing clonal lines or repeated cloning. We do consider this a useful method for producing twins and by doing so increasing the number of pregnancies per superovulation and 1ush of a valuable cow. 139

Cloning by Nuclear Transfer Somatic Nuclei. The most useful method for cloning high performance individuals would be the substitution of nuclei from somatic cells of high performance individuals for nuclei of mass produced low cost 1-cell embryos. At present this is not possible in mammals. Many genes of differentiated cells are highly methylated and inactive until selectively activated and expressed. An exception to this are teratocarcinoma cells (Silver et al., 1983) and in a highly controversial experiment lllmensee (1982) claims to have produced live mice from nuclear transfer of teratocarcinoma cells. -- Other potential sources of cells for nuclear transfer or substitution include spermatogonia and somatic cells undergoing differentiation in the adult such as primordial blood cells. Newer methods for controlling demethylation and redifferentiation of differentiated cells are being discovered (Jones et al., 1983; DiBerardino, 1980) and may some day allow use of somatic or premeiotic germ cells. Early Embryonic Nuclei. For the present the most promising donor cells are pronuclei of i cell embryos (McGrath and Solter, 1983) or cells of the inner cell mass of mouse blastocysts (lllmensee and Hoppe, 1981; Hoppe and lllmensee, 1982). It is our hope and expectation to develop a system for producing multiple clones by nuclear transfer which will allow repeated cloning of a clonal line after its performance in milk or meat production is characterized. We plan eventually to use the embryos derived from in vitro fertilization as a low cost source of recipient ova for transferred nuclei. Ultimate perfection of nuclear transfer for practical commercial use depends in part on the use of in vitro fertilization of in vitro matured bovine oocytes as a cheap supply of surrogate 1-cell recipient embryos and on 140

the development of methods for culturing bovine embryos from the 1-cell to blastocyst stage in vitr.. o (Figure i). Specifically the plan is to disaggregate the cells of the inner cell mass of a bovine blastocyst which contains 30 or more donor cells, remove the nuclei from these cells and substitute them for the pronuclei of recipient 1-cell fertilized eggs (Figure 3). Recipient eggs ultimately would be derived from in vitro fertilization. We then plan to develop the nuclear substituted eggs in culture to the blastocyst stage. We would then reclone all which became blastocysts. If i0 of the 30 original cells develop to blastocysts then recloning will produce a total of i00 genetically identical blastocysts. A sample, perhaps 20, cloned blastocysts will be transferred to recipient cows. We could expect around i0 live calves born which could be raised to maturity and production tested to characterize the genetic value of the clonal line. Meanwhile the remaining clones, perhaps 80 or more, would be maintained as frozen stored blastocysts. With present technology we expect 30 or more to survive freezing. After sorting clonal lines for sex and genetic value, blastocysts from selected lines would be disaggregated, recloned, developed to blastocysts and the process-repeated for as many times as the demand for embryos of that clonal line required. We visualize as many as 30,000 or more J clones might be derived from one original embryo. We would plan to use the trophoblast cells remaining after removal of the donor cells of the inner cell mass as a source of cells to determine karyotypically the sex of the embryo. This is important because we do not want to clone bulls but rather cows for the purpose of milk production. The model on which this research is based is from the mouse experiments I of lllmensee and Hoppe (1981). They reported the production of a small number of live cloned mice by substitution of blastocyst inner cell mass cells 141

of one strain of mouse for the pronuclei of 1-cell fertilized eggs of another strain. The steps in Figure 3 are based on their original model. While not repeating the exact experiment, in principal many aspects of these experiments were recently repeated in mice by McGrath and Solter (1983a) who transferred pronuclei from one strain of mice to another. They achieved a phenomenal 91% success rate in producing blastocysts of which 14% of the original number of embryos became live mice. In a later experiment their attempts to transfer nuclei of the 2-, 4- and 8-cell stage and inner cell mass of mouse blastocysts to pronuclei of recipient eggs by cell fusion resulted in -- successful nuclear transfer but the developmental fate of the transferred embryos is still unknown (McGrath and Solter, 1983b). Figure 3. There are five key steps with this method of cloning as indicated in Step i - Ability to microsur_ically separate the inner cell mass from the trophoblast cells of the bovine blastocysts_ and to disa_re_ate the cells and remove the nuclei, lllmensee and Hoppe (1981) did this successfully in mice and we have also accomplished this for murine and bovine blastocysts. Our efficiency with this step is approximately 80%. Step 2 - A supply of 1-cell recently fertilized embryos is required as recipient cells for nuclear substitution, lllmensee and Hoppe (1981) obtained large numbers of recipient cells by synchronizing females, timing ovulation, and ultimately killing mice for rapid and efficient egg recovery. For commercial use killing cattle is costly and inefficient. We have been developing methods for producing the recipient 1-cell embryos by maturing and fertilizing oocytes recovered from ovaries of cows slaughtered daily for meat at a local abattoir. At present these methods provide a large supply of ova and result in high levels of oocyte maturation (70-95%) and fertilization and 142

pronuclear development (60-80%). However, the frequency of fertilized ova becoming normal 2-cell embryos (15%) is reduced when immature oocytes are used (Table 2). Oocytes matured in vivo are normal and presently more useful for recipients in nuclear transfer (Brackett et al., 1982; Ball elt al., 1983a; Ball et al., 1983b; Leibfried et al., unpublished). Studies are under way to understand why the potential for development of immature oocytes is low. Step 3 - Ability to visualize pronuclei for removal. Eggs of domestic animals contain a dense granular cytoplasm filled with lipids which prevent < visualization of pronuclei by common forms of microscopy. In contrast, the eggs of mice, rats, hamsters and primates are clear and pronuc!ei are easily i visualized. Of the domestic species the rank in ease of visualization of pronuclei is caprine, ovine, bovine and porcine. To resolve th_s problem for I the bovine we have developed a fluorescence method which makes the pronuclei visible without seriously damaging the cells (Critser et al., 1983). A similar method was also published by Minhas et al. (1984). Unfortunately the i UV light required for use of this method damages nuclei. Alternate methods of clearing the cytoplasm and reducing exposure to UV light are being tested. Step 4 - Ability to remove pronuclei and to introduce the donor nucleus. I Based on the mouse studies of lllmensee and Hoppe (1981) an d McGrath and Solter (1983a,b) it is anticipated that the introduced nucleus will direct the i cell and subsequent embryo, fetus and offspring to be genetically and phenotypically like the donor. We have repeated the pronuclear removal and cell fusion procedures of McGrath and Solter (1983a,b) in mice and have found nuclear fusion to be highly efficient for introduction of nuclei and likely much more efficient than the microinjection methods used by lllmensee and Hoppe (1981). 143

144 Step 5 - Culture of 1-cell nuclear substituted embryos to the blastocyst stage. This step is important because it is the blastocyst stage that is easily transferred nonsurgically to recipient cows. This is also the developmental stage at which the embryos can best survive freezing and where they can be sexed. It is the essential stage for supplying donor nuclei cells for repeated cloning. Unfortunately, this step has never been developed for bovine embryos, although it can be accomplished in mice. Bovine embryos have been cultured from the 8-cell stage to blastocysts with reasonable success and from 1-cell to 2- or 4-cell embryos (Wright and Bondioli, 1981; Critser et al., unpublished). We are currently attempting to identify oviducts of laboratory species which could serve as incubators for the development of bovine embryos from 1-cell to blastocyst stages. Overall, cloning by nuclear transfer could provide at least three benefits to dairy and livestock production. I) Cloning each embryo and performance testing a sample of the clonal line while the rest of the line is stored frozen would allow a phenotypic identification of sperm-egg combinations with high milk or meat production potential. 2) Once identified it would provide a means for producing a large number of heifers of nearly identical characteristics and high milk or meat production. It is emphasized that we are attempting to move to a system of genetic improvement which can circumvent the Mendelian mechanism when necessary. Heritability estimates based on twin studies indicate that the total genotypic differences between animals may be many fold greater than the additive genetic differences between animals. 3) Frozen storage of a few embryos of a clonal line with the ability to multiply or "clone" the thawed embryos means only a small amount of storage space is required and the product (embryos) might be multiplied almost indefinitely to meet the market demand.

The critical question regarding this model is whether some of the cells r of the inner cell mass of the blastocyst can be dedifferentiated and redifferentiate in the recipient 1-cell embryo. The experiments of lllmensee and Hoppe (1981) and Hoppe and lllmensee (1982) suggest this can occur in mice but with low frequency. In invertebrates Briggs and King (1952) demonstrated that nuclei from developing frog embryos could be transplanted into 1-cell frog embryos with the resulting offspring containing the genotype of the donor nuclei. This pioneering experiment has been repeated with numerous modifications in fish and amphibia (McKinnell 1978, 1981; DiBerardino, 1980). In general, offspring have resulted from transferred nuclei originating from blastula or earlier stages. Transfer of nuclei from later stages has not resulted in offspring. The frequency of embryonic donor cells resulting in offspring is progressively reduced when cells from developmental stages proceeding from early stage donor cells to blastocysts are compared in amphibia. There are two possible reasons for this. First, it is known that the DNA of cells becomes progressively more inactivated by methylation as embryos progress to the blastocyst stage. Methods now available for promoting differentiation-by preventing methylation of these cells with chemotherapeutic agents such as 5-azacytidine (Jones et al., 1983) may allow increased efficiency of nuclear transfer. Secondly, the stage of the cell cycle of the donor nucleus may affect its suitability for redifferentiation. This too could be controlled. Additionally, these methods may be assisted by using the egg itself to cause dedifferentiation. It has been shown by DiBerardino (1980) that placement of a cell nucleus from a differentiated stage into oocyte cytoplasm enlarges the array of developmental expressions of which that nucleus is capable. If nuclear transfer is for the present not feasible using donor cells 145

from the blastocyst stage it may be that nuclear transfer using earlier stage embryos where the DNA is less methylated will be feasible and more efficient. The experiments of McGrath and Solter (1983a) and our own recent studies (Robl, unpublished) show this feasibility for pronuclear exchange. Efficiency of different stages are now being tested in our laboratory and at least one other. Unfortunately at the present time stages earlier than 16 cell are not usually or reliably recovered nonsurgically from donor cows. Of additional concern is the potential effect of recipient cytoplasm on genetic or phenotypic expression in the offspring. While cases of cytoplasmic inheritance are known (Hutchinson et al., 1974) phenotypic effects manifest through direct maternal control of development of mitochondria may also affect productivity traits (Wagasugi, 1974). The availability of offspring produced by nuclear transfer could allow research aimed at determination of the genetic and phenotypic contributions of cytoplasm. For the moment these and other potentially useful experiments utilizing multiple cloned animals must await confirmation of the original experiment of lllmensee and Hoppe (1981) and modifications which result in more efficient production of the multiple cloned offspring. Gene Transfer Background Gene cloning and gene transfer technology provide possibilities for three useful applications in livestock and poultry production. In the first case bacteria, usually _ coli, are transformed with a gene coding for a product or protein such as bovine growth hormone. These bacteria then multiply and with appropriate large scale culture produce pharmaceutical quantities of a useful product. A well-known example of this was accomplished by Itakura et al.

(1977) and the product produced from the cloned gene was somatostatin. Since then this technology has been used to produce a large array of products such as growth hormone, interferon, insulin, etc. for pharmaceutical and potentially agricultural use. Secondly, theoretically genes may be transferred into somatic cells of an individual animal for the purpose of correcting a genetic deficiency or altering characteristics of that individual. This is without expectation that the gene will be expressed in future generations unless perhaps it is incorporated into gametogenic cells. This introduction could be accomplished by removal of cells from the individual, introduction of the gene during cell culture in vitro and replacement of the removed cells into that individual. This gene therapy approach is being extensively studied for human use with powerful gene transfer agents such as replication defective retrovirus vectors showing promise for delivery of genes in the near future (Kolata, 1984; Fox, 1984). Similarly, genes such as growth hormone or genes for disease resistance might be introduced into liver or blood cells removed from and returned to domestic animals with the hope of increasing milk production, growth o_ disease resistance. Thirdly and of more importance to animal agriculture is the development of methods for introduction of genes directly into the replicating genome of embryos. This is done in anticipation that some of the offspring will contain a stably integrated new gene in their genome which is transferred to successive future generations. This indeed has been accomplished in mice (Palmiter et al., 1983). Historically, scientists realized early on the agricultural potential for production of animals engineered for increased production or for disease resistance by the insertion of one or more specific genes into their genome. 147

However, accomplishment of direct gene insertion was delayed until the year 1981. There were important experiments leading to this accomplishment. For example, in 1973 Graham and van der Eb (1973) developed a reliable system for DNA transfer into cultured animal, plant or microbial cells by coprecipitation of DNA with calcium phosphate. Pellicer et al. (1980) used this technique and thymidine kinase deficient teratocarcinoma cells to show that the cloned herpes virus thymidine kinase gene would transform these cells to produce thymidine kinase and thereby correct the deficiency. Another approach involved infecting early cleavage stage mouse embryos_with a retrovirus and demonstrating incorporation and expression in the mouse. However, viral mediated genes were not always incorporated into the genome of the mouse and when incorporated had errors of expression. The high efficiency with which retroviruses infect cleaving embryos suggest that portions of retroviruses might be useful for enhancing the transfer of other gene sequences (Gordon, 1983; Jaenisch as cited by Fox, 1984). A useful method for gene transfer was developed by Gordon et al. (1980) and subsequently used by several groups in 1981. They showed that a recombinant plasmid containing the herpes TK (thymidine kinase) gene a Hind III restriction fragment of SV (simian virus) 40 DNA in the commonly used plasmid vector pbr322 (cloned in E. coli) could be introduced into mouse embryos and found integrated in the chromosomal DNA of newborn mice by the Southern blot DNA hybridization technique. However, this did not prove that DNA would remain stably integrated in future generations or that it was expressed as new protein or products in the mouse. Their procedure involved microinjection of the DNA into pronuclei of 1-cell embryos followed by embryo transfer. The landmark year for gene transfer in intact animals was 1981. During 148

this year_ Gordon and Ruddle (1981, 1982) showed that the previously mentioned I gene was integrated into the mouse genome and called the resulting offspring transgenic mice. During this same year T. Wagner et al. (1981), E. Wagner et al. (1981), Constantini and Lacy (1981) and Brinster et al? (1981) all demonstrated incorporation of functional genes into the mouse genome. From I these and later experiments there are now mouse lines that contain stably integrated herpes simplex virus thymidine kinase genes (Gordon and Ruddle, 1981, 1982; E. Wagner et al. 1981). This same gene linked to the MTI (metallothionein I) gene promoter (Brinster et al., 1981; Palmiter et al., 1982a); rabbit _-globin genes (T. Wagner et al., 1981; Constantini and Lacy, 1981; Lacy et al., 1983); the human _-globin gene (E. Wagner, 1981) the gene i for rat growth hormone linked to the MTI promoter (Palmiter et al., 1982b) the gene for human growth hormone linked to MTI promoter (Palmiter et al., 1983) and a chicken transferrin gene (McKnight et al., 1983). Exogenous genes :I i resulting in transgenic offspring have also been introduced into frogs - a rabbit _-globin gene was introduced (Rusconi and Schaffner, 1981) and into Drosophila - a Xanthine dehydrogenase gene (Rubin and Spradling, 1982).._ Site and Mechanisms of Gene Incorporation. In all of these experiments the exogenous gene was microinjected into a pronucleus of the 1-cell recently fertilized egg. In most cases this was the male pronucleus. There is some opinion that incorporation and stable _ integration may result only from injection of the male pronucleus. The rationale for this view and the sensitivity of chromatin of the decondensed sperm to incorporation of new DNA has been studied and reviewed by Wagner et al. (1983). This view may not be entirely correct. A number of investigators have produced stably transformed clones from eukaryotic cells in culture. The calcium phosphate technique of Graham and van der Eb (1973), for instance, has 149

been used by Robins et al. (1982) to cause mouse flbroblasts to produce human growth hormone, and Capecchi (1980), Anderson e_.tt a l. (1980) and others have transformed cells in culture by microinjecting genetic material. In addition, a recent experiment in our laboratory suggests that expression of the thymidine kinase - metallothionein gene in embryos occurs from injection of blastomeres at the 2-cell stage as well as from injection of the gene directly into pronuclei (Lohse, Robl and First, unpublished). The chromosomal location, level of incorporation, effects on other genes and tissue sites of expression have been of interest to scientists studying introduction and expression of foreign genes. Evidence for chromosomal integration of introduced genes comes from in situ hybridization studies of Constantini and Lacy (1981) and Lacy et al. (1983) showing integration of tandem multiple copies of rabbit B-globin genes in mouse metaphase chromosomes. The copy number of new integrated genes varies in transgenic mice from one to two copies per genome to as many as 20 copies (Constantini and Lacy, 1981; E. Wagner et al., 1981; Palmiter et al., 1982b, 1983). Depending on the promoter and gene construct, the efficiency of gene expression achieved in the previously mentioned studies ranged from 3 to 40%. In general the injected genes locate randomly on chromosomes at different sites in different mice and usually but not exclusively at the end of chromosomes (Constantini and Lacy, 1981). Lacy et al. (1983) found that when the rabbit B-globin gene was introduced into five different strains of mice it integrated into one or two different chromosomal loci in each strain. Each locus contained 3 to 40 copies of the foreign DNA arranged in a tandem array and inherited as a simple Mendelian marker. While foreign genes are stably integrated into the genome they are often not expressed by tissues expected to express a given gene, they are frequently expressed by unlikely tissues and 150

often unregulated. Neither globin mrna nor polypeptides encoded by the rabbit 8-globin gene were detected in erythroid cells, the usual t location for _-globin. One line of mice expressed B-globin in skeletal muscle and another in the testes. These aberrant sites of expression were heritable in future generations and may result from gene integration at abnormal positions (Lacy et al., 1983) or from characteristics of the promoter system or gene used. il Mice integrating human growth hormone linked to the MTI promoter expressed the growth hormone gene in all of eight tissues examined in amounts roughly proportional to the endogenous metallothionein produced in each tissue (Palmiter et al., 1983). Insertion of a foreign gene may have damaging effects on the native genome. Insertional mutagenesis has been recognized as a spontaneous phenomenon in maize (McClintock, 1956), yeast (Roeder and Fink, 1980) and Drosophila (Kidwell et al., 1977). In mice insertional mutagenesis in the germ line has been caused by murine leukemia retroviruses. Mutation in one case resulted from spontaneous insertion of proviral DNA sequences in the dilute coat color locus (Jenkins et al., 1981) and in the other there was an experimental insertion of the clicollagen gene after injection of virus into a postimplantation embryo (Jaenisch, 1980 and Schnieke e ta l.,1983). E. Wagner et al. (1983) recently described insertional mutagenesis due to integration into a mouse germ line of the human growth hormone gene in a pbr322 plasmid. Two independent recessive prenatal lethal mutations were found in six mice. These mutants may have been caused by the gene per se or by the vector system ;I used to cause its integration. The most exciting, the most efficient, most successful and the most beneficial experiments of this group to animal agriculture are those of Palmiter e_t a l.(1982b, 1983) wherein the structural portion of a rat growth 151

hormone gene was linked to the powerful promoter for mouse metallothionein I in the first case and the structural portion of a human growth hormone gene similarly linked to the promotor from the metallothionein I gene in the second case. These and the previous experiments with thymidine kinase structural gene linked to metallothionein I promoter showed use of the metallothionein I promoter to result in the highest levels of gene incorporation, expression and integration thus far achieved. Additionally, this promoter allows regulation of the promoter gene complex because the expression of metallothionein is increased by heavy metals such as cadmium or zinc. When animals are reared on - diets deficient in heavy metals the linked gene is expressed at a low level and can be turned on by addition of heavy metal to the diet. Use of this promoter has allowed gene incorporation and expression to be as high as 20 to 40%. These authors have studied extensively the expression of both growth hormone genes. The human growth hormone gene was expressed in all of eight tissues examined. However, the ratio of human growth hormone messenger RNA to exogenous metallothionein I messenger RNA varied among different tissues in different animals suggesting that expression of the foreign gene was influenced by site of integration and tissue environment. Seventy percent of the mice that stably incorporated the fusion gene showed high concentrations of human growth hormone in their serum and grew significantly larger than control mice. Synthesis of human growth hormone was induced further by cadmium or zinc which increases transcription of the metallothionein gene. Transgenic mice that expressed human growth hormone also showed increased concentrations of insulin-like growth factor in their serum and histology of the pituitaries suggested that pituitary function and likely production of its own growth hormone had been suppressed by the exogenous growth hormone. Growth was not correlated with the number of copies of a gene or the amount of 152

growth hormone found in different mice. It was suggested that the introduced gene enhanced growth by perturbing the mechanisms regulating growth; that is the somatostatin, growth hormone releasing factor, growth hormone-insulin like growth factor I, regulatory system. An explanation given for the poor correlation between gene dosage and the level of expression was that one or a few genes in the tandem arrays were actually expressed and if these favorite genes were at the ends of the array they would be subject to neighboring chromatin influences. It was suggested that a more uniform Copy number and perhaps expression might be achieved by the use of longer DNA fragments of the MTI promoter sequence. Both the human growth hormone and the rat growth hormone genes were shown to be transmitted to future generations of mice. Since growth appears to be regulated similarly in mice, domestic animals and birds animal geneticists are eagerly looking forward to the insertion of one of these growth hormone metallothionein fusion genes into milk or meat producing animals or into poultry. Potential Uses for Gene Transfer in Livestock and Poultry Production In meat producing animals the economically important traits in order of importance are considered to be reproduction, growth efficiency and carcass value; in dairy cattle milk production and secondly reproductive efficiency; and in poultry egg production, broiler growth efficiency and carcass value are also the traits of importance. In general these traits are each influenced by several genes. Intuitively this suggests that little would be gained by insertion of single genes into livestock or poultry. This reasoning may not be entirely valid. There is evidence that manipulation of single influential genes can produce major changes in productivity responses in these multiple gene traits. For example, insertion of rat growth hormone (Palmiter et al., 1982b) or human growth hormone (Palmiter e_tta_._l.,1983) into the genome of mice 153 i'

perturbed the growth regulatory mechanisms sufficiently to cause as much as a two-fold increase in growth. In dairy cattle daily injection of growth hormone has resulted in an increase of approximately 15% in milk production (Bines et al., 1980; Peel et al., 1981a,b; Gorewit et al., 1982). The giant mouse gene identified by Bradford (1982) might also produce enhanced growth in livestock. Specific genes affecting components of reproduction have been identified. For example, the Booroola gene when present causes the Australian merino to increase ovulation rate and to produce multiple births rather than single lambs (Davis et al., 1982). In mice Spearow (1984) has also identified genes influencing ovulation rate which increase ovulation up to 6 fold. If the Booroola gene were inserted into cattle or the Spearow gene into swine it might be possible to reliably produce twins in cattle or increase litter size in swine. If this indeed occurs there will be a great need to identify other genes affecting reproduction or growth. For example, genes affecting uterine carrying capacity in swine or other regulatory points in growth control need to be identified. This task of gene identification will place great responsibility on quantitative geneticists. It may also be possible to impart resistance to specific diseases of animals or birds through transfer of genes. Resistance to some diseases of humans and mice (Bach, 1982) or Mareks disease in poultry (Payne, 1973) are known to be controlled by single gene loci. At the present time recombinant DNA technology is being used to produce vaccines for use in animal and poultry production (Trevis and Bertelsen, 1982). The tools of biotechnology show promise for the production of rumen microorganisms which might effectively digest the presently unused fibrous lignin and cellulose portions of plants (Smith and Hespell, 1983). Microbial enzymes with the capacity to digest lignin and cellulose are available in nature (Streeter et 154

a_._l.,1982). The problem is how to get these organisms to survive in the rumen. An alternate and perhaps more fruitful approach would be to clone the microbial genes coding for these enzymes and insert them into microorganisms which normally inhabit the rumen and might survive therein. The Development of Technology for Gene Transfer in Domestic Animals The mouse experiments of Palmiter e tal. (1982b, 1983) suggest that dramatic increases in growth of meat producing animals and birds should be possible. Unfortunately, to date there are no published papers showing the introduction, expression or integration of foreign genes into domestic species. For the eggs of cattle, sheep and swine the problem is visualization of the pronuclei. The fluorescent method of Critser et al. (1983) and Minhas et al. (1984) are useful for visualizing bovine pronuclei in the living state. However, exposure of eggs to the UV light needed to see the fluorescent pronuclei for time periods needed to accomplish gene insertion can damage the egg nucleus and survival of the egg. Additionally, this procedure is damaging to swine embryos. Better methods for visualizing pronuclei of domestic species and systems for injection of the 2 cell stage are _eing developed. Therefore, gene expression (approximately 25%) has occurred for a thymidine kinase-metallothionein gene injected into mouse pronuclei or into blastomeres of murine and porcine 2 cell embryos (Robl et al., unpublished). An additional problem requiring solution before gene transfer is routinely accomplished in domestic species is the inability to culture oocytes in vitro from the 1-cell to the blastocyst stage where they might be transferred easily to recipients. Once these limitations are overcome animal geneticists will have the responsibility for determining which genes are important for growth, reproduction, meat, milk and egg production in order that those genes might be cloned and inserted for study and possible livestock 155

improvement. Animal geneticists will also have the responsibility and the tools for characterizing the genome of each of our domestic species of animals and birds. This characterization could be accomplished by cloning a specific gene, introducing it into the native or a foreign species and examining alterations in the physiology of that animal as indicatative of gene expression. Overall, these are exciting times and the tools for manipulating and rapidly changing the genome of our food producing animals appear to soon be in hand. 156

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