Cell Culturing Technology as a Major Hurdle in the Commercialization of Genetically Altered Animals

Recent discoveries in animal cloning via nuclear transfer combined with advanced genomic manipulation have opened vistas of opportunity in livestock improvement, bioreactors for pharmaceuticals, and organ transplants, within a realistic commercial time frame. The Advanced Technology Program (National Institute of Standards and Technology, U. S. Department of Commerce) recently (September 22 - October 27, 1998) conducted an on-line discussion in 22 papers and commentary by 36 authors about near and mid-term Research and Development needs in this nascent industry.

Embryo Availability

Manipulation of the genome in animals has, until recently, been largely limited to mice. This was mainly due to three factors, 1) the availability of inbred strains improves functional analysis of rapidly acquired sequence information and gene distribution, 2) mouse gestation and post-natal maturation (indeed, the lifespan) is short and reasonably robust, 3) pre- and post-implantation development in the mouse is easily reproduced in vitro (1). Thus, the supply of embryos is not the rate limiting step in most mouse experiments. Using non-lethal genes and F2 eggs, competent laboratories can easily harvest 200 or more one-celled embryos of high quality per experimental day, with at least 75% surviving microinjection, 25% of these completing gestation, and 20 - 25% of the offspring containing the transgene. This produces an overall transgenesis efficiency of 1-10%, thus providing a reliable source of genetically modified animals (2, 3).

In contrast, generating and maintaining a sufficient number of pre-implantation embryos in livestock species currently involves at least as much art as science. Labor-intensive ultrasound technologies may optimize cattle oocyte harvest at 7 - 10 per collection (4). Ideal culture conditions for livestock embryos vary substantially from the well described maintenance of mouse embryos in vitro (5). Overall transgenic efficiencies via zygote microinjection are well under 0.1% in cattle, sheep, goats and pigs (6). Working with pig embryos may be particularly challenging due to the earlier onset of embryonic transcription, and a lack of techniques to address in vitro maturation and activation of oocytes and culture of embryos to blastocyst (7). Molecular markers for determining viable embryos and the differentiation state of cells and embroys will be particularly useful (5).

Chromosomal Manipulation Techniques

DNA microinjection is an inefficient and highly random vehicle for genetic manipulation, and may be complicated by transgene silencing, inappropriate transgene expression, and tissue variegation (8). Homologous recombination is even more inefficient, but highly specific, allowing discrete modification of a given locus (9). Parameters for maximizing a targeting event (such as amount and length of homology, DNA concentration, and physical form of the insert) have been established for murine embryonic stem (ES) cells (10). Homologous recombination technology, combined with the availability of these robust ES cells which can re-integrate into the inner cell mass (and thus contribute to all tissues of the resultant offspring) have secured the place of the mouse as the animal of choice in genomic manipulation research. However, commercial applications require access to ES cells in livestock species (9, 11). In addition, the conditions to allow such changes in the chromosomes of other commonly cultured cell types are largely unknown (11).

Mammalian artificial chromosomes have recently been used as a delivery system that circumvents the random integration problems faced with small fragment insertion (8, 12). In general, these structures are built up from the minimal genetic cis-elements necessary and sufficient for independent existence and replication within the target nucleus. Since one essential requirement is a minimum size, there is plenty of room for insertion of the gene of choice with neighboring DNA to negate positional effects. Transgenesis has been achieved with these vectors in several cell lines and founder mice. Germline integration has yet to be demonstrated (12).

Embryonic Stem Cells

ES cells have provided an invaluable tool for discrete, targeted genetic manipulation of whole organisms. However, the promise of these truly pluripotent cells is far greater. Under fairly straightforward culture conditions, mouse ES cells have been forced to differentiate down several pathways including the formation of neurons (13) and vascular endothelial cells (14). The fine definition of angiogenesis resulting from such investigations may also have benefits for organ transplantation since establishment of vascular connections is a critical limitation to current protocols. Establishment of the appropriate conditions to grow any differentiated cell type in vitro from such precursors could have tremendous therapeutic potential and revolutionize cellular medicine and transplantation (15). Another important application is the exploitation of the in vitro differentiation potential of ES cells for generating systems of sufficient complexity in the culture dish to mimic tissues and organs. Such cultures could rapidly replace some animal models and offer higher throughput screening assays for gene regulation and drug toxicity (16). The most significant roadblock to developing these technologies remains the inavailability of these self-renewing cells from non-rodent species.

Transgenesis in Poultry

While there are great challenges in developing transgenesis via microinjection and/or ES cell-mediated chimerism in livestock, the methods are extensions of techniques established in mice. Genetic manipulation in chickens is much more technically complex due to the inaccessibility of the earliest stages of embryo development (17). However, the commercial potential of transgenic chickens is great due principally to 1) their short generation time and ease of breeding, and 2) convenience and potential protein yield of the laid hen's egg -- the "bioreactor" producing a transgenic therapeutic protein of choice. As with other species, nuclear transfer protocols will be useful for increasing transgenic efficiencies and make the technology commercially viable (18).

Transgenesis via Nuclear Transfer

Early efforts at mammalian cloning via nuclear transfer succeeded only with early embryonic cells as donors (19). Using aged embryos as recipients, nuclei from sufficiently pluripotent cells could be used at any stage of the cell cycle. However, other factors severely decreased efficiency. Using more immature (metaphase II) embryos increased overall efficiency but required donor nuclei arrested in G1 or G0, and, most strikingly, allowed the use of terminally differentiated, adult cells as the nuclear donor (20). The reasons for these differences are not at all clear, and the nature of G0 arrest needs much more study (20, 21). An intriguing possibility may be that cultured cells in G0 arrest and near-death differentiated cells such as cumulus are prepared for demethylation and acetylation changes by their apoptotic condition (19). Nuclear transfer using differentiated (adult or fetal mid-gestational) cells has been accomplished in sheep (22), cows (23), and mice (24).

This ability to clone animals from differentiated cells has immediate advantages for production of transgenic livestock including: 1) all offspring will contain the transgene in the same location compared to random insertion at unpredictable rates using microinjection technology; 2) prescreening for the highest expression, genomic stability, etc., before generating offspring; 3) the opportunity to use gene targeting technology which is currently unavailable in species without robust ES cell lines; and 4) ability to produce identical livestock in the first generation, thus reducing herd production times (19, 20, 25). While genetically manipulated nuclei have already been used for nuclear transfer (26), the true test of the combined technology will be the generation of animals with complex deletions and/or additions. For example, the establishment of livestock herds lacking the target sequence for prion infection could improve the safety of cattle products for human consumption (11, 25). Also, one early target may be the production of human gamma globulin, which would require the knockout of the endogenous gene and the insertion of the human gamma globulin complex (27). Nuclear transfer may also be useful in reviving cell lines approaching exhaustion. Bovine fibroblast cell lines near senescence were used as nuclear donors, and the fibroblasts reestablished from the resulting fetuses had a life span equal to the original line (28).

There are also critical hurdles to be overcome before the technology can become routine. The most immediate challenges outline a veritable catalog of early development in different species. For example, fetal wastage and neonatal death occur more frequently with nuclear transfer than other embryonic manipulations. Early gestation mortality might easily be due to nuclear-cytoplasmic incompatibilities that prevent activation of critical developmental pathways. However, the high incidence of late gestation stillbirth and post-natal mortality, and the hypertrophy of some organs in young pups may have more subtle causes (29, 30). Some clues to understanding these issues may come from early studies examining the 2 -- 5% differences in RNA expression seen in blastocysts freshly harvested from the uterus, or produced from in vitro fertilization or nuclear transfer (30). In addition, mitochondrial matching between the nuclear donor cell and the recipient egg have not been studied, and may play a role since cloning in mice involved transfer of the nucleus only (19). Indeed, one near-term requirement is an understanding of totipotency and cellular differentiation at the molecular level (31). Finally, some species will present more intractable problems than others. Cloning via nuclear transfer has yet been shown in pigs, probably due to complexities in early development (21).

Other important considerations for optimizing transgenic technology include: increasing efficiency for homologous recombination and screening for targeted integrations in ES cells (21, 28, 29, 30), establishing conditions for gene targeting in fibroblasts and other cells types (19, 20, 25, 27, 29, 31), and understanding the limits to recloning (19, 27, 30).

Human Germ Line Engineering

While a firm prohibition precludes the use of federal funding for human embryo research, the time to examine and discuss the realistic benefits and challenges these new reproductive technologies embody is now, while they are still nascent (32). It will be important to see how each new experiment, which increases our understanding of animal developmental biology, may also enable genetic engineering in humans.

References

1. Hogan, B., F. Constantini, and E. Lacy (eds.). Manipulating the Mouse Embryo. 2nd edition. Cold Spring Harbor, NY (1994).

2. Brinster, R. , et al. Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. PNAS. 85: 846 (1985).

3. Wight, D. And Wagner, T Mutation Research 307: 429 (1994).

4. Yang, X. Cloning and other reproductive technologies for application in transgenics. Genetic Manipulation in Animals: Advanced Transgenesis and Cloning. Paper #22, http://www.atp.nist.gov/atc (1998).

5. Yaswen-Corkery, L. Cell culturing technology as a major hurdle in the commercialization of genetically altered animals. Genetic Manipulation in Animals: Advanced Transgenesis and Cloning. Paper #23, http://www.atp.nist.gov/atc (1998).

6. Wall, R., Hawk, H. And Nil, N. J. Cell. Biochem. 49: 113 (1992).

7. Hawley, R. And Greenstein, J. Application of nuclear transfer technology in the generation of donor pigs for xenotransplantation. Genetic Manipulation in Animals: Advanced Transgenesis and Cloning. Paper #9, http://www.atp.nist.gov/atc (1998).

8. Drayer, J. SATACs and transgenesis. Genetic Manipulation in Animals: Advanced Transgenesis and Cloning. Paper #4, http://www.atp.nist.gov/atc (1998).

9. Pati, S. Homologous recombination and genetic engineering of transgenic recombinant animals. Genetic Manipulation in Animals: Advanced Transgenesis and Cloning. Paper #13, http://www.atp.nist.gov/atc (1998).

10. Joyner, A. Gene Targeting, A Practical Approach. IRL Press, NY (1993).

11. Piedrahita, J. Gene targeting in the porcine and bovine species: challenges and opportunities. Genetic Manipulation in Animals: Advanced Transgenesis and Cloning. Paper #12, http://www.atp.nist.gov/atc (1998).

12. Vos, J-M. Mammalian Artificial Chromosomes for animal transgenesis. Genetic Manipulation in Animals: Advanced Transgenesis and Cloning. Paper #19, http://www.atp.nist.gov/atc (1998).

13. Gottlieb, D. ES cells make neurons in a dish. Genetic Manipulation in Animals: Advanced Transgenesis and Cloning. Paper #7, http://www.atp.nist.gov/atc (1998).

14. Gendron, R. Prospects and hurdles in optimizing the vascular support of engineered tissues. Genetic Manipulation in Animals: Advanced Transgenesis and Cloning. Paper #6, http://www.atp.nist.gov/atc (1998).

15. Mountford, P. Genomics: delivering cell culture systems for tissue therapy. Genetic Manipulation in Animals: Advanced Transgenesis and Cloning. Paper #10, http://www.atp.nist.gov/atc (1998).

16. Snodgrass, H. And Keller, G. ES cells offer a powerful tool for understanding the genetic control of tissue development and for screening potential therapeutic drugs. Genetic Manipulation in Animals: Advanced Transgenesis and Cloning. Paper #17, http://www.atp.nist.gov/atc (1998).

17. Love, J., et al. Biotechnology 12: 60 (1994).

18. Harvey, A. Nuclear transfer and gene targeting in domestic animals: bioreactors of the future. Genetic Manipulation in Animals: Advanced Transgenesis and Cloning. Paper #8, http://www.atp.nist.gov/atc (1998).

19. First, N. Concerns about gene transfer and nuclear transfer in domestic animals. Genetic Manipulation in Animals: Advanced Transgenesis and Cloning. Paper #5, http://www.atp.nist.gov/atc (1998).

20. Wilmut, I. Role of cell cycle. Genetic Manipulation in Animals: Advanced Transgenesis and Cloning. Paper #21, http://www.atp.nist.gov/atc (1998).

21. Bondioli, K. Technical challenges for cloning pigs for biomedical research. Genetic Manipulation in Animals: Advanced Transgenesis and Cloning. Paper #2, http://www.atp.nist.gov/atc (1998).

22. Wilmut, I. et al. Nature 385: 810 (1997).

23. Cibelli, J., et al. Science 280: 1256 (1998).

24. Wakayama, T. et al. Nature 394: 369 (1998).

25. Colman, A. Somatic cell nuclear transfer in mammals. Genetic Manipulation in Animals: Advanced Transgenesis and Cloning. Paper #3, http://www.atp.nist.gov/atc (1998).

26. Schnieke, A. et al. Science 278: 2130 (1997).

27. Robl, J. Nuclear transplantation in the cos: future challenges. Genetic Manipulation in Animals: Advanced Transgenesis and Cloning. Paper #15, http://www.atp.nist.gov/atc (1998).

28. Stice, S. Enhancing ytransgenics through cloning. Genetic Manipulation in Animals: Advanced Transgenesis and Cloning. Paper #16, http://www.atp.nist.gov/atc (1998).

29. Petersen, R. Nuclear transfer technology. Genetic Manipulation in Animals: Advanced Transgenesis and Cloning. Paper #11, http://www.atp.nist.gov/atc (1998).

30. Westhusin, M. Understanding developmental abnormalities in offspring produced by nuclear transplantation. Genetic Manipulation in Animals: Advanced Transgenesis and Cloning. Paper #20, http://www.atp.nist.gov/atc (1998).

31. Bishop, M., et al. From cell to production. Genetic Manipulation in Animals: Advanced Transgenesis and Cloning. Paper #1, http://www.atp.nist.gov/atc (1998).

32. Stock, G. and Campbell, J. Human germline engineering -- the prospects for commercial development. Genetic Manipulation in Animals: Advanced Transgenesis and Cloning. Paper #18, http://www.atp.nist.gov/atc (1998).

Return to ATC's Main Page