Production of transgenic farm animals as
bioreactors to make human re-combinant proteins in their milk or as
organ donors to transplant in humans with organ failure has been heavily
researched and invested in the last decade. However, the current efficiency
for producing transgenic animals particularly farm animals, is low
and the cost is high. Success in the production of transgenic farm
animals requires an adequate animal facility and dedicated teams of
embryologists, veterinarians, animal scientists and molecular biologists.
Improvement on the success rate of the transgenic technology largely
relies on the application of 1) various classical reproductive and
embryological technologies such as artificial insemination, superovulation
and reproductive management etc., and 2) newly emerged contemporal
technologies such cloning and other assisted reproductive technologies.
While the importance of classical technologies should not be ignored,
emphasis will be given to the newly emerged technologies in this presentation.
The Art of Making Transgenic Farm Animals Transgenic technology
has great potential in molecular breeding of farm animals, such
as development of disease resistance-transgenic animals, or animals
with more lean meat or better milk quality. Unfortunately, production
of transgenic farm animals is very costly and there has been little
support from the government or farmers to sponsor this line of research
for agriculture. Fortunately the pharmaceutical industry has been
investing heavily in the production of transgenic farm animals as
bioreactors or for xenotransplantation which has been keeping the
transgenic research of farm animals ongoing.
While almost all transgenic animal techniques are initiated in
mice, production of transgenic farm animals is not as simple as
that of transgenic mice. Technology for producing transgenic mice
has been well established and hundreds of transgenic animal facilities
worldwide now offer commercial production of transgenic mice at
a reasonable cost. By contrast, there are only a handful of groups
in the world who have a record of producing transgenic farm animals.
Compared to transgenic mice, the cost is high and the efficiency
is low for making transgenic farm animals. Estimates have been that
it would cost hundreds of thousands of dollars to produce a transgenic
farm animal. The worst news of all to a transgenic company is the
fact that there is no guanranty for producing a transgenic farm
animal even if you have the estimated amount of dollars available.
There are many factors which may affect the success rate of making
transgenic animals. Perhaps the biggest factor is the skill of the
technician, and the knowledge and experience of the team with a
particular species. A very experienced microinjection technician
who may have a successful record in producing transgenic mice may
become disappointedly helpless when asked to make a transgenic pig
or cow. The collective expertise of the transgenic group in embryo
culture, embryo transfer, reproductive management and animal husbandry
is also very critical for a very successful transgenic program (Wang
et al., 1996).
In cattle, a recent summary of the transgenic results from PPL
indicate that 9 transgenic calves were produced out of 25,023 microinjected
zygotes (overall efficiency = 0.04%). One of the bottlenecks of
the overall procedure was the poor development following microinjection
(5%). Our experience demonstrated that this efficiency may be improved
up to 5-fold (27%, unpublished results). The other bottleneck of
the technology was the low integration rate. Out of 134 calves produced
in the PPL study, only 9 were transgenic (6.7% transgenic rate per
birth). A great saving of cost may be achieved if the microinjected
embryos can be screened prior to embryo transfer so that only transgenic
positive embryos are transferred.
The Promises of Cloning
Cloning mammalian embryos has been researched extensively in the
last decade. This technology offers the possibility to duplicate
a valuable embryo into numerous identical copies. This has sparked
much interest in the cattle embryo transfer industry and several
companies were established worldwide in the late 80's and early
90's to explore commercialization of this technology. Unfortunately
those companies were soon out of business because of the poor efficiency
of the cloning technology and the abnormality of the cloned calves
(Yang, 1991). In 1997, the production of Dolly (Wilmut et al, 1997),
the first mammal cloned from an adult cell, sparked a second wave
of interest in the cloning technology. The promises behind this
new version technology are 1) the possibility to duplicate an unlimited
number of copies of animals using somatic cell lines; 2) the possibility
of genetic manipulations of the cell lines prior to cloning (Cibelli
et al., 1998), and 3) the feasibility of cloning a proven valuable
animal. However, the efficiency of the cloning technology remains
low and more research is needed before commercialization of this
technology.
Reproduction of Juvenile and Prepubertal Calves
Another technology which should be of interest to the transgenic
industry is to reproduce the valuable transgenic animal as early
as possible and as many as possible. Our recent breakthrough was
the birth of two healthly calves through the transfer of embryos
produced from a 2-month-old donor calf (Taneja et al., 1998). Three
more calves were born from oocytes of another 2-month-old donor
last monhts. To date, we have produced over 30 calves using oocytes
derived from pre-pubertal heifers. Estimates are that via this technology,
a calf may be able to produce several to dozens of pregnancies before
she reaches her normal breeding age.
In Vitro Fertilization (IVF) and Oocyte Pick-Up guided by
Ultrasound (OPU)
Upon a heifer reaching puberty at 11-12 months of age, her oocytes
may be retrieved weekly or even twice weekly for embryo production
and embryo transfer (Presicce et al., 1997, Yang et al., 1998).
One of the recent breakthroughs in the practical world of animal
reproduction is the combined application of the existing IVF technology
and the state-of-the-art OPU technique in cattle. Although, a cow
may ovulate only about 200 oocytes in her life time, there are tens
of thousands of oocytes in her ovaries as indicated earlier. Our
long-range goal is, through the application of various assisted
reproductive technologies, to improve the reproductive efficiency
of the genetically elite females, particularly cattle. While a cow
normally may can produce one viable oocyte during each estrus cycle
(ovulation), up to 50 antral follicles exist on the ovary at any
time of the estrus cycle. Via OPU, potentially a valuable donor
cow may yield 15-20 oocytes each week (twice weekly collection of
7-10 oocytes per collection) or about 700-1000 oocytes/year/cow.
Assuming a 30% blastocyst rate from those oocytes, and a 40% pregnancy
rate, a cow may potentially offer 200-300 blastocysts or 80-120
pregnancies each year. Therefore this relatively well-established
technology would enable the transgenic industry to expand their
transgenic herd very rapidly. However, to achieve this goal, a reliable
IVF system and a dedicated OPU team are needed.
Other Reproductive and Embryological Technologies
Other reproductive or embryological technologies which might be
of interest to the transgenic industries include embryo cryopreservation,
sperm or embryo sexing to produce desired transgenic animals of
known sex and possibly sperm injection. Detection of transgenic
positive embryos prior to transfer will be of importance to reduce
cost of transgenic production. Alternative methods to microinjection
such as sperm-mediated gene transfer should also be further explored.
References
Cibelli, J.B., S.L. Stice, P.J. Golueke, J.J. Kane, J. Jerry, C.
Blackwell, F. A. Ponce de Leon, and J.M. Robl. Cloned transgenic
calves produced from nonquiescent fetal fibroblasts. Science 280:1256-1258,
1998.
Eyestone, W.H., M. Gowallis, J. Monohan, T. Sink, S.F. Ball and
J.D. Cooper. Production of transgenic cattle expressing human %-lactalbumin
in milk. Theriogenology 49:386, 1998.
Presicce G.A., S. Jiang, M. Simkin and X. Yang. Age and Hormonal
Dependence of Acquisition of Oocyte Competence for Embryogenesis
in Prepubertal Calves. Biol. Reprod. 56:386-392, 1997.
Taneja, M., P.E.J. Bols, A. Van de Velde, J-C Ju, D. Schreiber,
M. Tripp, H. Levine, J. Riesen and X. Yang. Pregnancies from in
vitro produced embryos derived from calf oocytes collected at 10
to 15 weeks of age. Biol. Reprod. Suppl. (In press), 1998.
Wang, B., R. L. Page and X. Yang. Improved transgenic efficiency
in rabbits attributed to successful microinjection, embryo transfer
and animal husbandry procedures. Theriogenology 45:342, 1996.
Wilmut, I., A.E. Schnieke, J. McWhite, A.J. Kind, and K.H.S. Campbell.
Viable offspring derived from fetal and adult mammalian. Nature
27:385, 1997.
Yang, X., C. Kubota, H. Suzuki, M. Taneja, P.E.J. Bols and G.A.
Presicce. Control of oocyte maturation in cows - biological factors.
Theriogenology 49:471-482, 1998
Yang, X. Featured article: Embryo cloning by nuclear transfer in
cattle and rabbits. Intl Embryo Transfer Newsletter 9 (4):10-22,
1991.
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