Gene injection in the bovine: effect of time of microinjection and nuclear transfer technologies
Krisher, Rebecca L.
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Four experiments were conducted to investigate methods of producing transgenic bovine embryos entirely in vitro. Experiment 1 examined the effect of DNA microinjection at 11, 15 and 19 h after fertilization (haf) on survival rate and DNA detection frequency by polymerase chain reaction (PCR). There was no difference in transgene detection frequency between treatments (53% at 11; 50% at 15; 48% at 19 haf). Of all injected embryos developing to the morula or blastocyst stage after 7 d in culture, 89% tested positive for the presence of the transgene by PCR. Greater developmental efficiencies can be obtained when injection is performed early in pronuclear formation (7% (11/161) at 11; 4% (61159) at 15; 1 % (1/165) at 19 haf; p<0.05). Experiment 2 examined the effect of microinjection of DNA into the germinal vesicle (gv) of bovine oocytes on subsequent development and detection of the transgene. Injection of the transgene into the gv reduced developmental rates compared to controls (control=23% (89/384); non-injected=9% (23/250); GV injected=5% (12/259); p
0.05). Of the embryos developing from microinjected donors, 32% (12/37) were PCR positive. Microinjected embryos can be successfully used in a nuclear transfer program to produce more viable embryos, and the resulting embryos may be more reliably screened by PCR. The efficiency of producing viable bovine embryos positive for the injected gene may be increased by performing microinjection early in pronuclear formation, and entering the resulting embryos into a nuclear transfer program.
- Doctoral Dissertations