Title page for ETD etd-06062008-164748
|Type of Document
||Krisher, Rebecca L
||Gene injection in the bovine :effect of time of microinjection and nuclear transfer technologies
||Animal Science (Dairy)
|Gwazdauskas, Francis C.
|Akers, Robert Michael
|Lewis, Gregory S.
|Pearson, Ronald E.
|Saacke, Richard G.
|Vinson, William E.
|Wong, Eric A.
|Date of Defense
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); p0.05).
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.
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