Frequency of detection of exogenous DNA 7, 14, and 21 days after microinjection of bovine zygotes

Abstract

The frequency of PCR positive detection of exogenous DNA was determined in bovine embryos over a 21 day period of culture after microinjection. After in vitro maturation and in vitro fertilization, embryos were microinjected with one of two genomic DNA constructs. Approximately 5% of the injected embryos were prepared for PCR analysis immediately after injection (d 0). After culture for 7 d on Buffalo Rat liver (BRL) cells, embryos were assessed for stage of development. Embryos developing to the morula or blastocyst stage were randomly assigned to two groups; PCR analysis (d 7) or extended culture. Extended culture embryos were cultured for an additional 7 d. Embryos maintaining a blastocoel at d 14 were considered alive, and were assigned randomly to either further culture or PCR analysis. Of zygotes examined after d 7 of culture, 26% (189/733) of control and 12% (294/2482) of injected embryos had developed to the morula or blastocyst stage (control vs. injected; P<0.01). After 14 d of culture, 33% (10/30) of control and 51% (100/196) of injected embryos were alive (P>0.05). After 21 d of culture, 29% (2/7) of control and 14% (9/66) of injected embryos were alive (P>0.05). Analysis of d 0 injected embryos resulted in a transgene detection frequency of 100% (46/46). At d 7, detection frequency was 82% (51/62) of morulae and blastocysts, and was 84% (513/610) of dead embryos. Transgene detection frequencies decreased to 49% (18/37) and 50% (41/82) on d 14, and 38% (3/8) and 22% (5/23) on d 21 in live and dead embryos, respectively. Transgene detection frequency between live embryos on d 0, 7, and 14 was significantly different (P<0.01). DNA detection frequency in microinjected bovine embryos by PCR analysis does not give a reliable indication of live transgenic birth rates, based on reported rates, before embryo transfer must be performed.

Bovine Follicular Dynamics, Oocyte Recovery, and Development of Oocytes Microinjected with a Green Fluorescent Protein Construct

“…The higher developmental score of GFP-positive embryos from Grade A oocytes suggests that the quality of the oocyte affects DNA integration in cattle embryos. Conversely, in the present study, the number of embryos expressing integration of DNA was lower than the number of putative transgenic morulae and blastocysts that were identified using PCR procedures (2,3,20). The low DNA expression in bovine embryos in the present study could reflect true expression more so than overall integration of GFP.…”

“…The polymerase chain reaction ( PCR) was used to detect the transgene before embryo transfer to a recipient to decrease the time and production costs by screening embryos (19,20,23). However, PCR analysis only confirms the presence of microinjected DNA, regardless of its integration status.…”