DNA fragmentation, transgene expression and embryo development after intracytoplasmic injection of DNA-liposome complexes in IVF bovine zygotes

摘要

This study was designed to evaluate the quality and viability of bovine embryos produced by in vitro fertilization (IVF), after intracytoplasmic injection of pCX-EGFP-liposome complexes or pBCKIP2.8- liposome complexes (plasmids that codify the human insulin gene). Cleavage, blastocysts and expanded blastocysts rates of these both groups were not different from that of controls (IVF or IVF embryos injected with liposomes alone; IVF-L). The percentage of EGFP-positive (EGFP~+) blastocysts was 41.8%. In Experiment 2, the blastocysts obtained after injection of pCX-EGFP-liposome complexes that did or did not express the transgene, were analyzed by TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labelling) assay at days 6, 7 and 8 of culture in vitro(Bd6, Bd7 and Bd8), in order to evaluate DNA fragmentation. The EGFP~+ blastocysts showed different proportions of TUNEL-positive cells (T~+) at Bd6, Bd7 and Bd8 (91, 73.7 and 99.5%, respectively) while blastocysts without EGFP expression (EGFP~?) showed statistically lower numbers of fragmented nuclei (0, 44.6 and 85%, respectively; P<0.05). There was no evidence of DNA fragmentation in either Bd6 or Bd7 IVF and IVF-L control blastocysts, but T~+ nuclei were detected at Bd8 in both groups (66.4 and 85.8% respectively). Finally, IVF blastocysts (n=21) injected with insulin-liposome complexes, cultured for 6, 7 and 8 days, were transferred to recipient cows. Pregnancy rates of 18.2% (2/11) and 40% (2/5) resulted from the transfer of Bd6 and Bd7 cells, respectively. Two pregnancies developed to term but they were not transgenic for the insulin gene. In conclusion, EGFP expression affects DNA integrity but not embryo development. Moreover, additional transfers are required in order to overcome the drawbacks generated by in vitro culture length and transgene expression.