Transgenic animals have important applications in agriculture and human medicine; nevertheless the available techniques still remain inefficient and technically difficult. We have recently developed a novel method to transfect bovine embryos that consists of intracytoplasmic injection of exogenous DNA–liposome complexes (eDNA-LC) in IVF zygotes. This study was designed to evaluate the quality and viability of IVF bovine embryos, after intracytoplasmic injection of pCX-EGFP–liposome complexes (EGFP-LC) or pBCKIP2.8-liposome complexes (plasmid that codifies the human insulin gene, HI-LC). First, we evaluated embryo development and enhanced green fluorescent protein (EGFP) expression of IVF embryos injected with both plasmids separately. This treatment was analysed by Fisher's Exact test (P ≤ 0.05). Cleavage rates for EGFP-LC, HI-LC and IVF embryos injected with liposomes alone (IVF-L) and IVF control (IVF-C) were 62% (63/102), 67% (67/100), 66% (67/101) and 79% (98/124); blastocysts rates were 17% (17/102), 21% (21/100), 21% (21/101) and 23% (28/124), respectively. No statistical differences were seen among groups. The percentage of EGFP-positive embryos (EGFP+) after EGFP-LC injection was 42.9% after 3 days of culture and 41.8% at the blastocyst stage. In the second experiment, the blastocysts obtained, EGFP+ or EGFP-negative (EGFP–), were analysed by TUNEL assay at Day 6 (Bd6), 7 (Bd7) and 8 (Bd8) of in vitro culture, in order to evaluate the effect of the transgene and culture length, on DNA fragmentation. This treatment was analysed by the difference of proportions test (P ≤ 0.05) using statistical INFOSTAT software. All EGFP+ blastocysts showed TUNEL positive cells (T+). The percentage of T+ in Bd6, Bd7 and Bd8 were 91, 73.7 and 99.5%, respectively (P ≤ 0.05). EGFP– blastocysts showed lower fragmented nuclei (0, 44.6 and 85%, respectively; P ≤ 0.05). Groups IVF-L and IVF-C were also evaluated. In both groups, there was no evidence of DNA fragmentation in Bd6 and Bd7, but T+ were detected in Bd8 (66.4 and 85.8%, respectively; P ≤ 0.05). In the third experiment, bovine blastocysts obtained from the HI-LC group were individually transferred to recipient cows after 6 (n = 11), 7 (n = 5) and 8 (n = 5) days of culture post-IVF and HI-LC injection. The pregnancies obtained were from Bd6 [18.2% (2/11)] and Bd7 [40% (2/5)], although none of the recipients receiving Bd8 were diagnosed pregnant. Two pregnancies developed to term, one derived from Bd6 and the other from Bd7. Analysis by PCR determined that none of the born cows were transgenic. In summary, IVF bovine embryos could be easily transfected after the injection of eDNA-LC and the technique did not affect offspring viability. The results indicate that extended time in in vitro culture increases the percentage of fragmented nuclei in blastocysts. Moreover, this parameter increases in blastocysts with transgene expression compared with those without expression. Finally, more transfers are required in order to obtain the real efficiency of this new technique and to overcome the drawbacks generated by in vitro culture length and transgene expression.
An epigenetic gene silencing pathway selectively acting on transgenic DNA in the green alga Chlamydomonas
