Effect of Transgene Introduction and Recloning on Efficiency of Porcine Transgenic Cloned Embryo Production In Vitro

摘要

Retrovirus-mediated exogenous gene transfection of somatic cells is an efficient method to produce transgenic embryos by somatic cell nuclear transfer (SCNT). This study evaluated whether efficiency of transgenic embryos production, by SCNT using fibroblast cells transfected by retrovirus vector, is influenced by the introduced transgene and whether recloning could further improve its efficiency. Transgenic cloned embryos were produced by SCNT of porcine foetal fibroblast cells transfected by either LN beta-Z or LN beta-enhanced green fluorescent protein (EGFP) retrovirus vector and evaluated for their developmental ability in vitro. Blastomeres from four-cell stage porcine embryos, produced by SCNT of foetal fibroblast cells transfected with LN beta-EGFP retroviral vector, were subsequently recloned into enucleated metaphase II oocytes and evaluated for changes in chromatin configuration, in vitro embryo development and gene expression. Analysis of results showed that cleavage and blastocyst rates of porcine SCNT embryos, using LacZ (53.6 +/- 6.4%; 12.0 +/- 5.7%) or EGFP (57.5 +/- 6.3%; 10.1 +/- 4.1%) transfected fibroblasts, did not differ (p > 0.05) from those of non-transfected controls (60.9 +/- 8.2%; 12.3 +/- 4.0%). Recloning of blastomeres did not further improve the in vitro development rate. Interestingly, the nuclei of blastomere underwent slower remodelling process than somatic cell nuclei. Both cloned and recloned embryos showed 100% transgene expression and there were no evidence of mosaicism. In conclusion, our data shows that the efficiency of transgenic cloned embryos production by SCNT of somatic cells transfected with replication-defective retrovirus vector is not influenced by the transgene introduction into donor cells andrecloning of four-cell stage blastomere could not further improve its efficiency.