In this study, we demonstrated expression of enhanced green fluorescent protein (EGFP) and neomycin resistant (Neo(R)) genes in porcine embryos following nuclear transfer from porcine fetal fibroblasts (PFFs) transduced with the EGFP and Neo(R) genes by retrovirus-mediated infection. Nuclear transfer of the nonstarved transfected PFF into enucleated oocytes was accomplished by cell to cell fusion. Out of 188 porcine eggs reconstructed by nuclear transfer, 116 (61.7%) eggs cleaved and 25 (13.3%) developed to morula and blastocyst stages. Of these 25 morulae and blastocysts, 25 (100%) embryos emitted green fluorescence. Expression of the both EGFP and Neo(R) genes was detected as early as the 2-cell stage. As determined by EGFP gene expression, mosaicism was not observed in any embryo. These results suggest that porcine oocytes reconstructed by nuclear transfer with transfected PFFs can successfully develop to the blastocyst stage. In addition, this approach might be applicable to the production of transgenic pigs with complex genetic modifications.

译文

:在这项研究中,我们证明了从猪胚胎成纤维细胞(PFF)进行核转移后,猪胚胎中的绿色荧光蛋白(EGFP)和新霉素抗性(Neo(R))基因在猪胚胎中的表达通过逆转录病毒介导的感染。非饥饿转染的PFF向去核卵母细胞的核转移是通过细胞间融合来完成的。在通过核移植重建的188个猪卵中,有116个(61.7%)卵裂开,有25个(13.3%)卵发育到桑ula和胚泡期。在这25个桑ula和胚泡中,有25个(100%)胚胎发出绿色荧光。早在2细胞阶段就检测到了EGFP和Neo(R)基因的表达。如通过EGFP基因表达所确定的,在任何胚胎中均未观察到镶嵌现象。这些结果表明,用转染的PFF通过核转移重建的猪卵母细胞可以成功地发展到胚泡期。此外,这种方法可能适用于生产具有复杂基因修饰的转基因猪。

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