关键词: 胚胎干细胞, 转基因, 四倍体胚胎补偿技术, 小鼠

Abstract: Objective To use tetraploid embryo complementation combined with gene transfer to produce genetically modified embryonic stem cells (EsCs) clones. Methods In this study, EGFP was introduced into ESCs by electroporation, and transfected positive cells were selected by G418 resistance. The tetraploid embryos were obtained from diploid blastomere electrofusion which preformed at 2-cell stage. Afterwards, 19-21 EGFP-ESCs were inserted into each tetraploid blastocyst cavity by piezo drilled microinjection,then the injected blastocysts were transferred into the uterus of pseudo-pregnancy at 2.5-day or the oviduct of 0.5-day female mice. Results The transfected ESCs maintained normal karyotype even after long-term passage (2EM>n/EM>=40). The rate of fusion was 95.07%, and the developmental rate of tetraploid blastocyst was 95%.Totally 410 injected blastocysts were obtained. Unfortunately, we have not got any vital offsprings, except 151 implantation sites (pseudo-pregnancy 2.5 days:29.41%;the oviduct of half one day:64.37%). Furthermore, scattered EGFP expressions in transgenic fetus were observed under invert fluorescent microscope. Conclusion The transfected ESCs were observed in transgenic fetus, and the implantation rate in oviduct was higher than that in uterine.

Key words: Embryonic stem cell, Transgene, Tetraploid embryo complementation, Mouse