【摘要】 目的将四倍体胚胎补偿技术与转基因相结合,构建基因打靶载体转化的胚胎干细胞(ESCs),获得小概率的同源重组事件,进而直接得到所要的突变品系。方法通过电穿孔的方法将增强型绿色荧光蛋白(EGFP)表达质粒转入ESCs,用G418筛选获得EGFP-ESCs。另外,通过电融合获得四倍体囊胚细胞,显微注射法将19～21个EGFP-ESCs注入每个四倍体囊胚腔,分别移植到假孕2.5d雌鼠子宫或假孕0.5d的输卵管内。结果获得稳定表达的EGFP-ESCs,染色体数目正常(2n=40条);四倍体细胞融合率为95.07%,囊胚发育率为95%;共获得410个重构囊胚;移植后未得到出生小鼠,但共观察到了151个着床位点,子宫移植的着床率为29.41%,输卵管移植的着床率为64.37%,获得的胚胎中EGFP蛋白有散在的表达。结论稳定表达的EGFP-ESCs参与到了转基因胎鼠的发育中;本实验中输卵管移植的着床率高于子宫移植的着床率。更多还原

【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(2n=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.更多还原