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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。
OBJECTIVE: To combine the tetraploid embryo compensation technique with transgene to construct embryonic stem cells (ESCs) transformed by gene targeting vector and obtain the homologous recombination event with a small probability, and then obtain the desired mutant strain directly. Methods EGFP expression plasmids were transfected into ESCs by electroporation and EGFP-ESCs were screened by G418. In addition, tetraploid blastocysts were obtained by electrofusion. Nineteen to twenty-one EGFP-ESCs were injected into each tetraploid blastocyst by microinjection, and were transplanted into the uterus of female rats of 2.5 days pseudopregnancy or 0.5 days of pseudopregnancy respectively The fallopian tube. Results The number of stable EGFP-ESCs was normal (2n = 40). The tetraploid cell fusion rate was 95.07% and the blastocyst rate was 95%. A total of 410 reconstructed blastocysts were obtained. However, a total of 151 implantation sites were observed. The implantation rate of uterine transplantation was 29.41%. The implantation rate of tubal transplantation was 64.37%. The expression of EGFP protein in the obtained embryos was scattered. Conclusion Stably expressing EGFP-ESCs are involved in the development of transgenic fetal rats. In this experiment, the implantation rate of tubal transplantation was higher than that of uterine transplantation.