精子膜处理方法对精子介导的外源质粒转基因效率的影响  

Impact of Different Method in Sperm Membrane Treatment on Exogenous Plasmid Transgenic Efficiency by Sperm

作　　者：贾晓峰[1] 徐彦[1] 刘淼[1] 王亮[2] 施惠娟[1] 

机构地区：[1]上海市计划生育科学研究所国家卫生和计划生育委员会计划生育药具重点实验室,上海200032 [2]第二军医大学长征医院妇产科,上海200003

出　　处：《实验动物与比较医学》2013年第4期256-260,共5页Laboratory Animal and Comparative Medicine

基　　金：上海市基础研究重点项目（10JC1409900）和上海市重点学科建设项目（S30201）资助

摘　　要：目的探讨超声、NaOH和溶血卵磷脂三种小鼠精子膜处理方法对转染质粒与精子共孵育后卵胞质内单精子注射（ICSI）授精，观察质粒转染的效率。方法采用上述三种方法对小鼠精子进行膜处理，通过Transgreen／PI荧光染色，观察小鼠精子质膜的完整性；通过流式细胞仪检测小鼠精子DNA的完整性；将处理后的小鼠精子与带GFP的转染质粒共孵育，再采用ICSI与促排卵后成熟小鼠卵子授精，统计各组受精率、卵裂率及荧光囊胚率，观察三种方法对小鼠精子作为质粒载体ICSI后转基因效率的影响。结果溶血卵磷脂方法处理小鼠精子膜的效果显著优于超声和NaOH组（99．2％vs94．7％％，90．9）（P〈0．05）。三种方法对小鼠精子DNA结构的损伤无显著性差异（P〉0．05），行ICSI后三组小鼠胚胎发育率差异不显著（P〉O．05），各组之间荧光胚胎的形成率无显著性差异（P〉0．05o结论三种小鼠精子膜处理方法的效率均能达到90％以上，且对小鼠精子DNA的损伤、行ICSI后小鼠胚胎的发育率和转基因效率无显著差异，因此，三种方法均可用于ICSI法小鼠转基因研究。Objective To explore the effect of three methods in removing mouse sperm membrane including ultrasound, NaOH and lysolecithin on the efficiency of transgenic by intracytoplasmic sperm injection （ICSI）. Methods The mouse sperms were treated with the three methods above mentioned. Both of sperm membrane and DNA integrity of the sperm cells were evaluated by flow cytometry after died with Transgreen and propidium iodide （PI）. The treated sperms were incubated with GFP-carrying plasmid and then conducted ICSI. The effect of these methods on efficiency of transgenic was evaluated by the results of fertilization cleavage and blastocyst with fluorescence. Results The rate of removing sperm membrane by lysolecithin method was higher than that of ultrasound and NaOH methods （99.2% vs. 94.7%, 90.9%）（P〈0.05）, but the rates of sperm DNA damage, the development of embryos from ICSI and blastocyst with fluorescence were similar among these methods （P〉0.05）. Conclusion The three methods of mouse membrane treatment could be used to improve mouse transgenic efficiency by ICSI.

关 键 词：精子去膜法 卵胞质内单精子注射(ICSI) DNA完整性 转基因效率 小鼠 

分 类 号：Q95-33[生物学—动物学]