精原干细胞体内转染途径建立rsp3111转基因小鼠的可行性研究  

Study on the Feasibility of Establishing of rsp3111 Trangenic Mouse through in vivo Transfection of Spermatogonial Stem Cells

作　　者：周密[1] 贾晓峰[2] 史庭燕[2] 王健[2] 袁瑶[2] 施惠娟[2] 

机构地区：[1]复旦大学上海医学院,上海200032 [2]国家人口和计划生育委员会计划生育药具重点实验室,上海市计划生育科学研究所,上海200032

出　　处：《实验动物与比较医学》2008年第3期138-143,共6页Laboratory Animal and Comparative Medicine

基　　金：国家自然科学基金面上项目(No.30571968);上海市人口和计划生育委员会局管科技发展基金项目(05JG05009)资助

摘　　要：目的构建目的基因rsp3111与荧光蛋白融合表达的质粒,并且实现目的基因在体外受精胚胎及在体附睾成熟精子中表达。为进一步通过精原干细胞体内转染途径建立rsp3111转基因小鼠奠定基础。方法将rsp3111基因以XholI和BamHI酶切后插入经同样酶切处理的真核细胞表达质粒pDsRed-Monomer-N1中,重组质粒为pDsRed-Monomer-N1-rSP3111。采用共培养和脂质体介导方法将ICR小鼠精子与pDsRed-Monomer-N1-rSP3111质粒共孵育,再与成熟的卵母细胞体外受精;或用脂质体介导睾丸直接注射法,将pDsRed-Monomer-N1-rSP3111导入小鼠睾丸中,通过PCR及荧光显微镜观察体外受精后的受精卵及体内睾丸转染rsp3111基因后附睾精子中是否有外源rsp3111基因的表达。结果PCR结果表明,rsp3111基因成功地转入了附睾精子及受精卵中。荧光检测结果表明,目的基因rsp3111在体外受精胚胎及睾丸的曲细精管中均有表达。结论通过雄性生殖细胞载体法实现了大鼠配子特异性蛋白rSP3111在小鼠受精卵及睾丸、附睾精子中的表达;为进一步通过精原干细胞体内转染途径建立rsp3111转基因小鼠奠定了基础。Objective To construct a plasmid for fusion expression of rsp3111 protein and fluorescent protein, and to express rSP3111 protein in IVF embryos and in mature sperms in epididymis. Methods Expression plasmid pDsRed-Monomer-Nl-rSP3111 was constructed by subcloning the cDNA of rSP3111 protein pDsRed-Monomer-N1. Mouse sperms were transfected by expression plas- mid pDsRed-Monomer-Nl-rSP3111 by liposome-mediated methods, and fertilized with mature eggs. Alternatively, pDsRed-Monomer-N1-rSP3111 plasmid were introduced into testis by liposome-mediated injection. The expression level and pattern ofrSP3111 protein was examined by PCR assay and fluorescent microscopy. Results The result of PCR assays confirmed that rsp3111 gene was successfully tranfered into the sperms in epididymis and into fertilized eggs. The results of fluorescent microscopy shows the specific expression ofrsp311 lgene both in IVF embryos and in convoluted seminiferous tubules of the testis. Conclusion Specific expression of rat gamete-specific protein rSP3111 was achieved through sperm-mediated trasnfection, which undermines the establisment of rsp3111 trangenic mouse through in vivo transfection of spermatogonial stem cells.

关 键 词：雄性生殖细胞载体法 特异性基因rsp3111 体外受精 

分 类 号：Q78[生物学—分子生物学]