人乳铁蛋白乳腺特异性表达载体的构建及其功能的验证

Construction of mammary gland specific vector for expression of human lactoferrin

构件pbCNCS/LF36经微注射获得转基因小鼠,同时为了准确筛选出整合的单克隆细胞,增加1个Cre/LoxP-GFP-Neo系统构建成pAPLM,将其转染山羊胎儿成纤维细胞筛选阳性细胞。结果表明:小鼠受体妊娠率28.5%,产仔32个,PCR整合检测4只阳性(3♀、1♂,整合率12.5%)。构件产生的3只母鼠和4号后代母鼠乳汁中重组人乳铁蛋白(hLF)用ELISA方法检测,表达量分别为3.8、2.8、0.9mg.mL-1,4号后代3只母鼠表达量分别为4.2、3.9、3.6mg.mL-1。构件pAPLM片段转染细胞后,经G418筛选,挑取单克隆扩大培养,其中8株PCR阳性克隆细胞荧光显微镜观察100%都有GFP的表达。以上结果证实,双启动子能调控外源基因在乳腺中特异高水平表达hLF,双标记基因可提高筛选阳性供核细胞的准确性,这为进一步作体细胞转基因克隆奠定基础。

To screen transgenic donor cells for nuclear transfer,the cassette of LoxP-cre-GFP-Neo was ligated to 3′ end SV40 polyA sequence of pbCNCS/LF36 thus producing pAPLM.Fertilized mouse eggs（ICR） were microinjected with the expression cassette of pbCNCS/LF36 and transferred into pseudo-pregnant females.The pregnancy rate of pseudopregnancy mices were 28.5% and,32 and 43 founder mice were born respectively.4（3♀,1♂） transgenic mice were positive checked by PCR and the integration rate were 12.5%.F_1 mice from male were not transgenic.The expression level of hLF were 3.8,2.8,0.9 mg·mL-1in the G0 mice and 4.2,3.9,3.6 mg·mL-1 in the milk of 3 F_1 mice of 4number G0 mice checked by ELISA.Fetal fibroblast cells were transfected with mammary gland specifical expression vector of pAPLM and then screened with G418.The single colony was selected and expanded.Genomic DNA were analysed by PCR.8 lines of fetal fibroblast cells expressed GFP and these transgenic cells would be used to produce hLF transgenic goat.These results indicated that milk promoter combined with CMV could direct high expression of hLF cDNA specifically to the mammary gland in the transgenic mice and the dual selectable marker genes of pAPLM could be used to produce transngenic donor cells for nuclear transfer.