敲除β珠蛋白β^(maj)及β^(min)基因的C129小鼠ES-D3细胞系建立

Construction and selection of β^(maj) & β^(min) of β-globin gene knock-out in ES-D3 lines

目的通过构建C129系小鼠β珠蛋白基因内源性珠蛋白βmaj及βmin基因打靶载体,建立C129小鼠敲除β珠蛋白基因内源性珠蛋白βmaj及βmin基因ES-D3细胞系,为制作基因敲除嵌合体小鼠提供稳定的胚胎干细胞系。方法根据C129系小鼠β珠蛋白基因分子结构基础,用基因克隆方法在βmaj和及βmin两个同源序列两侧分别将1.7kb和7.0kb的片段插入pNTK质粒中,构建置换型打靶载体β-pNTK,用新霉素抗性基因(Neo)和单疱病毒腺苷激酶(TK)抗性基因作为正负筛选标记。用SalⅠ酶切将打靶载体β-pNTK线性化,采用电穿孔法在ES-D3细胞水平进行打靶,通过G418和GAN进行正负筛选,获得生长良好的胚胎干细胞克隆;用PCR和Southern印迹杂交方法鉴定阳性克隆基因组DNA整合结果,用碱性磷酸酶实验、体外胚体形成实验以及畸胎瘤成瘤实验鉴定细胞生物学特性。结果构建了C129系小鼠置换型打靶载体,经Neo-Major鉴定引物PCR扩增、BamHⅠ及EcoRⅠ酶切及部分测序分析,所插入方向正确,结构合理,达到所预期的目的;获得了8个阳性克隆珠,阳性克隆细胞株经PCR和South-ern印迹杂交方法鉴定外源基因整合到ES细胞基因组DNA中;经胚胎干细胞特性鉴定所获细胞具有良好的未分化特性和多能性。结论构建了C129系小鼠β珠蛋白基因内源性珠蛋白βmaj及βmin基因置换型打靶载体;该载体在胚胎干细胞水平进行基因打靶后,获得了稳定的敲除β珠蛋白内源性珠蛋白βmaj及βmin基因的胚胎干细胞系,为制作基因敲除嵌合体小鼠奠定了基础。

AIM： To construct a knock - out vector for β^maj ＆ β^min gene of β - globin gene and establish embryonic stem cell lines by gene knock - out in ES level for generating gene knock - out animal model. METHODS： Two homologous sequences 1.7 kb and 7.0 kb were inserted into basic plasmic vector pNTK, which contains positive and negative selection system of the neomycin resistance （ Neo ） and herpes simplex virus thymidine kinase （TK） resistant gene to construct a gene knock- out recombinant plasmic vector nominated β - pNTK, which was certified by polymerase chain reaction, enzyme digested and DNA sequencing. The β - pNTK that contains two homologous sequence was linearized with Sal 1 and introduced into the D3 line of ES cells by electroporation. Positive cloning of ES cells was selected with positive and negative selection system of G418 600 mg/L and 2.5 mol/L Gancyclovir for four weeks. ES cells clone were picked, explanded and certified by polymerase chain reaction, Southern blotting. Examination of the undifferentiated state and pluripotential characteristics of the cell lines were made with the alkaline phosphatase （AKP） staining and embryoid bodies formation test and teratomas formation in nude mice. RESULTS： A knock- out recombinant plasmic vector for β^maj ＆ β^min gene of β - globin gene, β- pNTK, was constructed in C129 mouse, which contains 8.7 kb length inserted homologous sequence and deleted most part of theβ^maj ＆ β^min gene. 8 strains of positive ES cells cloning were obtained, all of them were testified positive with polymerase chain reaction and Southern blotting. The results of AKP staining, embryoid bodies formation in vitro and teratomas formation in nude mice showed undifferentiated state and phiripotential characteristic of cell line. CONCLUSIONS： A knock - out recombinant plasmic vector of β^maj ＆ β^min of β - globin gene in C129 mouse was constructed, in which a knock - out β^maj ＆ β^min gene of β- globin gene ES - D3 cell line was established by electroporation, characterization of these cells show a prospect utility in producing gene knock - out mice in blastocysts stages.