人抗药基因在小鼠ES细胞中的表达及嵌合体小鼠的研究  

EXPRESSION OF HUMAN MULTIDRUG RESISTANCE GENE(mdr 1)cDNA IN MURINE ES CELLS AND IN CHIMERIC MICE

作　　者：丛笑倩[1] 李秀兰[1] 徐洁 沈鼎武 姚錱 

机构地区：[1]中国科学院上海细胞生物学研究所

出　　处：《实验生物学报》1993年第4期429-439,共11页Acta Biologiae Experimentalis Sinica

基　　金：国家自然科学基金

摘　　要：本文用磷酸钙沉淀法将含人mdrl 基因表达质粒(pHaMDR 1/A)转染到小鼠胚胎干细胞(ES-5),得到了四个能稳定地在含有200 ng/ml 秋水仙素培液中生长传代的细胞克隆。经Southern 印迹杂交及RNA 印迹杂交证明人mdrl 基因已整合到ES 细胞基因组,并有表达。其中两个克隆杂交信号较强,抗药基因可随秋水仙素浓度提高而扩增,表达量也随之增加。用抗该基因编码的p170糖蛋白抗体对ES-mdr 1细胞作免疫荧光染色,证实p170蛋白分布于细胞表面。未发现ES-mdr 1细胞的体内、外发育潜能与亲代细胞的有何差异。但是mdr1细胞不能被RA 和HMBA 化学药物诱导分化,表明这些细胞已与亲代ES-5细胞不同,具有拮抗化学药物诱导分化的作用,其机理尚待研究。因此,ES-mdr1细胞可作为在细胞水平研究p170糖蛋白作用机理的体系,也可用以建立体外筛选拮抗抗约基因作用新手段的模型。我们也得到了含人mdr1基因的嵌合小鼠,并对它们的嵌合情况作了分析。Human multidrug resistance gene(md-rl)was introduced into mouse embryonicstem cells(ES-5 line)by calcium phosp-hate mediated transfection,and transfectedES-5 cells were then s elected by stepwiseincrease in colchicine concentration(30,50,100,and 200ng/ml respectively).Fi-nally,we obtained 4 clones mat could bestably grown in culture medium with col-chicine at 200ng/ml and designated asES-mdrl clones A,B,C,and D.Southernblot analysis of DNA from ES-mdr 1 Aand D cells digested by Hind Ⅲ and hyb-ridized with mdrl cDNA 5 A probe wasshown in Fig.3.Characteristic 4.8 and2.4kb fragments of mdrl gene were foundas expected and their amplification underincreased concentration of colchicine in cu-lture medium was also evident from thefigure.Slot blot and Northern analysis oftotal RNA and poly A^+ RNA extractedfrom ES-mdrl cells were shown in Fig.4and 5,demonstrating that ES-mdrl cellscould express mdrl mRNA.Indirect immu-nofluorescence analysis with antibodiesagainst p170 glycoprotein indicated thatP170 protein translated from mdrl mRNAwas present at the surface of ES-mdrlcells(Plate Ⅰ,Fig.2).The biologicalcharacteristics of ES-mdrl cells cultured inmedium containing 200ng/ml colchicinewere investigated.The cells maintainedtheir undifferentiated morphology and grewin nests(Plate Ⅰ,Fig.1),like the pa-rental ES-5 cells.When ES-mdrl cellswere cultured in suspension in vitro,thesecells were still capable of producing simpleand cystic embryoid bodies.ES-mdrl cellsinjected subcutaneously into 129 mice for-med tumor-like outgrowths giving a greatvariety of cell types(Plate Ⅰ,Fig.4).These results indicated that the integrationand expression of human mdrl gene andselection against colchicine did not affectthe pluripotency of ES-mdrl cells both invitro and in vivo.However,ES-mdrl cel-ls,unlike their parental ES-5 cells,couldno longer be induced to differentiate byeither RA or HMBA(Plate Ⅰ,Figs.3a,3b),indicating that the human mdrl genetransfected ES cells had changed their com-petence of inducible response to differentia-ti

关 键 词：人抗药基因 胚胎干细胞 嵌合体鼠 

分 类 号：R－332[医药卫生]