【摘要】 目的:建立HSF1-/-,HSF1+/+两种基因型小鼠胚胎成纤维永生化细胞系,为HSF1的功能研究提供实验模型。方法:用脂质体介导的基因转染法将pSV 3 neo质粒导入HSF1-/-,HSF1+/+两种基因型小鼠胚胎成纤维细胞,经G 4 1 8筛选,抗性克隆扩大培养,建立永生化细胞系;用PCR检测两种细胞株中目的基因的整合,用RT-PCR法鉴定SV 4 0T基因在转染细胞中的表达;用W estern b lot检测所建细胞株的诱导型热休克蛋白7 0的表达情况。结果:有3个细胞克隆已扩大培养稳定传代达6个月,经鉴定SV 4 0 T抗原已整合到两种细胞中且稳定表达,HSF1-/-胚胎成纤维细胞热休克蛋白7 0的诱导表达消失。结论:成功建立永生化HSF1-/-,HSF1+/+两种基因型小鼠胚胎成纤维细胞。更多还原

【Abstract】 Objective To establish immortalized embryonic fibroblast lines in heat shock transcription factor 1（HSF1） HSF1^-/-and HSF1^+/+ mice and to provide experimental models to study the function of HSF1.Methods A mammalian expression vector（pSV3neo） containing the SV40 large T antigen was used to transfect the HSF1^-/- and HSF1^+/+mouse embryonic fibroblast using Lipofectamine^TM 2000.Colonies were screened by G418 and expanded to immortalized cell lines.PCR was used to detect the integration of the large T antigen with genome in the mouse embryonic fibroblast.Expression of SV40 large T antigen gene in expanded cells was identified by RT-PCR.HSP70 expression was examined by Western blot in the embryonic fibroblast lines.Results The stable growth and serial propagation were observed in the HSF1^-/- and HSF1^+/+ cell lines for six months.The mRNA of SV40 T antigen gene expressed in the two cell lines.HSP70 expression could not be induced in the heat-treated HSF1^-/-mouse embryo fibroblasts.Conclusion The immortalized cells of HSF1^+/+ and HSF1^-/- mouse embryo fibroblasts are successfully established.更多还原