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背景:建立具有抗药(新霉素或潮霉素)性的饲养层细胞是转染外源目的基因胚胎干细胞阳性克隆的筛选必备条件。建成的具有抗药性的NIH3T3细胞系可用于胚胎干细胞其他目的基因的筛选。目的:建立具有G418抗性的NIH3T3细胞系,用于转染pTet-on基因的胚胎干细胞阳性细胞克隆筛选的饲养层。设计:细胞培养观察及DNA检测。单位:中国医科大学实验动物部。材料:NIH3T3细胞由中国医科大学细胞生物教研室惠赠,pWL/neo质粒为美国哈佛大学医学院金壮教授惠赠。G418、白血病抑制因子(胚胎干细胞GRO106U/mL)、DMEM为GIBCOBRL产品,丝裂霉素-C、DIG标记及检测试剂盒为Roche产品,脂质体为Invitrogen产品,均购自沈阳联星生物技术有限公司。方法:①通过脂质体转染的方法,将含有neo基因的质粒pWL/neo导入NIH3T3细胞中。②将细胞传至25mL培养瓶中,加入梯度浓度的G418继续培养,通过观察细胞生长及死亡情况测定G418对NIH3T3细胞最小致死量。③将转染的细胞进行传代,挑取单个细胞的克隆至24孔培养板继续进行筛选扩增,选用正常的NIH3T3细胞加入相同剂量的G418的选择性培养基作为筛选的阴性对照。④采用较低的细胞密度铺板,加入含最小致死量G418的DMEM培养基进行再次筛选;同时选用正常的NIH3T3细胞为对照,使用含有最小致死量G418及未加G418的DMEM培养基进行培养,采用光学显微镜对G418抗性NIH3T3细胞进行观察。⑤选用具有G418抗性的NIH3T3细胞和小鼠胚胎成纤维细胞分别作为饲养层细胞进行传代,采用光学显微镜对培养后胚胎干细胞-D3细胞进行观察,并制备细胞DNA,对其进行PCR和southernblot鉴定。主要观察指标:①G418对NIH3T3细胞最小致死量的测定结果。②G418抗性NIH3T3细胞的筛选结果。③G418抗性NIH3T3细胞的生长观察结果。④胚胎干细胞-D3细胞生长观察结果及G418抗性NIH3T3细胞的鉴定。结果:①G418对NIH3T3细胞最小致死量为500mg/L。②经过500mg/LG418压力筛选后,获得了抗G418细胞克隆。③抗性NIH3T3细胞的形态和生长速度与正常NIH3T3细胞没有差异。④饲养层培养的胚胎干细胞呈集落形状生长,边缘光滑,保持未分化的状态。用特异性核苷酸引物检测抗性细胞基因组DNA,可以扩增出对应的核苷酸片段,Southernblot鉴定结果表明neo基因片段已整合入G418抗性NIH3T3细胞中。结论:成功地培育了G418抗性NIH3T3细胞,生长在G418抗性NIH3T3细胞饲养层上胚胎干细胞细胞基本保持正常胚胎干细胞细胞特征。
BACKGROUND: Establishing feeder cells with drug resistance (neomycin or hygromycin) is a prerequisite for selection of positive clones of embryonic stem cells transfected with exogenous gene of interest. The constructed drug-resistant NIH3T3 cell line can be used for the screening of other target genes of embryonic stem cells. OBJECTIVE: To establish a G418-resistant NIH3T3 cell line for selection of feeder layers of embryonic stem cell positive cell clones transfected with pTet-on gene. Design: Cell culture observation and DNA testing. Unit: Experimental Animal Department of China Medical University. MATERIALS: NIH3T3 cells were kindly donated by Department of Cell Biology, China Medical University, and pWL / neo plasmid was donated by Professor Jin Zhuang from Harvard Medical School. G418, leukemia inhibitory factor (embryonic stem cell GRO106U / mL), DMEM is GIBCOBRL product, mitomycin-C, DIG labeling and detection kit is Roche product and liposome is Invitrogen product, both purchased from Shenyang Lianxing Biotechnology Limited. Methods: ① The plasmid pWL / neo containing neo gene was introduced into NIH3T3 cells by lipofection method. ② The cells were transferred to 25mL culture flasks, adding gradient concentration of G418 continue to culture, by observing cell growth and death measured G418 NIH3T3 cells minimum lethal dose. (3) The transfected cells were passaged, picked single cell clones to 24-well culture plates to continue screening and amplification, selection of normal NIH3T3 cells to join the same dose of G418 selective medium as a screening negative control. ④ The cells were plated at a lower cell density, and DMEM medium containing the minimal lethal dose of G418 was selected for screening again. Meanwhile, normal NIH3T3 cells were used as a control and cultured with DMEM medium containing the minimal lethal dose G418 and without G418. G418-resistant NIH3T3 cells were observed under an optical microscope. ⑤ NIH3T3 cells with G418 resistance and mouse embryonic fibroblasts were selected as feeder cells for passage respectively. The cultured embryonic stem cells -D3 cells were observed under optical microscope and DNA was prepared. PCR and southern blot were performed on the cells. . MAIN OUTCOME MEASURES: ①The results of G418 assay on the minimum lethal dose of NIH3T3 cells. ② G418 resistant NIH3T3 cells screening results. ③ G418-resistant NIH3T3 cells observed the results of the growth. ④ Embryonic stem cell-D3 cell growth observation and identification of G418-resistant NIH3T3 cells. Results: ① The minimum lethal dose of G418 to NIH3T3 cells was 500mg / L. ② After 500mg / LG418 pressure screening, anti-G418 cell clone was obtained. ③ resistant NIH3T3 cell morphology and growth rate and normal NIH3T3 cells no difference. ④ feeder layer cultured embryonic stem cells were colony shape growth, smooth edges, remain undifferentiated state. Southern blot analysis showed that the neo gene fragment was integrated into G418-resistant NIH3T3 cells by using specific nucleotide primers to detect the resistant cell genomic DNA. CONCLUSION: G418-resistant NIH3T3 cells have been successfully cultivated and embryonic stem cells growing in the feeder layer of G418-resistant NIH3T3 cells have basically maintained normal characteristics of ES cells.