PLEKHQ1基因敲除小鼠胚胎成纤维细胞永生化细胞系的建立

Establishment of immortalized cell line of mouse embryonic fibroblasts derived from PLEKHQ1 gene knockout mice

目的:建立PLEKHQ1基因敲除小鼠胚胎成纤维细胞(MEF)永生化细胞系,为全面研究PLEKHQ1基因功能提供细胞实验材料。方法:采用慢病毒感染的方法将猿猴病毒40(SV40)大T抗原基因导入已鉴定出基因型的MEF中,建立永生化细胞系;利用聚合酶链反应(PCR)检测SV40大T抗原基因在MEF中的整合情况,利用反转录PCR及凝胶成像的方法观察SV40大T抗原基因在MEF中的表达。结果:SV40大T抗原基因已整合至MEF中,且此类MEF已扩大培养及稳定传代半年之久。结论:成功建立的PLEKHQ1基因敲除MEF永生化细胞系,可为进一步研究PLEKHQ1基因功能提供细胞实验材料。

Objective:To establish an immortalized mouse embryonic fibroblast(MEF)cell line of PLEKHQ1gene knockout mice in order to comprehensively research function of PLEKHQ1gene and provide cell experiment materials.Methods:The method of slow virus infection was adopted to transfect gene of simian virus40(SV40)large T antigen into MEF cell which have been identified genotype to establish an immortalized cell lines.Polymerase chain reaction(PCR)was used to detect the integration of the genome of SV40large T antigen in the mice embryonic fibroblast cell.Expression of SV40large T antigen gene in MEF cell was identified by reverse transcription PCR and gel imaging.Results:The mRNA of SV40T antigen gene has been integrated in cells of the immortalized cell line,and through observation,stable growth and serial propagation of the immortalized MEF cell have achieved for half the year.Conclusion:Successful establishment of the PLEKHQ1knockout immortalized MEF cell line can provide cell experimental material for further study of PLEKHQ1function.