小鼠H1foo逆转录病毒载体的构建及表达

Construction and expression of retroviral vector pMX-H1foo in mouse embryonic fibroblast

【目的】构建小鼠pMX-H1foo逆转录病毒载体,并使其在小鼠胚胎成纤维细胞(MEF)中异位表达,为进一步研究H1foo在重编程方面的作用奠定理论基础。【方法】根据NCBI公布的小鼠H1foo基因的mRNA序列设计引物,以小鼠卵巢组织总RNA为模板,RT-PCR扩增小鼠H1foo CDS序列,并连接到逆转录病毒载体pMX上。经双酶切鉴定和测序比对正确后,利用磷酸钙转染包装细胞plat-E的方法制备H1foo病毒液并感染MEF细胞,进一步收集感染的细胞进行半定量RT-PCR和免疫荧光染色检测,分析其在细胞中的表达情况。【结果】克隆得到915bp的H1foo基因,构建得到逆转录病毒载体pMX-H1foo。该载体制备的病毒能够感染MEF细胞,并且H1foo在MEF细胞中与细胞核共定位,而对照组绿色荧光蛋白(GFP)遍布整个细胞。【结论】成功构建了pMX-H1foo逆转录病毒载体,并在MEF中进行表达。

【Objection】This study constructed and expressed recombinant retroviral plasmid pMXH1 foo in mouse embryonic fibroblast(MEF)cells to further investigate the function of H1 foo in the induced pluripotent stem cell.【Method】Primers of H1 foo were designed according to the published mRNA sequence of mouse oocyte-specific linker histone(H1foo:NM_138311.2)in NCBI database.The CDS of H1 foo was amplified from RNA of mouse ovary using RT-PCR and then cloned into the retroviral vector pMX.The recombinant retroviral plasmid was named as pMX-H1 foo after being confirmed by BamHⅠand SalⅠ double digestion and DNA sequencing.Then it was packaged in plat-E cells using the method of calcium phosphate transfection,and MEF was infected by the obtained virus after collecting viral supernatant.The expression of pMX-H1 foo was identified by RT-PCR and immunofluorescence.【Result】The H1 foo gene was amplified and the recombinant retroviral plasmid pMX-H1 foo was constructed successfully and infected into MEF cells.Semi-quantitative RT-PCR and immunofluorescence staining proved that H1 foo was expressed in the nuclei of MEF cells while GFP was located everywhere in MEF cells.【Conclusion】The construction and expression of recombinant retroviral plasmid pMX-H1 foo in MEF cells was success-ful in this study.