小鼠BMPRⅠb基因慢病毒载体构建及转染神经干细胞

Construction of lentivirus vectors carrying BMPRⅠb genes and transfection of neural stem cells

目的:构建携带小鼠BMPRⅠb基因的慢病毒载体,并转染神经干细胞,为研究BM-PRⅠb在BMPs调控神经干细胞分化中作用奠定基础。方法:利用RT-PCR从小鼠脑组织中获得BMPRⅠb基因,然后定向插入慢病毒表达质粒,进行双酶切及测序鉴定。将鉴定成功的重组慢病毒质粒和另外两种辅助质粒共转染包装细胞,收集、浓缩慢病毒并检测其滴度。利用获得的慢病毒载体转染NSCs,并观察目的基因表达情况。结果:RT-PCR产物经电泳及测序证实克隆成功BM-PRⅠb基因,酶切及测序鉴定成功构建重组慢病毒质粒,共转染293T细胞72h后大部分细胞表达绿色荧光,病毒滴度为5×108 TU/ml。慢病毒感染后的NSCs表达绿色荧光且BMPRⅠb mRNA表达上升。结论:成功构建BMPRⅠb基因慢病毒载体,并成功转染NSCs。

Objective： To construct lentivirus vectors carring mouse Type Ⅰb bone morphogenetic protein receptor gene and transfect neural stem cells.Method： The gene fragment of BMPRⅠb was obtained by using rt-pcr method with total RNA extracted from mouse brain tissue.Gene recombinant technology was employed to chlone BMPR1b gene to lentivirus vector to construct a recombinant lentivirus plasmid.The plasmid was transfected respectively with two other plasmids into packaging cells.The lentiviral vector lentiCMV-BMPRⅠb/CMVeGFP were collected and tittered.The lentiviral vector lentiCMV-BMPRⅠb/CMVeGFP was used to infect NSCs.After infection,the expression of BMPR Ⅰb was indentified by using rt-pcr.Results： A 1600bp band was seen by rt-pcr amplification.Result： The gene sequencing result confirmed that BMPRⅠb gene was successfully cloned.The recombinant lentivirus plasmid was identified by double-digested with BamHI/XhoI.After transfection,a large number of 293T cells expressed green fluorescence.The concentration of virus titer was 5×108TU/ml.After infection,the expression of BMPR Ⅰb mRNA increased.Conclusion： mouse BMPR Ⅰb gene expression lentiviral vectors are successfully constructed and can transfect neural stem cells.