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BMPRⅠb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BMPRⅠb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5×108TU/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 earring mouse Type I b bone morphogenetic protein receptor gene and transfect neural stem cells. Method: The gene fragment of BMPR I b was obtained by using rt-pcr method with total RNA extracted from mouse brain tissue. Gene recombinant technology was employed to chlone BMPRlb 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- BMPR I b/CMVeGFP were collected and tittered. The lentiviral vector lentiCMV- BMPR I b/CMVeGFP was used to infect NSCs. After infection , the expression of BMPR I b was indentified by using rt-pcr. Results: A 1600bp band was seen by rt-pcr amplification. Resu The gene sequencing result confirmed that BMPR I b 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 5X 108TU/ml. After infection, the expression of BMPR I b mRNA increased. Conclusion: mouse BMPR I b gene expression lentiviral vectors are successfully constructed and can transfect neural stem cells.
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