小鼠T-bet逆转录病毒载体的构建与功能验证

Construction and characterization of a novel recombinant retroviral vector expressing mouse T-bet

摘要:

为在小鼠细胞中表达并研究T-bet功能，首先构建了含有报告基因Thy1.1的小鼠T-bet逆转录病毒载体，并将构建的载体质粒转染病毒包装细胞系包装成重组病毒，再利用重组病毒分别感染NIH-3T3和D9细胞系检测其感染能力。之后，使用该重组病毒感染T-bet敲除小鼠的CD4+ T淋巴细胞，流式细胞术检测T-bet及其下游靶基因Ifng的表达情况。经验证，重组的逆转录病毒感染T-bet敲除小鼠T淋巴细胞后可以在细胞中表达T-bet，并进一步引起下游靶基因Ifng的表达上调，证明本研究中外源表达的转录因子T-bet具有正常功能。综上所述，本实验成功构建了含有报告基因的小鼠T-bet重组逆转录病毒载体，为进一步在小鼠细胞中研究T-bet功能奠定了基础。

Abstract:

In order to study T-bet function in mouse cells, a novel retroviral vector expressing mouse T-bet and reporter gene Thy1.1 was constructed. Retrovirus particles were then produced by transfection of the recombinant retroviral plasmid into a packaging cell line Platinum-E. The recombinant retrovirus played considerable infection ability. T-bet expression was then identified by FACS after infection of CD4+ primary T cells from T-bet knockout mouse with recombinant retrovirus. To determine if exogenous expressing T-bet has normal function, we checked the expression level of T-bet target gene, Ifng. IFN-γ expression was upregulated in the T-bet knockout T cells infected with recombinant retrovirus. In conclusion, we successfully constructed an effective mouse T-bet recombinant retroviral vector.