College of Life Science and Health, Wuhan University of Science and Technology, Wuhan 430065, China.
College of Life Science and Health, Wuhan University of Science and Technology, Wuhan 430065, China
Biosci Rep. 2019 May 2;39(5). doi: 10.1042/BSR20182383. Print 2019 May 31.
One of the conventional methods to study the effects of gene mutations is that gene mutants are transfected into mammalian cells, and the dominant effects of gene mutants in the cells are examined. However, the result obtained using this method is not always satisfactory due to the interference of endogenous expression. Whether there is a better method to investigate the effects of gene mutations in cells remains to be examined. In the present study, a novel dual expression lentiviral vector was constructed using a shRNA-expressing lentiviral vector and combined techniques. Using this dual expression system, the vectors expressing both transcription factor IIA γ (TFIIAγ) shRNA and HA-TFIIAγ or its mutants were generated, and the effects of gene mutations on transcription and protein-DNA interaction were investigated. We show that the transfection of the vector expressing TFIIAγ shRNA and fusion gene was able to silence the expression of endogenous gene but not affect that of exogenous fusion gene in either transiently transfected cells or stable cell lines. Mutations in the conservative domain between AA and AA in TFIIAγ inhibit the activities of promoters and endogenous gene expression, and reduce TFIIAγ binding to AdML core promoter compared with wild-type (WT) TFIIAγ. ChIP-qPCR data suggest that the TFIIAγ N63A mutant inhibits insulin-like growth factor 2 (IGF2) transcription by reducing the recruitments of TFIIAγ, polymerase II (Pol II), TATA box-binding protein (TBP), and TBP associated factor 1 (250 kDa) (TAF1) at its promoter. Our study provides a novel method that is used to investigate the effects of gene mutations at the cellular level.
研究基因突变影响的一种传统方法是将基因突变体转染到哺乳动物细胞中，并检查基因突变体在细胞中的显性效应。然而，由于内源性表达的干扰，使用这种方法获得的结果并不总是令人满意。是否有更好的方法来研究细胞中基因突变的影响仍有待检验。在本研究中，使用表达 shRNA 的慢病毒载体和组合技术构建了一种新型的双表达慢病毒载体。使用这种双表达系统，生成了表达转录因子 IIAγ (TFIIAγ) shRNA 和 HA-TFIIAγ 或其突变体的载体，并研究了基因突变对转录和蛋白-DNA 相互作用的影响。我们表明，转染表达 TFIIAγ shRNA 和融合基因的载体能够沉默内源性基因的表达，但不会影响瞬时转染细胞或稳定细胞系中外源融合基因的表达。TFIIAγ 中 AA 和 AA 之间保守结构域的突变抑制了启动子和内源性基因表达的活性，并且与野生型（WT）TFIIAγ 相比，TFIIAγ 与 AdML 核心启动子的结合减少。ChIP-qPCR 数据表明，TFIIAγ N63A 突变通过减少 TFIIAγ、聚合酶 II (Pol II)、TATA 框结合蛋白 (TBP) 和 TBP 相关因子 1（250 kDa）（TAF1）在其启动子上的募集来抑制胰岛素样生长因子 2 (IGF2) 转录。我们的研究提供了一种新的方法，用于在细胞水平上研究基因突变的影响。
