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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Validation-based insertional mutagenesis identifies lysine demethylase FBXL11 as a negative regulator of NFκB
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Validation-based insertional mutagenesis identifies lysine demethylase FBXL11 as a negative regulator of NFκB

机译：基于验证的插入诱变可确定赖氨酸脱甲基酶FBXL11为NFκB的负调节剂

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摘要

We describe a highly efficient use of lentiviral validation-based insertional mutagenesis (VBIM) to generate large populations of mammalian cells in which a strong promoter is inserted into many different genomic loci, causing greatly increased expression of downstream sequences. Many different selections or screens can follow, to isolate dominant mutant clones with a desired phenotypic change. The inserted promoter can be excised or silenced at will, to prove that the insertion caused the mutation. Cloning DNA flanking the insertion site identifies the locus precisely. VBIM virus particles are pseudotyped with VSV G protein, allowing efficient infection of most mammalian cell types, including non-dividing cells, and features are included that give high yields of stable virus stocks. In several different selections, useful mutants have been obtained at frequencies of approximately 10~(-6) or higher. We used the VBIM technique to isolate mutant human cells in which the F-box leucine-rich protein 11 (FBXL11), a histone H3K36 demethylase, is shown to be a negative regulator of NFkB. High levels of FBXL11 block the ability of NFkB to bind to DNA or activate gene expression, and siRNA-mediated reduction of FBXL11 expression has the opposite effects. The H212A mutation of FBXL11 abolishes both its histone H3K36 demethylase activity and its ability to inhibit NFkB. Thus, we have used a powerful tool for mutagenesis of mammalian cells to reveal an aspect of the complex regulation of NFKB-dependent signaling.
机译：我们描述了基于慢病毒验证的基于插入诱变（VBIM）的高效使用，以生成大量的哺乳动物细胞，其中强启动子插入许多不同的基因组基因座，导致下游序列的表达大大增加。随后可以进行许多不同的选择或筛选，以分离具有所需表型改变的显性突变体克隆。插入的启动子可以随意切除或沉默，以证明插入引起了突变。插入位点侧翼的克隆DNA可以精确识别基因座。 VBIM病毒颗粒是用VSV G蛋白假型化的，可以有效感染大多数哺乳动物细胞类型，包括非分裂细胞，并且所包含的功能可以稳定地产生高产量的病毒原种。在几种不同的选择中，已经获得了有用的突变体，其频率大约为10〜（-6）或更高。我们使用VBIM技术分离突变的人类细胞，其中富含F-box的亮氨酸蛋白11（FBXL11）（组蛋白H3K36脱甲基酶）被证明是NFkB的负调控因子。高水平的FBXL11会阻断NFkB与DNA结合或激活基因表达的能力，而siRNA介导的FBXL11表达的降低则具有相反的作用。 FBXL11的H212A突变消除了其组蛋白H3K36脱甲基酶活性和其抑制NFkB的能力。因此，我们已经使用了一种强大的工具来诱变哺乳动物细胞，以揭示NFKB依赖性信号传导复杂调控的一个方面。

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    Tao Lu; Mark W. Jackson; Aatur D. Singhi; Eugene S. Kandel; Maojing Yang; Yi Zhang; Andrei V. Gudkov; George R. Stark;

  • 作者单位

    Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195;

    Department of Pathology, Case Western Reserve University, Cleveland, OH 44106;

    Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195;

    Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, NY 14263;

    Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195;

    Howard Hughes Medical Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599;

    Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, NY 14263;

    Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
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  • 关键词

    cre recombinase; lentivirus; promoter insertion;

    机译：cre重组酶;慢病毒启动子插入;

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