利用CRISPR-Cas9技术构建TLE1 S284点突变AML12细胞系
首发时间:2022-11-21
摘要:目的:用CRISPR-Cas9基因编辑技术,通过DNA同源重组修复将TLE1蛋白S284位点的密码子AGC(Ser)突变为GCC(Ala)/GAC(Asp),并研究TLE1 S284位点磷酸化的作用。 方法:首先在S284密码子位点附近设计sgRNA,构建切割载体,再将位点突变的模板重组至pL451质粒中,构建donor载体。其次将两种质粒与Cas9表达载体共转至AML12细胞系中,加入抗生素药筛后,挑取单克隆细胞,并通过测序、Western blot、qPCR进行验证后,表明细胞构建成功,最后通过RNA-seq分析TLE1 S284磷酸化参与的信号通路及生物学过程。 结果:成功构建了AML12 TLE1 S284A(无磷酸化)及AML12 TLE1 S284D(模拟磷酸化)两种点突变细胞系,并通过RNA-seq分析发现TLE1 S284可参与代谢、细胞增殖与分化、癌症、炎症、免疫、生物节律等过程。
关键词: CRISPR-Cas9 点突变 蛋白磷酸化 RNA-seq分析
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Construction of TLE1 S284 point mutation AML12 cell line by CRISPR-Cas9 technique
Abstract:Objective: Biallelic homo-mutation at TLE1\'s S284 in AML12 cell line was made to explore its function in phosphorylation. Methods: First, a cutting vector adopted the sequence of sgRNA around S284 site, and the donor vector was built in pL451 plasmid with the mutant template. Secondly, the vectors of cutting, donor and Cas9 were co-transfected into AML12 Cell line. The successful monoclonal cells were acquired through the anti-biotic screening and the verification of the sequencing, Western blot, and qPCR. Finally, RNA-seq was done for analysis of the signaling pathways and biological processes involved in TLE1 S284 phosphorylation. Results: Successfully constructed two AML12 cell lines with biallelic homo-mutation at TLE1 S284A (no phosphorylation) and AML12 TLE1 S284D (mimicking phosphorylation) respectively, and RNA-seq analysis revealed that TLE1 S284\'s potential implications in metabolism, cell proliferation and differentiation, cancer, inflammation, immunity , biological rhythms, etc.
Keywords: .CRISPR-Cas9 point mutation protein phosphorylation RNA-seq analysis
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利用CRISPR-Cas9技术构建TLE1 S284点突变AML12细胞系
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