Efficient Modulation of TP53 Expression in Human Induced Pluripotent Stem Cells

Q2 Biochemistry, Genetics and Molecular Biology

Current Protocols in Stem Cell Biology Pub Date : 2019-12-28 DOI:10.1002/cpsc.102

Constanze Uhlmann, Lisa-Maria Kuhn, Julia Tigges, Ellen Fritsche, Ulf Dietrich Kahlert

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引用次数: 4

Abstract

TP53 point mutations are found in 50% of all cancers and seem to play an important role in cancer pathogenesis. Thus, human induced pluripotent stem cells (hiPSCs) overexpressing mutant TP53 are a valuable tool for the generation of in vitro models of cancer stem cells or for in vivo xenograft models. Here, we describe a protocol for the alteration of gene expression in hiPSCs via overexpression of a mutant form of the TP53 (R249S) gene using lentiviral transduction. A high amount of TP53 protein is detected 1 week after transduction and antibiotic selection. Differentiation of transduced hiPSCs gives insight into better understanding cancer formation in different tissues and may be a useful tool for genetic or pharmacologic screening assays. © 2019 The Authors.

Basic Protocol 1: Production and concentration of third-generation lentivirus

Support Protocol 1: Cloning of gene of interest into modulation vector

Support Protocol 2: Preparation of DMEM GlutaMAX™ with 10% fetal bovine serum and 1% penicillin-streptomycin

Basic Protocol 2: Transduction of human induced pluripotent stem cells and selection of positively transfected cells

Support Protocol 3: Preparation of Matrigel^®-coated plates

Support Protocol 4: Preparation of mTeSR™1 medium

Abstract Image

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人诱导多能干细胞中TP53表达的有效调控

TP53点突变在50%的癌症中发现，似乎在癌症发病中起重要作用。因此，过表达突变体TP53的人诱导多能干细胞(hiPSCs)是生成体外癌症干细胞模型或体内异种移植模型的有价值的工具。在这里，我们描述了一种通过慢病毒转导过表达TP53 (R249S)基因突变形式来改变hiPSCs中基因表达的方案。在转导和选择抗生素后1周检测到大量TP53蛋白。诱导hipsc的分化有助于更好地了解不同组织中癌症的形成，并可能成为遗传或药理学筛选分析的有用工具。©2019作者。基本方案1:第三代慢病毒的生产和浓缩支持方案1:将感兴趣的基因克隆到调制载体上支持方案2:用10%胎牛血清和1%青霉素-链霉素制备DMEM GlutaMAX™基本方案2:人诱导多能干细胞的转导和阳性转染细胞的选择支持方案3:制备Matrigel®包被板支持方案4:制备mTeSR™1培养基

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Current Protocols in Stem Cell Biology Biochemistry, Genetics and Molecular Biology-Cell Biology

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期刊介绍： Published in affiliation with the International Society for Stem Cell Research (ISSCR), Current Protocols in Stem Cell Biology (CPSC) covers the most fundamental protocols and methods in the rapidly growing field of stem cell biology. Updated monthly, CPSC will constantly evolve with thelatest developments and breakthroughs in the field. Drawing on the expertise of leading researchers from around the world, Current Protocols in Stem Cell Biology includes methods and insights that will enhance the progress of global research.