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Curr Protoc Stem Cell Biol. 2020 Mar;52(1):e102. doi: 10.1002/cpsc.102.

Efficient Modulation of TP53 Expression in Human Induced Pluripotent Stem Cells.

Author information

1
Department of Neurosurgery, University Hospital Düsseldorf, Düsseldorf, Germany.
2
Department of Pediatric Oncology, Hematology and Clinical Immunology, Medical Faculty, Center of Child and Adolescent Health, Heinrich-Heine University, Düsseldorf, Germany.
3
IUF-Leibniz Research Institute for Environmental Medicine, Düsseldorf, Germany.
4
German Cancer Consortium (DKTK), partner site Essen, Düsseldorf, Germany.

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.

KEYWORDS:

TP53; hiPSC; lentiviral transduction; molecular alteration

PMID:
31883435
DOI:
10.1002/cpsc.102

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