Open AccessEfficient Modulation of TP53 Expression in Human Induced Pluripotent Stem Cells
Author(s) -
Uhlmann Constanze,
Kuhn LisaMaria,
Tigges Julia,
Fritsche Ellen,
Kahlert Ulf Dietrich
Publication year - 2020
Publication title -
current protocols in stem cell biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.658
H-Index - 28
eISSN - 1938-8969
pISSN - 1941-7322
DOI - 10.1002/cpsc.102
Subject(s) - biology , induced pluripotent stem cell , matrigel , stem cell , transfection , transduction (biophysics) , microbiology and biotechnology , cell culture , signal transduction , cancer research , gene , genetics , embryonic stem cell , biochemistry
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