Efficient Modulation of TP53 Expression in Human Induced Pluripotent Stem Cells

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