title: Derivation of genetically modified human pluripotent stem cells with integrated transgenes at unique mapped genomic sites.
abstract: Many applications in human pluripotent stem cell (PSC) research require the genetic modification of PSCs to express a transgene in a stable and dependable manner. Random transgene integration commonly results in unpredictable and heterogeneous expression. We describe a protocol for the derivation of clonal populations of human embryonic stem cells or induced pluripotent stem cells (iPSCs) expressing a transgene from a single copy of an integrated lentiviral vector that is mapped to the genome. Using optimized transduction conditions, followed by single-cell subcloning and a round of antibiotic selection, we find that approximately half of the colonies retrieved contain a single vector copy. After expansion, the majority of these are confirmed to be clonal. The vector/genomic DNA junction is sequenced and the unique integration site is mapped to the genome. This protocol enables the efficient derivation of genetically modified PSCs containing an integrated transgene at a known genomic site in ∼7 weeks.
authors: Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA. papapete@mskcc.org
authors: Eirini P
authors: EP
authors: Papapetrou
authors: Michel
authors: M
authors: Sadelain
mesh_terms: D001483:Base Sequence; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002460:Cell Line; D002874:Chromosome Mapping; D053595:Embryonic Stem Cells / Q000166:cytology*; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D015894:Genome, Human; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D016086:Lentivirus / Q000235:genetics; D008969:Molecular Sequence Data; D014161:Transduction, Genetic / Q000379:methods; D019076:Transgenes; D014776:Virus Cultivation