Derivation of genetically modified human pluripotent stem cells with integrated transgenes at unique mapped genomic sites.

Overview

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.