Gene Targeting in Human Pluripotent Cells

Abstract

Mouse embryonic stem cells (mESCs) have the ability to differentiate into any cell type and can generate chimeric mice when transplanted into a host blastocyst. This remarkable potential, together with the development of robust gene targeting strategies in mESCs, were essential for establishing the mouse as the most widely used model organism in biomedical research. Recent advances have allowed the isolation of human embryonic stem cells and the derivation of induced pluripotent stem cells. Genetic tools similar to those proven routine in the mouse system are needed to realize the full potential of human pluripotent cells as disease models and putative therapeutics. Gene targeting in human cells, however, has proven to be more difficult, more time-consuming, and less robust than in mESCs. In this chapter, we discuss the strategies that have been used to allow specific genetic modifications in human pluripotent cells. We focus on the novel application of custom-engineered zinc-finger nucleases for gene targeting, which has promise to become a robust tool for efficient genetic manipulation of human pluripotent cells.

Genetic and molecular identification of three human TPP1 functions in telomerase action: recruitment, activation, and homeostasis set point regulation

“…To conduct a genetic analysis of human TPP1 requirements for telomerase action, we used genome-editing technologies that we previously established for the efficient genetic manipulation of human pluripotent stem cells (Hockemeyer and Jaenisch 2010;Hockemeyer et al 2009Hockemeyer et al , 2011. Specifically, we used zinc finger nucleases (ZFNs) that target the second exon of TPP1 in combination with a donor plasmid to facilitate homology-directed repair of the endogenous TPP1 locus in WIBR#3 hESCs.…”

Advances in the Development of Gene-Targeting Vectors to Increase the Efficiency of Genetic Modification

“…9 kb or more in length, as reported by other groups. 37,38) Once the targeting strategy has been decided, the next step is vector construction. Following PCR amplification of each homology arm, targeting vectors may be constructed by standard molecular biology techniques utilizing restriction enzymes and DNA ligase.…”

Endogenous Telomerase Reverse Transcriptase N-Terminal Tagging Affects Human Telomerase Function at Telomeres In Vivo

“…With the advent of genome editing in human pluripotent stem cells (reviewed in reference 37), an experimental system became available that can overcome these challenges. Robust protocols to genetically modify human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) (37)(38)(39)(40)(41), collectively referred to as hPSCs, have recently become available. With these technical developments, epitope tags or fluorescent reporter genes can now be inserted into the hPSC genome to endogenously mark cells for imaging or biochemical purification.…”