GAPTrap: A Simple Expression System for Pluripotent Stem Cells and Their Derivatives

The ability to reliably express fluorescent reporters or other genes of interest is important for using human pluripotent stem cells (hPSCs) as a platform for investigating cell fates and gene function. We describe a simple expression system, designated GAPTrap (GT), in which reporter genes, including GFP, mCherry, mTagBFP2, luc2, Gluc, and lacZ are inserted into the GAPDH locus in hPSCs. Independent clones harboring variations of the GT vectors expressed remarkably consistent levels of the reporter gene. Differentiation experiments showed that reporter expression was reliably maintained in hematopoietic cells, cardiac mesoderm, definitive endoderm, and ventral midbrain dopaminergic neurons. Similarly, analysis of teratomas derived from GT-lacZ hPSCs showed that β-galactosidase expression was maintained in a spectrum of cell types representing derivatives of the three germ layers. Thus, the GAPTrap vectors represent a robust and straightforward tagging system that enables indelible labeling of PSCs and their differentiated derivatives. [Display omitted] •GAPTrap vector system targets transgenes to the ubiquitously expressed GAPDH locus•Targeting transgenes to the GAPDH locus yields reliable transgene expression•Transgenes at this locus are robustly expressed in differentiated cells•Generation of GAPTrap targeted human PSC lines is simple and efficient In this article Stanley, Elefanty, and colleagues describe a simple vector system, GAPTrap, for constitutively expressing transgenes in pluripotent stem cells and their differentiated derivatives. GAPTrap vectors target transgenes to the GAPDH locus, ensuring robust and reliable expression in a wide variety of cell types, and providing an ideal base for cell tagging and gene overexpression experiments.