GAPTrap: a novel gene expression system for pluripotent stem cells and their differentiated derivatives

© 2017 Dr. Ming-Hui (Tim) KaoHuman induced pluripotent stem cells (iPSCs or iPS cells) are derived by the in vitro reprogramming of somatic cells and resemble embryonic stem cells (hESCs) derived from the inner cell mass of the pre-implantation blastocyst stage embryo. In vitro, both iPSCs and hESCs can be directed to differentiate towards specific lineages and therefore represent a tractable platform to study important events during the specification of early human cell types. The ability to reliably express either fluorescent reporters or other genes of interest is critical for using pluripotent stem cells to investigate cellular differentiation pathways and gene function. We designed a simple expression system in which genes of interest can be inserted into the ubiquitously expressed GAPDH locus. In this system, transgenes are fused in frame with GAPDH via a T2A linker sequence that results in the generation of two separate proteins from a single GAPDH mRNA. Some versions of these vectors contain an additional element that enables targeted clones to be selected using antibiotic resistance. Independent PSC clones harbouring variations of this vector, which we have named GAPTrap, express remarkably consistent levels of the associated reporter genes. In vitro differentiation experiments, which generated cells representing the three embryonic germ layers, mesoderm, endoderm and ectoderm, showed that GAPTrap transgene expression remained consistent in a number of different cell types, including cardiomyocytes, blood cells and neurons. In addition, analysis of teratomas derived from iPSCs harbouring a GAPTrap-LacZ transgene showed that beta-galactosidase expression was maintained in a variety of differentiated cell types. Examination of cells containing a single allele insertion of the GAPTrap vector revealed that GAPDH activity was not reduced in undifferentiated transgenic PSCs. Nevertheless, western blot analysis showed that particular vector configurations adversely affected the level of GAPDH expression derived from the modified allele. Using this information, we redesigned the vectors to increase the level of transgene expression and of the upstream GAPDH protein. These new configurations subsequently enabled the isolation of doubly targeted cell lines: PSCs in which both alleles of GAPDH contained a different GAPTrap vector. The ability to independently target both GAPDH alleles provides further flexibility in designing more sophisticated experiments requiring multiple elements. Indeed, we used this information to create iPSCs harbouring two GAPTrap vectors encoding BCL-2 and MYC, each with independent regulatory elements to enable either protein to be activated in controllable manner. In summary, the GAPTrap vectors are a promising and straightforward system for expressing genes of interest in pluripotent stem cells and their differentiating derivatives