Development of a positive and negative drug selection system towards gene correction in medaka fish haploid ES cells

This project aimed to develop an improved positive-negative selection system for potential use in basic research and clinical applications. The NTR-CB1954 system was chosen, where introduced expression of the E. Coli derived gene product nitroreductase (NTR) confer sensitivity on cells to prodrug CB1954. Recent studies generated a human-codon optimized ntr sequence and introduction of 3 individual substitutions. The superiority to the original ntr gene in drug selection efficiency was demonstrated, highlighting the need and possibility to further improve the efficiency by enhancing NTR expression, activity and cellular distribution. My work consisted in optimizing the codon usage of the ntr gene for efficient expression in vertebrate cells and introducing 3 independent amino acid substitutions towards a higher activity. The obtained ntro3m was inserted in frame between puromycin aceltyltransferase (puromycin resistance) and rfp, leading to pCVpnr towards the constitutive expression of the fusion gene pnr from the cytomegalovirus (CV) promoter. Furthermore, the pGCtyr vector was constructed for targeted gene correction (GC) by homologous recombination (HR) in medaka haploid ES cells (HX1), where CVpnr is flanked by two genomic sequences of the medaka tyrosinase gene (tyr). The tyr gene will be a convincing control to prove that HR occurred by rescuing pigmentation as the vector will recombine in HX1 cells derived from the i1 albino Medaka strain. Preliminary tests showed that the pGNTRo and the pGNTR vector seemed efficient. Their effect could be observed after only 2 to 3 days. The CVpnr and pGCtyr are functional but their efficacies still have to be determined. In conclusion, this work has generated vector systems incorporating several advantageous modifications towards improved drug selection efficiency. Furthermore, the functionality and utility of these vectors have been evaluated by using stem cell cultures