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A Special Issue on 5th Annual Meeting of GSZ.
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Vol. VIII Issue: 2 (Special Issue) : (JSRM Code: 008020700061)

Efficient ZFN-based gene targeting in transgenic human iPS cells as a model for gene editing in patient-specific cells
S. Merkert1, K. Kahn2, K. Schwanke1, J. Meyer2, T. Cathomen2, U. Martin1
(PGSSCR 2011-055)

1 Hannover Medical School, LEBAO, Hannover, Germany,
2 Hannover Medical School, Experimental Hematology, Hannover, Germany

Gene targeting by homologous recombination via customized zinc-finger nucleases (ZFN) is a powerful method to manipulate the genome and correct genetic defects. Although efficiency of ZFN based homologous recombination has been shown to be significantly higher than by means of conventional gene targeting, the selection of suitable clones still requires cells that proliferate in culture. Clinically applicable ZFN-based gene correction in patient-specific cells was hardly possible so far, due to the inability to sufficiently expand most adult (stem and progenitor) cells in vitro. However, the availability of human induced pluripotent stem (hiPS) cells with their almost unlimited potential for proliferation and differentiation now offers novel opportunities for the development of patient-specific regenerative therapies.

As a first step towards ZFN-based gene targeting, a non-viral gene-transfer in human iPS cells with transfection rates of up to 80% and high cell vitality was established. Aiming at the development of a general ZFN-based recombination approach, transgenic human iPS cell clones stably expressing eGFP under an ubiquitous promoter were generated to investigate the functionality of an eGFP specific ZFN. For proof of principle we initially knocked out the eGFP via non-homologous end joining and achieved up to 3% eGFPneg cells. For the establishment of a general applicable protocol for ZFN based site-specific recombination an appropriate eGFP targeting vector encoding for RedStar was generated. Successful gene targeting resulted in eGFPneg RedStarpos human iPS cells and could be achieved with an efficiency of up to 1%. Hence we generated eGFPneg RedStarpos human iPS cell clones. So far these clones show stable RedStar expression for up to 20 passages and could be verified for specific integration via PCR analysis.

Ultimately, the development of a generally applicable protocol for ZFN based site-specific recombination and gene correction in patient-specific hiPS cells may enable the development of cellular therapies for various genetic diseases.

** 6th Annual Meeting of the Germany Society for Stem Cell Research

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