Gene targeting refers to the practice of introducing genetic modifications to cells with the goal to inactivate a specific gene or alter its biochemical properties. Over the last twenty years, this has been a major approach that investigators use to delineate in vivo function of genes in the mouse, the mammalian genetic model organism. Unfortunately, gene targeting technologies for human ES cells are still immature. Here we propose to develop and optimize the method for gene targeting in several widely used human ES cell lines. We will compare a conventional method routinely used for gene targeting in mouse ES cells and an recombinant Adeno-Associated Virus (rAAV)-based method that is increasingly being used for gene targeting in human somatic cell but not yet in human ES cells. We will first compare the time requirement, experimental complexity and efficiency of these two methods in knocking out one test gene in five human ES cell lines. We will experiment with various parameters in an attempt to optimize stable transfection and gene targeting frequency across different human ES cell lines. Once we identify the best protocol for gene targeting, we will apply the method to targeting of eight genes in human ES cells, to determine the robustness and generality of our gene targeting method. Successful completion of this project will lead to a robust and generally applicable gene targeting method for human ES cells. Such a tool will be critical for understanding the functional roles of various genes in ES cell proliferation and differentiation. It will also greatly facilitate the development of ES cell based therapeutics by, for example, enabling the repairing of faulty genes in patient specific ES cells or induced pluripotent stem (iPS) cells.
The overall aim of this application is to develop a robust and generally applicable gene targeting method for use in human ES cells. Development of such technology is highly significant if we are to achieve the full potential of human ES cells as a tool for both therapeutic development and basic research. It will enable the repairing of faulty genes in patient specific ES cells or induced pluripotent stem (iPS) cells, thereby speeding up the development of ES cell based therapeutic strategies. Further, the ability to inactivate or modify gene function in human ES cells will permit scientists to define roles of genes in self-renewal or differentiation of these cells, which is crucial for a clear understanding of the ES cell biology and ultimately developing better diagnostics and therapeutic tools.