Developing dopaminergic reporter hESC lines by homologous recombination
The ability of cells to continue to divide in prolonged culture allows researchers an unprecedented opportunity to introduce genes into the genome and select clones with the appropriate genetic modification. The development of a variety of vectors and strategies has allowed perhaps the most dramatic examples of this in non-human stem cell populations – the use of mouse embryonic stem cells (mESCs) to obtain truly remarkable modifications of the mouse genome. In principle, these various strategies could be adapted to human stem cells. The human stem cell field gained momentum when human embryonic stem cells (hESCs) were successfully isolated in 19981. hESCs present an unparalleled opportunity to fully benefit from the advantages of genetic manipulations in a controlled manner, in particular, gene targeting via homologous recombination. In theory, by developing techniques for gene targeting in hESCs, it is possible to analyze the function of any gene and to develop in vitro models for developmental studies or for the purification of cells of specific somatic lineages.
However, the techniques that have been successfully used in targeting mESCs have met unexpected challenges when adapted to hESCs, and to date, only three genes have been targeted in hESCs. One major difficulty in targeting hESCs is the complex DNA constructions required for homologous recombination. We believe that elaborate targeting vector construction is a prerequisite for successful gene targeting in hESCs. To generate such targeting vectors rapidly, more efficient cloning techniques are required. For example, instead of the traditional strategy of restriction digestion and ligation, which is often laborious and ineffective for cloning large DNA fragments required for homologous recombination, ET cloning approach via homologous recombination allows for direct isolation of human genomic DNA from BACs or yeast artificial chromosomes, thereby eliminating the step of prior isolation of large DNA fragments.
In this proposal, we will develop novel targeting vectors utilizing ET cloning directly from human BACs for homologous recombination in hESCs. In particular, we will develop a GFP knock-in approach to target key genes involved in dopaminergic differentiation for which we have a well-defined system for characterization. We plan to target three transcription factors that are essential for specification and maturation of dopaminergic neurons in mice.
The ability to target a specific gene in mouse embryonic stem cells via homologous recombination has revolutionized the study of mammalian gene function. Our proposed research will develop novel tool/technique for targeting human embryonic stem cells via homologous recombination. In particular, we will target genes that are know to play an important role in the development of dopaminergic neurons, neurons that are selectively and progressively degenerated in patients with Parkinson’s disease. Such targeted cells will be very useful for studying the roles of these genes in dopaminergic differentiation of hESCs which may provide insights into pathology and treatment of Parkinson’s disease as well as new therapy development. Therefore, the proposed research will also benefit California residents suffering from PD.