Development of Optimal Approaches for Genomic Engineering of Human Stem Cells

Funding Type: 
Tools and Technologies I
Grant Number: 
ICOC Funds Committed: 
Public Abstract: 

This application describes the development of tools that will be helpful to many CIRM supported stem cell projects. While none of the methods are entirely novel, there are only sporadic reports describing the efficiency and specificity of human stem cell genome engineering. Genome engineering is important to introduce reporter genes to track or optimize differentiation. In addition, manipulation of gene expression through targeted mutagenesis of specific genes will provide a means for studying gene functin at key differentiation stages. The proposal represents a collaboration between three CIRM sites with complementing expertise: the laboratories of [REDACTED]) and (as consultant [REDACTED]. The [REDACTED] effort will mostly focus on vector construction and the subsequent optimization of homologous targeting procedures. The [REDACTED] lab will demonstrate the feasibility and utility of (conditional) bi-allelic knockouts of a set of master genes involved in epigenetic regulation of differentiation: the DNAMethylase genes DNMT1, DNMT3A and DNMT3B. [REDACTED] will provide advice and assistance with respect to “zincfinger nucleases” (ZFNs), as reagents to enhance homologous recombination.
At the end of this project we expect to have better defined the conditions for homologous recombination and streamlined procedures for gene targeting. We will also have demonstrated in hES cells the usefulness and feasibility of conditional knockouts for three DNA methylase genes. This newly gained technical knowledge is important: it will facilitate applications in other laboratories to create new cell lines, useful for pharmaceutical screening of new drugs. By being able to perturb the normal developmen of stem cell with precise mutations, scientists will learn about the regulating mechanism which control normal development.

Statement of Benefit to California: 

The state of California has one of the highest number of institutions, both private and public, working on stem cell research. A brief survey of previous and current CIRM-funded projects reveal much efforts in understanding the biology of stem cells, the mechanisms that determine pluripotency and differentiation into a particular lineage, and possible applications of stem cell technology in drug discovery, and regenerative medicine. Many such studies would benefit from having an efficient system to introduce transgenes or modify a particular cell's genotype.

The satisfactory gene targeting rates in mouse ESCs have resulted in a wealth of information on the function of genes. From some of these studies, candidate drugs have been identified and some of these have even reached clinical trials. Nevertheless, the intrinsic differences in cellular pathways between human and mouse still continues to limit the pace of drug discovery, and efforts to understand the changes that occur in the normal and disease state. Through the systematic study of the parameters that govern homologous recombination we aim to provide California researchers with reliable and powerful tools to modify the human ES cell genome. Moreover, the genetically modified hESCs generated by this study will be distributed to qualified California researchers for a wide range of applications.