Tools and Technologies I
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.
This application focuses on the development of improved methods for targeting specific loci in the genome of human embryonic stem cells (hESC). First, the applicants will prepare a series of constructs to target several housekeeping loci as well as various epigenetic regulators. Next, the applicants propose to explore various parameters for improving the efficiency and specificity of homologous recombination, such as inclusion of zinc finger nucleases (ZFN), manipulation of DNA enzymes, and variations in the homology arms of the constructs. Finally, the applicants propose to demonstrate the validity of the engineered lines that would emerge from this effort. The reviewers were enthusiastic about the proposed technology as it addresses an important deficiency in the field, a lack of robust and reliable methods for genetic modification of hESC. If successfully realized, the proposed tools could be enormously useful, benefiting a wide range of studies and improving the overall pace of discovery. The reviewers lauded the capabilities of the research team to conduct the described research, and found the preliminary data to be very convincing. Although the proposal was poorly written, the experimental design was straightforward and the milestones were clearly defined. Nonetheless, a number of weaknesses were noted that cast doubt on its overall feasibility. Of significant concern was the choice of hESC lines for gene targeting, especially because one of the proposed lines displayed karyotypic instability following previous targeting attempts by others. The applicants did not provide a rationale for selecting these lines. Moreover, the strategy for detecting off target DNA cleavage by ZFNs was considered to be inadequate. In their homology arm analysis, the applicants failed to address a potential drawback, homologous recombination between co-transfected fragments, which may confound their comparative analysis of gene targeting efficiencies. Reviewers further questioned the rationale for the choice of some of the non-isogenic controls. In addition to these conceptual deficiencies, the proposal was found to be lacking in experimental details, particularly with regard to the use of ZFNs and the manipulation of DNA enzymes, although the latter was noted to be an interesting aspect of the proposal. The applicants were described as very well qualified to conduct the proposed research. The principal investigator is an established, well-published scientist with a proven track record in mouse ESC modification. The reviewers were particularly impressed with the strength of the collaboration, a key asset that spoke to the merits of this effort. In summary, while the technology described in this proposal could be tremendously useful, the reviewers remained unconvinced of its potential to be realized.