Site-Specific Genome Engineering in Mouse Primary Fibroblasts
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Site-specific genome engineering is a powerful tool for medical therapeutics and basic scientific research. As the name implies, site-specific genome engineering describes the ability to add or subtract nucleic acid information in a precise, controlled manner within the genome. This technology has developed as a result of two key discoveries. The first is that a cell’s double strand break machinery can be highjacked to affect a desired change in the genome. Double stranded breaks are typically repaired by a pathway called non-homologous end joining (NHEJ) which can allow for disruption of an endogenous locus, or the pathway of homologous recombination (HR) which can allow for insertion of sequences if a homologous donor is supplied. The second major discovery is that chimeric enzymes can be engineered to create site-specific double stranded breaks, and that these enzymes can dramatically stimulate the frequency of gene targeting at a given locus. Recently, there has been an outpouring of studies performing site-specific genome engineering in human cell lines and primary cells, including embryonic stem cells, induced pluripotent stem cells and CD34+ hematopoietic stem cells. However, very little has been accomplished in terms of animal modeling of these principles. The work described in this thesis seeks to address some of these issues in a reporter mouse model that we have developed to study genome engineering. With this model we have demonstrated gene correction of an endogenous locus and also site-specific gene addition through a novel strategy which does not require disruption of the gene product at the site of the insertion. We have accomplished gene correction in several cell types including embryonic and adult fibroblasts, astrocytes and embryonic stem cells. For gene addition, we have demonstrated site-specific insertion of multiple transgenes including human growth hormone and human platelet derived growth factor-B, as well as a surface selectable marker (the truncated nerve growth factor receptor) and drug selectable marker. The tantamount goal for ex vivo genome engineering is to be able to modify a patient’s cells and then re-transplant them into the patient for a therapeutic outcome. In our mouse model, we have described a strategy where primary fibroblasts can be modified and then transplanted back into a recipient mouse. We are currently investigating the ability of these fibroblasts to serve as vehicles for local protein delivery to augment biological processes, such as tissue repair and wound healing. This work contributes to the body of knowledge that will enable translation of genome engineering from a scientific endeavor into a clinical reality.