SSCs are also similar to many other stem cell types in that they are rare and difficult to identify definitively through expression of particular proteins. Rather, rodent SSCs can be most strictly defined functionally based on their ability to home to a niche and colonize a recipient’s testes following transplantation, and then undergo meiosis and differentiate into sperm. Following years of intensive effort by multiple laboratories, conditions were eventually discovered for enriching for SSCs and maintaining them essentially indefinitely in vitro. The cultured cells, termed “germline stem cells”, have properties of untransformed primary cells that can be propagated long-term because of the self-renewal of SSCs. Importantly, putative SSCs can also be identified and cultured in vitro from human testes, although the duration for which human SSCs can be kept in vitro remains controversial and conditions for long term culture need to be SP600125 optimized. Given the robust nature of the rodent GS cell propagation system, we chose to model the process of ex vivo genome editing using mouse GS cells. We decided to test one of the more challenging genome editing approaches whereby homologous recombination is used to modify an existing mutation in the genome because this approach creates the precise modifications necessary for the most powerful research and therapy applications. For research purposes genome modification by homologous recombination greatly decreases the possibility of heterogeneous phenotypes from uncontrolled random integration; that is, with the latter, a transgene’s expression may be variable or silenced depending on where it integrates. For therapeutic purposes HR is potentially safer because of the elimination of random insertions, which in certain settings have been shown to lead to cancer through the process of insertional oncogenesis. While the frequency of HR with an exogenous DNA repair substrate in most cell types is too low to be therapeutically useful, the frequency can be increased by several orders of magnitude by introducing a double strand break at the site in the chromosome to be modified. Creation of a DSB can be accomplished using custom designed nucleases, including zinc finger nucleases, TAL effector nucleases or RNA guided endonucleases. ZFNs and TALENs are chimeric proteins comprising a nuclease domain from the type II restriction enzyme Fok I and a DNA binding domain engineered to recognize a specific sequence. ZFNs and TALENs have been demonstrated to stimulate homology directed repair of a DSB using an exogenous DNA repair substrate, or “gene targeting”, in a wide variety of contexts. For example, correction of a point mutation in interleukin 2 receptor, gamma was accomplished in K562 cells, an immortalized myelogenous leukemia human cell line, as well as in primary human T-cells and human CD34+ cells. Also, ZFNs and TALENs have been shown to simulate gene targeting in mouse and human embryonic stem cells and induced pluripotent stem cells. Still, genome engineering in the context of adult primary-like stem cells, which are likely to more closely resemble cells that will be used in therapy, is relatively unexplored. Moreover, the use of ZFN or TALEN stimulated HR to modify the genome in GS cells has not been described.