? Applied Research The genetics underlying human biology and disease is complex and will require the use of ?system genetics? to dissect the myriad of interactions that control and regulate biological processes. Tools for human studies have advanced greatly yet there remains a necessary reliance on mammalian models. To this end new strains of mice have been created that better emulate the extent of human genetic variation. These strains include Collaborative Cross (CC), Diversity Outbred (DO) and recombinant inbred mice (e.g. BXD). The CC/DO and BXD platforms harness the natural inbred strain variation needed for systems genetics studies. However, the use of these strains typically requires additional genetic engineering of the mice to create ?tool strains? to allow specific types of genetic manipulations. Such strains already exist for standard inbred strains (e.g. C57BL/6J), but ?tool strains?, that carry commonly needed genetic manipulations, are not presently available for genetically diverse inbred strains such as CC/DO, BXD, or all of the founder inbred strains used to create these RI panels. The use of the powerful CC/DO/BXD strains is growing rapidly and to facilitate efficient use of these mice we propose to create a series of tool strains and mouse embryonic stem cells (mESC). To do this, we will take advantage of diverse inbred strains that harbor novel, high efficiency recombinase-based docking sites to facilitate the use of CRISPR-cas9 editing for creation of the new strains. Validated new tool strains will be made available through the Special Mouse Strain Resource (SMSR). To achieve this goal we will: Derive and validate germ line competent mESC lines from existing Bxb1 integrase strains. The goal here is to create a panel of genetically diverse mESC lines harboring Bxb1 integrase sites, as well as LSL- Cas9-FLAG. These mESCs will provide tools for engineering loci that are difficult to engineer in zygotes or for validation experiments that can be accomplished in vitro. Generate Rosa26-LSL-Cas9-FLAG tool strains for CAST/EiJ, PWK/EiJ, and DBA/2J inbred strains. These strains will enable CRISPR/cas9 editing in the absence of exogenous Cas9. This facilitates engineering in zygotes and allows for easy, precise engineering of somatic cells/tissues. The goal is to create and validate this important tool strain for the three high priority inbred strain backgrounds listed above. Generate germ line deletion tool strains for CAST/EiJ and PWK/EiJ inbred strain backgrounds. Removal of specific DNA sequences or engineering stop codons is frequently accomplished using flanking loxP sites that serve as catalytic domains for cre recombinase. Implementation of this approach frequently uses ?germ line cre? strains, that are not currently available on genetically diverse strain backgrounds. Thus, the goal here is to take advantage of our Bxb1 docking site strains to create Rosa26 Sox2-cre knock-in alleles on CAST/EiJ and PWK/EiJ.
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