Modeling Disease in Swine by Transplantation of Gene Targeted Germ Cells

PROJECT SUMMARY Swine are important in biomedical research for the study of human diseases that are poorly recapitulated by rodent speciesfor the development and testing of preclinical therapeutics in humanized disease models and as potential sources of xenogeneic or allogeneic organs and tissuesHoweverthe creation and propagation of biomedical swine is plagued by inefficiencies related to animal developmentreproduction and lethal phenotypesProduction breeding programs for swine model propagation are inadequate when the models have severe disease associated phenotypes that reduce long term viabilitythe ability to sexually reproduce or segregate numerous allelesFor regenerative medicine purposesthe development and propagation of organogenesis deficient animals also requires an alternative to standard breedingOur solution is to develop a platform technology based on germline stem cell transplantationGSTand blastocyst complementation in swine to rescue the germline of valuable lines and permit for the first time efficient propagation of congenital disease and organogenesis deficient allelesApplication of GST or blastocyst complementation relies on the generation of a strain of pigs that cannot produce their own gametesIn Phase Iwe generated germ cell deficient pigs by creating homozygous null mutations in the Deleted in Azoospermia likeDAZLgeneWeandapos ve also established a breeding herd of heterozygous DAZL animals that are phenotypically normal and fertileIn this Phase II grantwe will optimize the conditions for GST in DAZL null boars by evaluating germ cell engraftment and sperm characteristics after transplantation of limiting dosages of germline stem cellsSecondlysince germline stem cells from severe disease models could be limited due to high morbiditywe will quantify germline stem cell yield fromwk toweek old testes and develop strategies for expansion of these cellsAs proof of conceptwe will conduct germline transplantation from our severe model of dilated cardiomyopathyDCMthat is inefficiently produced by standard breedingBy rescuing the germline of homozygous DCM boarswe will double our production of this model without the need to expand our sow herdIn the last aim of this grantwe will use blastocyst complementation to demonstrate phenotypic rescue and fertility of our RAGIL Rg knockoutSCIDimmunodeficient swineDAZL null donor cells will rescue the immunodeficient phenotype but are unable to contribute to the germline of chimerasSuccessful implementation of this approach would increase the production rate of SCID animals from the currentby intercross of heterozygotes towith DAZLenabled intercross of homozygotesHencethis proposal will have immediate impact on our ability to produce and sell two swine models with increased marginsDCM and SCIDwhile creating a novel DAZL breeding platform that numerous other models and organogenesis deficient lines will rely on for propagation and scaleupPROJECT NARRATIVE This SBIR proposes to build a novel breeding platform for difficult to propagate swine models using our germline ablated Deleted in Azoospermia likeDAZLknockout pigs developed in PhaseTo demonstrate the impactwe will rescue the germline of our severe dilated cardiomyopathy model by germline stem cell transplantationpredicted to double production of this in demand model of heart failureSecondlywe will use blastocyst complementation to generate chimeras between our DAZL null and immunodeficient line to phenotypically rescue the immunodeficiency phenotype whileof the germline remains immunodeficient host derivedBreeding these models using the DAZL platform approaches increases production two and up to sixteen foldrespectivelyenabling reliable and economical provision of these models and several more to the biomedical community