Modeling Disease in Swine by Transplantation of Gene Targeted Germ Cells

项目摘要 猪在生物医学研究中很重要，因为人类疾病的研究很难通过 啮齿类动物，用于开发和测试人源化疾病模型中的临床前治疗剂， 作为异种或同种异体器官和组织的潜在来源。然而，创造和传播 生物医学猪的发展受到与动物发育，繁殖和致死相关的效率低下的困扰 表型猪模型繁殖的生产育种计划是不够的， 有严重的疾病相关表型，降低长期生存能力，有性生殖能力，或 分离许多等位基因。为了再生医学的目的，开发和传播 器官发生缺陷动物也需要标准育种的替代品。我们的解决方案是开发一个 基于生殖系干细胞移植（GST）和囊胚互补的平台技术， 猪拯救有价值的种系，并允许第一次有效繁殖先天性 疾病和器官发生缺陷等位基因。 GST或胚泡互补的应用依赖于猪品系的产生， 不能产生自己的配子。在第一阶段，我们通过创建纯合子， 无精子症样缺失（DAZL）基因的无效突变。我们还建立了一个繁殖群 表型正常且能生育的杂合DAZL动物。在第二阶段，我们将优化 通过评估DAZL基因敲除公猪的生殖细胞植入和精子特性， 有限剂量的生殖系干细胞移植。其次，由于生殖系干细胞来自严重的 由于高发病率，疾病模型可能受到限制，我们将从1周到10周量化生殖系干细胞产量 一周大的睾丸，并开发这些细胞的扩增策略。作为概念验证，我们将进行 从我们的严重扩张型心肌病（DCM）模型中进行生殖细胞移植， 通过标准的饲养。通过拯救纯合子DCM公猪的种系，我们将使我们的产量增加一倍， 这种模式不需要扩大母猪群。在这项资助的最后一个目标中，我们将使用囊胚 互补以证明我们的RAG 2/IL 2 Rg敲除（SCID）的表型拯救和生育力， 免疫缺陷猪无DAZL供体细胞将拯救免疫缺陷表型，但不能 有助于嵌合体的生殖细胞。成功实施这一办法将增加 SCID动物的生产率从目前的6.3%，通过杂合子的互交提高到100%， 使纯合子能够杂交。因此，这项建议将对我们的生产能力产生直接影响。 销售两种利润增加的猪模型，DCM和SCID，同时创造一种新的DAZL育种方法， 平台，许多其他模型和器官发生缺陷系将依赖于繁殖和规模- 起来 0

PROJECT SUMMARY Swine are important in biomedical research for the study of human diseases that are poorly recapitulated by rodent species, for the development and testing of preclinical therapeutics in humanized disease models and as potential sources of xenogeneic or allogeneic organs and tissues. However, the creation and propagation of biomedical swine is plagued by inefficiencies related to animal development, reproduction and lethal phenotypes. Production breeding programs for swine model propagation are inadequate when the models have severe disease-associated phenotypes that reduce long-term viability, the ability to sexually reproduce or segregate numerous alleles. For regenerative medicine purposes, the development and propagation of organogenesis-deficient animals also requires an alternative to standard breeding. Our solution is to develop a platform technology based on germline stem cell transplantation (GST) and 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 alleles. Application of GST or blastocyst complementation relies on the generation of a strain of pigs that cannot produce their own gametes. In Phase I, we generated germ cell-deficient pigs by creating homozygous null mutations in the Deleted-in-Azoospermia-like (DAZL) gene. We've also established a breeding herd of heterozygous DAZL animals that are phenotypically normal and fertile. In this Phase II grant, we 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 cells. Secondly, since germline stem cells from severe disease models could be limited due to high morbidity, we will quantify germline stem cell yield from 1wk to 10 week-old testes and develop strategies for expansion of these cells. As proof of concept, we will conduct germline transplantation from our severe model of dilated cardiomyopathy (DCM) that is inefficiently produced by standard breeding. By rescuing the germline of homozygous DCM boars, we will double our production of this model without the need to expand our sow herd. In the last aim of this grant, we will use blastocyst complementation to demonstrate phenotypic rescue and fertility of our RAG2/IL2Rg knockout (SCID), immunodeficient swine. DAZL-null donor cells will rescue the immunodeficient phenotype but are unable to contribute to the germline of chimeras. Successful implementation of this approach would increase the production rate of SCID animals from the current 6.3% by intercross of heterozygotes to 100% with DAZL- enabled intercross of homozygotes. Hence, this proposal will have immediate impact on our ability to produce and sell two swine models with increased margins, DCM and SCID, while creating a novel DAZL breeding platform that numerous other models and organogenesis-deficient lines will rely on for propagation and scale- up. 0