Modeling Disease in Swine by Transplantation of Gene Targeted Germ Cells

项目总结 猪在研究人类疾病的生物医学研究中很重要，这些疾病没有被概括为 啮齿动物物种，用于人性化疾病模型和临床前治疗的开发和测试 作为异种或异体器官和组织的潜在来源。然而，创作和传播 与动物发育、繁殖和致死有关的低效率困扰着生物医学猪的发展 表型。生猪模范繁育的生产繁育方案不完善 有严重的疾病相关表型，降低长期生存能力，有性繁殖能力或 分离出大量的等位基因。为了再生医学的目的，开发和推广 器官发生缺陷的动物还需要一种替代标准育种的方法。我们的解决方案是开发一种 基于生殖系干细胞移植(GST)和囊胚互补的平台技术 挽救猪种系的宝贵品系并首次允许先天性的高效繁殖 疾病和器官发生缺陷等位基因。 GST或囊胚补充的应用依赖于产生一种品系的猪 不能产生自己的配子。在第一阶段，我们通过创造纯合子来产生生殖细胞缺陷猪 类无精子缺失(DAZL)基因零突变。我们还建立了一群繁育的 表型正常且有生育能力的杂合子DAZL动物。在这笔第二阶段的赠款中，我们将优化 从生殖细胞植入和精子特性评价DAZL缺失型公猪GST的条件 生殖系干细胞有限剂量移植。其次，由于生殖系干细胞来自严重的 由于发病率高，疾病模型可能会受到限制，我们将量化生殖系干细胞产量从1周到10周 一周大的睾丸，并制定这些细胞的扩张策略。作为概念证明，我们将进行 从我们的严重扩张型心肌病(DCM)模型中进行生殖系移植，该模型生产效率低下 通过标准的繁育。通过挽救纯合子DCM公猪的种系，我们将使我们的产量翻一番 这种模式不需要扩大我们的母猪群。在这笔赠款的最后一个目标中，我们将使用囊胚 互补以证明我们的RAG2/IL2RG基因敲除(SCID)的表型拯救和生育能力， 免疫缺陷猪。DAZL缺失的供体细胞将挽救免疫缺陷表型，但无法 为嵌合体的种系做出贡献。这一方法的成功实施将增加 杂合子杂交的SCID动物产蛋率从目前的6.3%提高到100%，DAZL- 允许纯合子的杂交。因此，这项提议将对我们的生产能力产生直接影响 并销售两种利润率更高的猪型号DCM和SCID，同时创造了一个新的DAZL育种 许多其他模型和器官发生缺陷品系将依赖的平台来繁殖和扩大规模- 向上。 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