Creation of Knockout Laboratory Opossums

项目摘要/摘要 实验室负鼠是唯一可以大量用于生物医学研究的有袋类动物。它是 一种独特或专门的模型，用于研究许多人类疾病和发育过程以及 用于比较生物学和比较基因组学目的。PI保持着最大的繁殖量和 世界上这一物种的研究群体。充分发挥研究潜力的最关键障碍是 实验室负鼠是美国任何人在建立基因编辑程序方面缺乏成功的地方 物种。最近，一个日本团队克服了对该物种进行基因编辑的技术障碍， 并成功地敲除了随机繁殖种群中的酪氨酸酶(Tyr)基因。实施和实施 在国际和平研究所实验室对他们的方法进行优化，将为实现长期目标铺平道路， 是建立一个能够高效、经济地创造基因编辑的国家研究资源 美国调查人员需要负鼠来解决这种实验动物的重要生物医学问题 独一无二地适合寻址。 母项目的具体目标是1)通过以下方式在我们的实验室建立专业知识和原则证明 在实验室负鼠的完全近亲繁殖品系中靶向干扰Tyr，以及2)进行靶向干扰 同一近交系负鼠的磷酸酶和张力蛋白同源基因(Pten)。击倒负鼠 将CRISPR-Cas9技术应用于34小时前收集的负鼠胚胎 交配，当蛋壳变得太硬而无法穿透时。将通过使用 压电致动器。敲除基因的确认将通过基因组DNA分析来确定 从处理过的胚胎以及从这些胚胎产生的后续动物后代中断奶的后代 后代。 补充剂的具体目的是有针对性地干扰编码 γ？T细胞受体二聚体的？多肽。所有哺乳动物都有αβ和γδT细胞受体，但只有 有袋类动物具有µ基因和γµT细胞受体。它的功能尚不清楚。创建了一个淘汰赛 µ基因将为T细胞受体促进细胞免疫的机制提供新的见解。 这一目标将使用父项目中正在开发的技术来实现。 在父母资助和补充结束后，将培育出带有被破坏的µ基因的动物 生产一种将用于研究γ？T细胞受体功能及其功能的试剂盒 与αβ和γδT细胞受体的关系。因为T细胞受体对免疫应答是至关重要的 对于病原体和癌症的免疫监控，缺乏γ？T细胞受体的负鼠可能起到 在提高我们对T细胞受体功能机制的理解中的重要作用，并最终 制定预防和治疗传染病和癌症的新战略。

PROJECT SUMMARY/ABSTRACT The laboratory opossum is the only marsupial that is available in large numbers for biomedical research. It is a unique or specialized model for research on many human diseases and developmental processes, as well as for comparative biology and comparative genomics purposes. The PI maintains the largest breeding and research colony of this species in the world. The most critical barrier to fulfilling the full research potential of the laboratory opossum is the lack of success of anyone in the US in establishing gene-editing procedures for this species. A Japanese group recently overcame the technological impediments to gene editing of this species, and succeeded in knocking out the tyrosinase (Tyr) gene in a random-bred stock. The implementation and optimization of their methods at the PI’s laboratory will pave the way for achieving the long-term objective, which is to establish a national research resource that is capable of efficiently and economically creating gene-edited opossums required by US investigators to address important biomedical questions that this laboratory animal is uniquely suited to address. The specific aims of the parent project are 1) to establish expertise and proof-of-principle in our laboratory by targeted disruption of Tyr in a fully inbred strain of laboratory opossums, and 2) to conduct targeted disruption of the phosphatase and tensin homolog gene (Pten) in opossums of the same inbred strain. Knockout opossums will be created by applying CRISPR-Cas9 technology to opossum embryos collected prior to 34 hours after copulation, when the egg shell becomes too hard to penetrate. Penetration will be enhanced via the use of a piezoelectric actuator. Confirmation of knockout genotype will be established by genomic DNA analysis of progeny weaned from the treated embryos, and from subsequent generations of animals produced from those progeny. The specific aim of the supplement is to conduct targeted disruption of the µ gene that encodes the µ polypeptide of the γµ T cell receptor dimer. All mammals have the αβ and γδ T cell receptors, but only marsupials have the µ gene and the γµ T cell receptor. Its function is not known. Creating a knockout of the µ gene will provide new insights into the mechanisms by which T cell receptors contribute to cellular immunity. This aim will be accomplished using the techniques that are being developed in the parent project. After the conclusion of the parent grant and the supplement, the animals with the disrupted µ gene will be bred to produce a stock that will be used for research on function of the γµ T cell receptor and its functional relationships with the αβ and γδ T cell receptors. Since T cell receptors are critical to the immune response to pathogens and to immunosurveillance for cancers, opossums lacking the γµ T cell receptor may play an important role in improving our understanding of mechanisms by which T cell receptors function, and ultimately in developing new strategies for preventing and treating infectious diseases and cancer.