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。这是一个 研究许多人类疾病和发育过程的独特或专门模式，以及 用于比较生物学和比较基因组学目的。到目前为止，PI保持着最大的繁殖和 世界上这一物种的研究群体。实现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的负鼠的酪氨酸酶(Tyr)基因。实施和实施 在国际和平研究所的实验室中优化他们的方法将为实现长期目标铺平道路， 就是建立一个国家研究资源，高效、经济地创造基因-- 美国调查人员为解决重要的生物医学问题所需的编辑负鼠 实验动物是唯一适合寻址的动物。虽然对这个物种的研究数量在任何一个单独的 机构不能证明建立一个大群体和基因编辑技术的成本是合理的，集中化 资源将能够以最低的成本满足所有国家的需求。具体目标是：1) 通过在完全近交系中有针对性地破坏Tyr，在我们的实验室建立专业知识和原则证明 实验室负鼠的菌株，以及2)进行有针对性的破坏磷酸酶和紧张素 同源基因(Pten)在同一近交系的负鼠中。击倒负鼠将通过应用 CRISPR-Cas9技术将负鼠胚胎在交配后30-34小时采集，之后将卵 贝壳变得太硬，无法穿透。通过使用压电式致动器，渗透性将得到增强。 敲除基因型的确认将通过对断奶仔猪的基因组DNA分析来确定 经过处理的胚胎，以及从这些后代产生的后续动物的胚胎。PTEN-/- 纯合子有望导致胚胎死亡，但杂合子有望成为新的模式 在自闭症谱系障碍的比较医学研究中。我们计划建立一个研究项目 在两年项目期之后，在生产了足够数量的+/-动物之后，再讨论这一主题。 一些其他研究计划的例子，这些计划将通过创建 渴望合作的调查人员在支持信中提供了具体的基因敲除。 例子包括结合使用Pten+/-负鼠和Pten基因敲除负鼠ipscs(+/-和-/-) 用于体内和体外神经发育过程的比较研究 (Whitworth)，使用Pten+/-负鼠来研究听觉功能和 再生(Walters)，鉴定负鼠对蛇毒具有显著抵抗力的基因 (Sanchez)，和T细胞亚群的功能，其中有袋类动物有一种在真兽类中没有发现的(Miller)。

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 by far the largest breeding and research colony of this species in the world. The most critical barrier to fulfilling the 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 random-bred opossums. 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 will efficiently and economically create gene- edited opossums required by US investigators to address important biomedical questions that this laboratory animal is uniquely suited to address. While the volume of research with this species at any single institution cannot justify the cost of establishing a large colony and gene editing technologies, a centralized resource will be capable of serving all of the national needs at minimal cost. The specific aims 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 30 - 34 hours after copulation, after which 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. Pten -/- homozygotes are expected to embryonic lethal, but heterozygotes are expected to serve as new model in comparative medicine research on Autism Spectrum Disorder. We plan to establish a research project on that topic beyond the 2-year project period, after a sufficient number of +/- animals have been produced. Some examples of other research programs that will be made possible or highly enhanced by the creation of specific knockouts are provided in the letters of support from investigators who are eager to collaborate. Examples include the use of Pten +/- opossums in conjunction with Pten knockout opossum iPSCs (+/- and -/-) from the same inbred strain for comparative research on in vivo and ex vivo neurodevelopmental processes (Whitworth), the use of Pten +/- opossums to investigate mechanisms involved in auditory function and regeneration (Walters), identification of genes that confer remarkable resistance of opossums to snake venom (Sanchez), and function of T cell subsets, of which marsupials have one that is not found in eutherians (Miller).