Creation of Knockout Laboratory Opossums

淘汰赛实验室负鼠的创建

• 批准号: 10648854
• 负责人: JOHN L VANDEBERG
• 金额: $ 18.5万
• 依托单位: UNIVERSITY OF TEXAS RIO GRANDE VALLEY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10648854
• 关键词: Address; Animal Model; Animals; Area; Auditory; Behavior; Behavioral; Biomedical Research; Breeding; CRISPR/Cas technology; Characteristics; Collaborations; Comparative Biology; Copulation; DNA analysis; Development; Developmental Process; Didelphidae; Ear; Egg Shell; Embryo; Embryology; Experimental Models; Female; Future; Generations; Genes; Genetic Engineering; Genetically Modified Animals; Genome; Genomic DNA; Genotype; Grant; Heterozygote; Homozygote; Hour; Human; Implant; Inbred Strain; Institution; Japanese; Japanese Population; KDM1A gene; Knock-out; Laboratories; Laboratory Animals; Letters; Literature; Mammals; Marsupialia; Medicine; Methods; Modeling; Monodelphis; Monodelphis Domestica; Monophenol Monooxygenase; Mus; Natural regeneration; Neurodevelopmental Disorder; Outcome; PTEN gene; Penetration; Preparation; Procedures; Process; Protocols documentation; Research; Research Personnel; Research Project Grants; Resistance; Resources; Rodent; Rodent Model; Role; Science; Siblings; Snake Venoms; T-Lymphocyte Subsets; Technology; Time; Weaning; Work; animal breeding; autism spectrum disorder; comparative; comparative genomics; cost; experimental study; external ear auricle; genomic locus; human disease; in vivo; induced pluripotent stem cell; innovation; knockout gene; mosaic; neurodevelopment; next generation sequencing; novel; programs; reproductive; success; transmission process

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).

项目摘要/摘要 实验室负鼠是唯一的有袋类动物，可在大量的生物医学研究。这是一个 用于研究许多人类疾病和发育过程的独特或专门模型，以及 用于比较生物学和比较基因组学目的。PI保持着迄今为止最大的育种和 世界上最大的研究群体实现研究潜力的最关键障碍是 实验室负鼠是缺乏成功的任何人在美国建立基因编辑程序， 物种日本的一个研究小组最近克服了对这一物种进行基因编辑的技术障碍， 并成功敲除随机繁殖的负鼠中的酪氨酸酶（Tyr）基因。执行和 在PI实验室优化他们的方法将为实现长期目标铺平道路， 这是建立一个国家的研究资源，将有效和经济地创造基因， 编辑负鼠所需的美国调查人员，以解决重要的生物医学问题， 实验室动物是唯一适合解决。尽管对这个物种的研究量在任何一个 机构不能证明建立一个大的殖民地和基因编辑技术的成本，一个集中的 资源将能够以最低的成本满足国家的所有需求。具体目标是：（1） 通过在完全近交系中靶向破坏Tyr，在我们的实验室中建立专业知识和原理验证 实验室负鼠的菌株，和2）进行磷酸酶和张力蛋白的靶向破坏 同源基因（Pten）。淘汰负鼠将通过应用 CRISPR-Cas9技术对交配后30 - 34小时收集的负鼠胚胎进行处理， 外壳变得太硬而无法穿透。穿透将通过使用压电致动器来增强。 将通过对断奶后代的基因组DNA分析来确定敲除基因型的确认。 处理的胚胎，以及从这些后代产生的动物的后代。Pten -/- 纯合子有望成为胚胎致死模型，而杂合子有望成为新的模型 自闭症谱系障碍的比较医学研究。我们计划建立一个研究项目 在生产了足够数量的+/-动物之后，在2年项目期之后就该主题进行研究。 其他研究计划的一些例子，将成为可能或高度加强的创建， 渴望合作的调查人员在支持信中提供了具体的淘汰办法。 实例包括Pten +/-负鼠与Pten敲除负鼠iPSC（+/-和-/-）结合使用。 用于体内和离体神经发育过程的比较研究 （Whitworth），使用Pten +/-负鼠研究涉及听觉功能的机制， 再生（Walters），鉴定赋予负鼠对蛇毒显著抗性的基因 （Sanchez），以及T细胞亚群的功能，其中有袋类动物有一个在真兽目动物中没有发现的亚群（米勒）。