Creation of Knockout Laboratory Opossums

淘汰赛实验室负鼠的创建

基本信息

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
项目类别：
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

项目摘要

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可用于生物医学研究的有袋动物。是一个用于许多人类疾病和发育过程的研究独特或专业模型，以及用于比较生物学和比较基因组学目的。 PI维持最大的育种和该物种在世界上的研究殖民地。实现研究潜力的最关键的障碍实验室负鼠是在美国建立基因编辑程序的任何人缺乏成功的成功一个日本群体最近克服了该物种基因编辑的技术障碍，并成功地击倒了随机繁殖果中的酪氨酸酶（Tyr）基因。实施和在PI实验室中优化其方法将为实现长期目标铺平道路这是建立一个国家研究资源，该资源将有效，经济上创建基因美国调查人员需要编辑的骨头，以解决重要的生物医学问题实验室动物非常适合解决。在任何单个物种的研究量机构不能证明建立大型殖民地和基因编辑技术的成本是合理的，这是一个集中的资源将能够以最低的成本满足所有国家需求。具体目的是1）通过针对完全近亲的塔利（Tyr）有针对性的破坏在我们的实验室中建立专业知识和原理证明实验室负鼠的菌株，以及2）进行磷酸酶和tensin的靶向破坏相同近交菌株的骨中的同源基因（PTEN）。敲除ossums将通过应用创建交配后30-34小时收集的CRISPR -CAS9技术到负鼠胚胎，之后鸡蛋壳变得太难穿透了。通过使用压电执行器，将增强穿透性。确认基因型的确认将通过对从中断奶的后代的基因组DNA分析确定经过处理的胚胎，以及随后从那些后代产生的动物。 PTEN - / - 预计纯合子将胚胎致死，但杂合子有望用作新模型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2年期间的该主题上，已经产生了足够数量的+/-动物。其他研究计划的一些示例将通过创建而成为可能或高度增强渴望合作的调查人员的支持信中提供了具体的淘汰赛。示例包括使用PTEN +/-负鼠与PTEN基因淘汰赛Opossum IPSC（+/-和 - / - ）结合使用。从相同的近交菌株进行体内和体内神经发育过程的比较研究（惠特沃思），使用PTEN +/-负鼠研究听觉功能涉及的机制和再生（Walters），识别ossums蛇毒毒液的基因的识别（Sanchez）和T细胞子集的功能，其中有袋动物具有在Eutherians（Miller）中没有发现的。