Creation of Knockout Laboratory Opossums

项目概要/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。是 一个独特的或专门的模型，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， 世界上最大的研究群体最关键的障碍，以履行充分的研究潜力， 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， 并成功地敲除了随机繁殖的牛的酪氨酸酶基因。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， 是建立一个国家研究资源，能够有效和经济地创造基因编辑， 美国研究人员要求负鼠解决重要的生物医学问题，这种实验室动物是 唯一适合的地址。 母项目的具体目标是：1）通过以下方式在我们的实验室中建立专业知识和原理证明 靶向破坏实验室负鼠的完全近交系中的Tyr，和2）进行靶向破坏 同一近交系负鼠的磷酸酶和张力蛋白同源基因（Pten）。敲除负鼠 将通过将CRISPR-Cas9技术应用于34小时前收集的负鼠胚胎来创建， 交配，当蛋壳变得太硬而无法穿透时。将通过使用 压电驱动器通过基因组DNA分析确定敲除基因型， 从处理过的胚胎断奶的后代，以及从这些胚胎产生的后代动物， 后代 该补充剂的具体目的是对编码以下蛋白质的µ基因进行靶向破坏： γµ T细胞受体二聚体的µ多肽。所有哺乳动物都有αβ和γδ T细胞受体，但只有 有袋动物有µ基因和γµ T细胞受体。其功能尚不清楚。创建一个击倒的 µ gene将为T细胞受体促进细胞免疫的机制提供新的见解。 这一目标将使用母项目中正在开发的技术来实现。 在父母补助金和补充金结束后，将繁殖具有破坏的µ基因的动物 以生产用于研究γµ T细胞受体功能及其功能的储备液， 与αβ和γδ T细胞受体的关系。由于T细胞受体对免疫应答至关重要， 病原体和癌症的免疫监视，缺乏γµ T细胞受体的负鼠可能发挥作用， 在提高我们对T细胞受体功能机制的理解方面发挥着重要作用， 制定预防和治疗传染病和癌症的新策略。

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.