Developing techniques to generate the first transgenic opossums

项目摘要 灰色短尾负鼠(Monodelphis Home Tica)是一种小型无袋南方负鼠 美国有袋类动物是生物医学和进化研究中广泛使用的模型系统。 它是早产的，有发育良好的前肢，可以爬到母亲的乳头上 完成它的发展。后肢、间盘和许多其他器官 系统在诞生之初就处于形成的初级阶段。因为四肢不均 负鼠在出生时就形成了，长期以来一直被用作研究肢体的天然突变体 队形。在胎盘和胎盘之间进行了大量的进化比较 具有这种研究系统的有袋类哺乳动物。此外，负鼠还被用于 癌症研究(特别是黑色素瘤)，高胆固醇研究，脊髓再生， 伤口愈合和出生缺陷(特别是沙利度胺和维甲酸畸形)，以 仅举几个例子。已经为其畜牧业开发了许多资源，胚胎 收集、免疫组织化学、原位杂交、组织培养。它的基因组也是 通过RNA-Seq分析产生最近测序的和器官特异的转录本。这个 负鼠的体型也很小，在实验室里很容易饲养，而且相对温顺。 尽管有这些优点，但有袋类动物模型的主要缺点是缺乏转基因 或者是被淘汰的动物。转基因小鼠已经存在了几十年，而且变得更容易 随着技术的进步而制造。此外，转基因和其他工程突变体 在老鼠和其他家养哺乳动物中变得普遍，特别是农场动物和非家畜 人类灵长类动物。以前试图产生转基因负鼠的努力有限 成功。我们建议基于一种方法来产生第一只转基因负鼠 已经习惯于在其他哺乳动物身上取得巨大成功，尤其是啮齿动物。精原干细胞 负鼠(SSCs)将从睾丸中采集，在体外培养和扩增， 用携带记者的结构转化，并移植到供体雄性负鼠体内。如果 这项工作的成功，不仅将为有袋类和胎盘类提供有价值的比较 哺乳动物SSC生物学，但将为突变和转基因奠定基础 负鼠模型。从理论上讲，将有可能利用日益流行的 在负鼠SSCs中构建CRISPR/Cas9系统，并将其移植到供体雄性体内。为了更多的人 对于有袋类模型的使用者来说，转基因有袋类模型的可用性将是一个关键 添加到不断增长的工具包中。这项提案将是一项概念验证，用于生成 表达外源DNA的负鼠。

Project Summary The grey short-tailed opossum (Monodelphis domestica) is a small pouchless South American marsupial is a widely used model system for research into biomedicine and evolution. It is born prematurely, with well-developed forelimbs that permit it to climb to the mother's teat to complete its development. The hindlimbs, intervertebral disc, and a number of other organ systems are at a rudimentary stage of formation at birth. Because of the disparity in limb formation at birth, the opossum has long been used as a `natural' mutant for the study of limb formation. A number of evolutionary comparisons have been made between placental and marsupial mammals with this study system. In addition, the opossum has also been used in cancer research (particularly melanoma), studies of high cholesterol, spinal cord regeneration, wound healing, and birth defects (particularly thalidomide and retinoic acid teratogenesis), to name just a few. A number of resources have been developed for its husbandry, embryo collection, immunohistochemistry, in situ hybridization, and tissue culture. Its genome was also recently sequenced and organ-specific transcriptomes generated by RNA-Seq analysis. The opossum is also small-sized, fairly easy to raise in a laboratory setting, and relatively docile. Despite all these advantages, the main drawback to the marsupial model is a lack of transgenic or knockout animals. Transgenic mice have been available for decades, and become easier to make as technology improves. In addition, transgenic and other engineered mutants are becoming common for rats and other domestic mammals, particularly farm animals and non- human primates. Previous attempts to generate a transgenic opossum have had limited success. We propose to generate the first transgenic opossums based on a method that has been used to great success in other mammals, rodents especially. Spermatogonial stem cells (SSCs) from opossums will be harvested from the testes, cultured and expanded in vitro, transformed with a reporter-carrying construct, and transplanted into donor male opossums. If successful, this work will not only provide valuable comparisons of marsupial vs. placental mammal SSC biology, but will build the foundation for mutagenesis and transgenesis in an opossum model. Theoretically, it will be possible to utilize the increasingly popular CRISPR/Cas9 system in opossum SSCs and transplant them to donor males. For the many users of marsupial models, the availability of a transgenic marsupial model would be a critical addition to a growing toolkit. This proposal will be a proof-of-concept for the generation of opossums expressing exogenous DNA.