Establishment of xenopus stem cell lines

项目摘要 非洲爪蟾是一个了不起的模型系统，几十年来一直被用来回答细胞中的基本问题， 发展和进化生物学。几个诺贝尔奖被授予开创性的研究， 爪蟾爪蟾胚胎的器官发育和形态与哺乳动物系统相当，但 具有能够再生成人组织的额外益处，所述成人组织例如视神经、透镜、脊髓， 肢体组织多年来，非洲爪蟾已被用于模拟几种人类疾病和综合征，包括 先天性心脏病、内脏异位、胃肠道和胰腺疾病、内分泌疾病、肾脏 疾病、癌症、纤毛病、口面缺陷和神经发育障碍。 非洲爪蟾是一个强大的体内模型系统，但强大的补充体外工具仍然有限。而 动物帽和组织外植体可以很容易地从非洲爪蟾胚胎中分离出来，并在体外培养，这些细胞 无法长期存活。此外，细胞内和细胞内过程通常难以进行。 记录和分析体内。这些限制促使在20世纪90年代建立了几种细胞系， 但它们在更适合哺乳动物的体外系统中失宠。从那时起，非洲爪蟾群落 依赖于使用人或小鼠细胞系，包括胚胎和诱导干细胞，其引入 由于物种差异而产生的固有变异性。尽管脊椎动物的发展蓝图 物种在很大程度上是保守的，细胞，组织和器官生物学的几个方面具有物种特异性 特色其中一个例子是，在非洲爪蟾的神经嵴发育过程中， 转录因子Snai1和Snai2，而小鼠神经嵴仅表达Snai1和鸡神经嵴 只有Snai2。这些差异限制了实验中跨物种系统的使用。因此，我们和 其他人正在努力扩大非洲爪蟾群落的体外工具箱。我们建议生成 新的非洲爪蟾干细胞系，以加强非洲爪蟾社区目前和未来的研究项目。 干细胞代表一种正常的生理状态，它们的基因组缺乏大多数人通常发现的异常。 组织培养系，并且它们可以分化成许多不同的细胞类型和类器官。而且有 非洲爪蟾干细胞提供的强大优势将是有效进行基因组编辑的机会， 以及体细胞核转移（SCNT）以产生F0纯合无效动物以产生新的突变体。 目前，干细胞系通过两种方法获得：从内细胞分离胚胎干细胞， 大量哺乳动物囊胚期胚胎和通过重编程获得的诱导多能干细胞 以重新启动内源性多能性程序。在本申请中，我们建议生成 胚胎干细胞系来自在囊胚期分离的动物极细胞， 来源于蝌蚪组织的原代细胞系以产生诱导的多能干细胞。

PROJECT SUMMARY Xenopus laevis is a remarkable model system used for decades to answer fundamental questions in cell, developmental, and evolutionary biology. Several Nobel prizes were awarded for groundbreaking research in Xenopus. Xenopus embryos have comparable organ development and morphology to mammalian systems, but with the added benefit of being able to regenerate adult tissues, such as the optic nerve, lens, spinal cord, and limb tissue. Over the years, Xenopus has been used to model several human diseases and syndromes, including congenital heart disorders, heterotaxia, gastrointestinal and pancreatic diseases, endocrine disorders, kidney disease, cancer, ciliopathies, orofacial defects, and neurodevelopmental disorders. Xenopus is a powerful in vivo model system, but robust complementary in vitro tools are still limited. While animal caps and tissue explants can be easily isolated from Xenopus embryos, and cultured in vitro, these cells do not survive for extended time periods. Furthermore, cellular and intracellular processes are often difficult to document and analyze in vivo. These limitations prompted the establishment of several cell lines in the 1990's, but they fell out of favor for the more amenable mammalian in vitro systems. Since then, the Xenopus community has relied on the use of human or mouse cell lines, including embryonic and induced stem cells, which introduce inherent variability due to species differences. Even though the blueprint of vertebrate development across species is largely conserved, several aspects of cellular, tissue, and organ biology have species specific characteristics. An example among many, during development Xenopus neural crest expresses both the transcription factors Snai1 and Snai2, while mouse neural crest only expresses Snai1 and the chick neural crest only Snai2. These differences constrain the use of cross species systems experimentally. As a result, we and others are engaged in efforts to expand the in vitro toolbox of the Xenopus community. We propose to generate new Xenopus stem cell lines to enhance current and future research projects for the Xenopus community. Stem cells represent a normal physiological state, their genomes lack abnormalities typically found in most tissue culture lines, and they can be differentiated into many different cell types and organoids. Furthermore, a strong advantage offered by Xenopus stem cells would be the opportunity to perform genome editing efficiently, as well as somatic cell nuclear transfer (SCNT) to generate F0 homozygous null animals to create new mutants. Currently, stem cell lines are obtained via two methods: embryonic stem cells isolated from the inner cell mass of mammalian blastocyst-stage embryos, and induced pluripotent stem cells obtained via reprogramming of mature cells to re-initiate endogenous pluripotency programs. In this application we propose to generate embryonic stem cell lines from animal pole cells isolated at the blastula stage and reprogram newly generated primary cell lines derived from tadpole tissues to produce induced pluripotent stem cells.