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 blastul...