Production and characterization of monoclonal antibodies to Xenopus Proteins

项目总结： 两栖动物胚胎，特别是非洲爪蛙是一个杰出的模型，用于 世界各地的胚胎学家和细胞生物学家。这个模型系统引领了轴形成的发现 (前侧/后侧和背侧/腹侧)、组织诱导和核重新编程等。 基因组测序、蛋白质组学和基因沉默方面的进展使这一模式重新焕发活力。在……里面 在其最新的白皮书中，非洲爪哇社区发现缺乏足够的资源 研究非洲爪哇蛋白的抗体，并建议优先生产这种资源。我们 提议生产至少100种完全特征化的转录单抗 因子、细胞表面蛋白，包括信号受体和配体以及组织特异性 记号笔。为了实现这一目标，我们将用这两种精选的重组蛋白免疫小鼠。 在哺乳动物细胞和非洲爪哇胚胎中分离的复杂蛋白提取物中表达。 重组蛋白质靶标将由非洲爪哇组成的指导委员会挑选 基于蛋白质在发育和疾病过程中的重要性以及 没有交叉反应的商业抗体。从这些免疫接种中，我们将产生和 将10,000多个表达克隆性免疫球蛋白的氢化瘤存档，这些肿瘤将通过免疫荧光进行筛查- 发光法、全包埋免疫染色和免疫沉淀法鉴定单抗 到天然蛋白质。产生的抗该复合体的单抗的最终鉴定 胚胎提取物将通过质谱学和免疫荧光技术在 重组假定靶点(S)。这些单抗，连同鉴定数据 将通过发育杂交瘤研究库和Xenbase免费分发给社区。

PROJECT SUMMARY: The amphibian embryo and in particular the frog Xenopus laevis is an outstanding model used by embryologist and cell biologist worldwide. This model system has led the discovery of axis formation (antero/posterior and dorso/ventral), tissue induction and nuclear reprograming among others. Advances in genome sequencing, proteomics and gene silencing have rejuvenated this model. In its most recent White Paper, the Xenopus community has identified a lack of adequate resource of antibody to study Xenopus proteins and proposed the production of such resources a priority. We propose to produce at least a hundred fully characterized monoclonal antibodies to transcription factors, cell surface proteins including signaling receptors and ligands as well as tissue specific markers. To achieve this goal we will immunize mice with both selected recombinant proteins expressed in mammalian cells and complex protein extracts isolated from Xenopus embryos. The recombinant protein targets will be selected by a steering committee composed of Xenopus investigators based on the importance of the proteins in developmental and diseases processes and the absence of cross-reactive commercial antibodies. From these immunizations we will produce and archive more than 10,000 clonal IgG-expressing hyrbidomas that will be screened by immunofluor- escence, whole mount immunostaining and immunoprecipitation to identify monoclonal anti-bodies to native proteins. Final characterization of monoclonal antibodies produced against the complex embryonic extracts will be performed by mass spectrometry followed by immuno-fluorescence on the recombinant putative target(s). These monoclonal antibodies, together with the characterization data will be freely distributed to the community via the developmental hybridoma study bank and Xenbase.