Production and characterization of monoclonal antibodies to Xenopus Proteins

项目概要： 两栖动物胚胎，特别是青蛙非洲爪蟾是一个杰出的模型， 胚胎学家和细胞生物学家。这个模型系统导致了轴形成的发现 （前/后和背/腹）、组织诱导和核重编程等。 基因组测序、蛋白质组学和基因沉默的进展使这一模型重新焕发了活力。在 在其最新的白色文件中，非洲爪蟾社区已经确定缺乏足够的资源， 抗体，研究爪蟾蛋白质，并提出生产此类资源的优先事项。我们 我计划生产至少一百种完全表征的单克隆抗体， 细胞表面蛋白，包括信号受体和配体以及组织特异性 标记。为了实现这一目标，我们将用两种选定的重组蛋白免疫小鼠 表达于哺乳动物细胞和从非洲爪蟾胚胎分离的复合蛋白提取物中。 重组蛋白靶点将由非洲爪蟾组成的指导委员会选择 研究人员基于蛋白质在发育和疾病过程中的重要性， 缺乏交叉反应的商业抗体。从这些免疫中，我们将产生和 存档超过10，000个克隆IgG表达的汗血瘤，这些汗血瘤将通过免疫荧光筛选， 荧光、整体包埋免疫染色和免疫沉淀以鉴定单克隆抗体 天然蛋白质。针对复合物产生的单克隆抗体的最终表征 胚胎提取物将通过质谱法进行，然后在 重组推定靶标。这些单克隆抗体，连同表征数据 将通过开发杂交瘤研究库和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.