Expanding the Xenopus ORFeome to genome-scale by de novo cloning of protein-coding gene models

我们建议生成、验证和分发第二个版本的Xenopus ORFeome，我们将其定义为一组完全测序的、经过验证的非洲爪哇cdna克隆 包含基因组中编码的每个开放阅读框(ORF)中的一个 任何ORF序列都可以通过重组工程轻松转换为 表达载体的不同阵列。这一套试剂将大大降低 时间来表征任何蛋白质在无数的功能分析中由整个 非洲爪哇社区。但最重要的是，ORFeome集合将允许高吞吐量 体内功能基因组筛选的方式目前还不可行，这是一种特殊的 非洲爪哇系统的优势，其中功能屏幕一直是 基本生物学观测的次数。我们的联合体完成了第一阶段 在该项目中，我们将90%的可用全长cDNA克隆移至Gateway 兼容的矢量。然而，由于原始Xenopus EST项目的限制， 这只占基因组中所有ORF的一半。在这个项目中，我们将从头开始 将剩余的非洲爪哇ORF克隆到Gateway供体载体以生成唯一的 超过一半的基因组都有可用的cdna克隆。这些克隆人将可供使用， 不受限制地向世界各地的研究人员提供。非洲爪哇社区全力支持 在最近的一次PI会议上(MBL(Woodshole)，2015年9月) ORFeome被选为非洲爪哇所需资源的重中之重。

We propose to generate, validate and distribute the second version of the Xenopus ORFeome, which we define as a fully sequenced, validated set of Xenopus cDNA clones containing one each of every open reading frame (ORF) encoded in the genome, in a format in which any ORF sequence can be easily transferred using recombineering into a diverse array of expression vectors. This one set of reagents will greatly decrease the time to characterize any protein in the myriad functional assays carried out by the entire Xenopus community. But most importantly, an ORFeome set will allow high-throughput in vivo functional-genomic screening in manner currently not feasible, which is a particular strength of the Xenopus system where functional screens have been the basis of a number of fundamental biological observations. Our consortium completed the first phase of this project where we moved 90% of the available full-length cDNA clones to Gateway compatible vectors. However, because of limitations of the original Xenopus EST projects this represents only half of all of the ORFs in the genome. In this project we will de novo clone the remaining Xenopus ORFs to a Gateway donor vector to generate the only available cDNA clones for over half the genome. These clones will be made available, without restriction, to researchers worldwide. The Xenopus community is fully supportive of this project and at a recent PI meeting (MBL (Woodshole), September 2015) the ORFeome was voted as the Top priority of needed Xenopus resources.