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Xenopus的第二个版本 ORFeome，我们将其定义为一组完全测序的，经过验证的非洲爪蟾cDNA克隆 包含基因组中编码的每个开放阅读框（ORF）中的每一个， 任何ORF序列都可以使用重组工程技术容易地转移到一个 多种表达载体。这一套试剂将大大减少 时间来表征任何蛋白质在无数的功能测定进行了整个 非洲爪蟾群落但最重要的是，ORFeome集将允许高通量 以目前不可行的方式进行体内功能基因组筛选，这是一个特别的 Xenopus系统的优势，其中功能屏幕一直是 一些基本的生物学观察。我们的联合体完成了第一阶段 我们将90%的可用全长cDNA克隆转移到Gateway 相容向量然而，由于原始Xenopus EST项目的局限性， 这仅代表基因组中所有ORF的一半。在这个项目中，我们将重新 将剩余的非洲爪蟾ORF克隆到Gateway供体载体上， 超过一半基因组的可用cDNA克隆。这些克隆人将被提供， 不受限制地提供给全世界的研究人员。Xenopus社区全力支持 在最近的PI会议（MBL（Woodshole），2015年9月）上， ORFeome被选为非洲爪蟾资源需求的最高优先级。

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.