Group II Intron-Based Gene Targeting Methods for Xenopus

描述（申请人提供）：我们拟开发一种高效的非洲爪蟾和热带爪蟾II类内含子基因靶向方法。可移动的II组内含子通过一种独特的机制，即内含子RNA直接反向剪接到一条DNA链上，而相关的内含子编码蛋白则切割另一条DNA链，并使用切割后的3'端作为插入的内含子RNA逆转录的引物。该机制由内含子编码蛋白和切除的内含子RNA的RNP复合物介导，并利用两者进行DNA靶位点识别，其中大部分特异性来自内含子RNA与靶序列的碱基配对。这一特性与它们非常高的插入频率和特异性相结合，使得将移动的II族内含子开发成高效的细菌基因靶向载体（“靶子”）成为可能，只需修改内含子RNA，就可以对其进行重新编程，将其插入所需的DNA目标位点。本研究的目的是为非洲爪蟾开发类似的II族内含子基因靶向方法。具体目标是：(1)建立将II类内含子RNPs注射到非洲爪蟾卵母细胞和/或卵中的基因靶向方法。(2)通过修改常用的非洲爪蟾转基因方案，建立去致密化非洲爪蟾精核中II族内含子基因的靶向方法。(3)建立从注射DNA和/或RNA构建物中表达II组内含子RNPs的非洲爪蟾基因靶向方法。(4)将Aims 1-3中开发的方法扩展到热带爪蟾，并利用II组内含子在热带爪蟾中产生基因敲除和gfp融合敲入动物系。本项目将为非洲爪蟾发育和细胞生物学的遗传研究提供重要的新技术。这也将是我们最终目标的重要一步，可用于真核生物的基于II族内含子的基因靶向系统，在基因工程，功能基因组学和基因治疗方面具有潜在的广泛应用。

DESCRIPTION (provided by applicant): We propose to develop efficient group II intron-based gene targeting methods for Xenopus laevis and Xenopus tropicalis. Mobile group II introns insert site-specifically into DNA target sites by a remarkable mechanism in which the intron RNA reverse splices directly into one DNA strand, while the associated intron-encoded protein cleaves the opposite strand and uses the cleaved 3' end as a primer for reverse transcription of the inserted intron RNA. This mechanism is mediated by an RNP complex that contains the intron-encoded protein and the excised intron RNA and uses both for DNA target site recognition, with most of the specificity coming from base pairing of the intron RNA to the target sequence. This feature combined with their very high insertion frequencies and specificity have made it possible to develop mobile group II introns into highly efficient bacterial gene targeting vectors ("targetrons"), which can be reprogrammed to insert into desired DNA target sites simply by modifying the intron RNA. The objective of the proposed research is to develop analogous group II intron-based gene targeting methods for Xenopus. Specific aims are: (1) To develop methods for gene targeting via injection of group II intron RNPs into Xenopus laevis oocytes and/or eggs. (2) To develop methods for group II intron-gene targeting in decondensed Xenopus laevis sperm nuclei by modifcation of commonly used Xenopus transgenesis protocols. (3) To develop methods for gene targeting in Xenopus laevis by expressing group II intron RNPs from injected DNA and/or RNA constructs. (4) To extend the methods developed in Aims 1-3 to Xenopus tropicalis and to use group II introns to generate gene knockout and GFP-fusion knock-in animal lines in Xenopus tropicalis. We anticipate that this project will provide important new technologies for genetic studies of developmental and cell biology in Xenopus. It will also be a major step toward our ultimate goal of generalizable group II intron- based gene targeting systems for eukaryotes, with potentially broad applications in genetic engineering, functional genomics, and gene therapy.