Group II Intron-Based Gene Targeting Methods for Xenopus

描述（由申请人提供）：我们建议开发针对非洲爪蟾和热带爪蟾的有效的基于II组内含子的基因靶向方法。移动的II组内含子通过一种显著的机制位点特异性地插入DNA靶位点，其中内含子RNA直接反向剪接成一条DNA链，而相关的内含子编码的蛋白质切割相对的链，并使用切割的3'端作为引物逆转录插入的内含子RNA。该机制由RNP复合物介导，该复合物含有内含子编码的蛋白质和切除的内含子RNA，并将两者用于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。该特征与它们非常高的插入频率和特异性相结合，使得有可能将移动的II组内含子开发成高效细菌基因靶向载体（“靶向子”），其可以简单地通过修饰内含子RNA而被重编程以插入所需的DNA靶位点。拟议的研究的目的是开发类似的第二组内含子为基础的非洲爪蟾基因打靶方法。具体目标是：（1）开发通过将II组内含子RNP注射到非洲爪蟾卵母细胞和/或卵中进行基因打靶的方法。(2)通过改进常用的非洲爪蟾转基因方法，建立去致密非洲爪蟾精子核中II组内含子基因的打靶方法。(3)通过从注射的DNA和/或RNA构建体中表达II组内含子RNP，开发非洲爪蟾基因靶向的方法。(4)将目的1-3中开发的方法扩展到热带爪蟾，并使用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。我们预计，这一项目将提供重要的新技术，非洲爪蟾的发育和细胞生物学的遗传研究。这也将是我们朝着真核生物的可推广的基于II组内含子的基因靶向系统的最终目标迈出的重要一步，在基因工程，功能基因组学和基因治疗中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.