Group II Intron-Based Gene Targeting Methods for Xenopus

描述(由申请人提供)：我们建议开发有效的基于第二组内含子的基因打靶方法，用于非洲爪哇和热带非洲爪哇。移动的第二组内含子通过一种显著的机制将位点特异性地插入DNA靶点，在这种机制中，内含子RNA直接反向拼接到一条DNA链上，而相关的内含子编码的蛋白质切割相反的链，并使用被切割的3‘端作为引物来逆转录插入的内含子RNA。这一机制是由RNP复合体介导的，RNP复合体包含内含子编码的蛋白质和切除的内含子RNA，并用于DNA靶点识别，其中大部分特异性来自内含子RNA与靶序列的碱基配对。这一特征与其非常高的插入频率和特异性相结合，使得将第二组可移动内含子开发成高效的细菌基因靶向载体成为可能，通过修改内含子RNA，可以重新编程以插入到所需的DNA靶点。这项研究的目的是为非洲爪哇开发类似的基于第二组内含子的基因打靶方法。具体目标是：(1)通过将第二组内含子RNPs注射到非洲爪哇卵母细胞和/或卵中，建立基因打靶的方法。(2)对常用的非洲爪哇转基因方法进行改进，建立非洲爪哇去浓缩精子核中第二组内含子基因打靶的方法。(3)建立非洲爪哇基因打靶的方法，从注射的DNA和/或RNA中表达第二组内含子RNPs。(4)将AIMS 1-3中发展的方法推广到热带非洲爪哇，并利用第二组内含子建立热带非洲爪哇基因敲除和GFP融合敲入动物系。我们期待该项目将为非洲爪哇发育生物学和细胞生物学的遗传学研究提供重要的新技术。这也将是朝着我们的最终目标迈出的重要一步，即可推广的基于真核生物的第二组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.