Group II Intron-Based Gene Targeting Methods for Xenopus

基于第二组内含子的非洲爪蟾基因打靶方法

• 批准号: 7364153
• 负责人: ALAN M. LAMBOWITZ
• 金额: $ 24.75万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7364153
• 关键词: Animal Model; Animals; Base Pairing; Catalytic RNA; Cell Nucleus; Cleaved cell; Complex; DNA; Developmental Cell Biology; Eukaryota; Eukaryotic Cell; Frequencies; Gene Targeting; Gene Transfer Techniques; Genes; Genetic; Genetic Engineering; Goals; Human; Injection of therapeutic agent; Introns; Knock-in Mouse; Knock-out; Mediating; Methods; Modification; Oligonucleotides; Oocytes; Organism; Positioning Attribute; Proteins; Protocols documentation; RNA; RNA Interference; RNA Splicing; Range; Research; Reverse Transcription; Ribonucleoproteins; Sales; Site; Specificity; System; Technology; Vertebrates; Xenopus; Xenopus laevis; base; desire; egg; functional genomics; gene therapy; homologous recombination; knock-down; knockout gene; new technology; novel; sperm cell; vector

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.

描述（由申请人提供）：我们建议开发用于Xenopus laevis和xenopus tropicalis的有效基于II组内含子的基因靶向方法。移动组II内含子通过一种显着的机制将位点特异性插入到DNA靶位点中，其中内含子RNA直接将剪接直接剪接到一个DNA链中，而相关的内含子编码的蛋白质将相反的链切开，并将相反的3'端用作插入插入式内含子RNA的反向转录。该机制是由RNP复合物介导的，RNP复合物包含内含子编码的蛋白和切除的内含子RNA，并将两者都用于DNA靶位点识别，其中大多数特异性来自内含子RNA的碱基配对到目标序列。该特征结合其非常高的插入频率和特异性，使将移动II组内含子开发为高效的细菌基因靶向矢量（“靶标”），可以将其重新编程以通过修饰含有内含子RNA插入所需的DNA目标位点。拟议的研究的目的是开发基于II组内含子内含子的基因靶向爪蟾的方法。具体目的是：（1）通过将II组内含子RNP注射到Xenopus laevis卵母细胞和/或卵中开发用于基因靶向的方法。 （2）通过修饰常用的Xenopus转基因方案来开发对Xenopus laevis精​​子核中Xenopus laevis精​​子核中的II组内基因靶向的方法。 （3）通过从注射的DNA和/或RNA构建体中表达II组内含子RNP来开发Xenopus laevis中基因靶向的方法。 （4）将目标1-3中开发的方法扩展到热带异常的方法，并使用II组内含子在Xenopus tropicalis中生成基因敲除和GFP融合敲除动物系。我们预计该项目将为Xenopus的发育和细胞生物学遗传研究提供重要的新技术。这也将是朝着我们迈向真核生物的基于II组内含子基因靶向系统的最终目标的重要一步，在基因工程，功能基因组学和基因治疗中可能广泛应用。