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: 7169700
• 负责人: ALAN M. LAMBOWITZ
• 金额: $ 25.26万
• 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/7169700
• 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）：我们建议为非洲爪蟾和热带爪蟾开发有效的基于 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 DNA 靶位点，其中内含子 RNA 直接反向剪接至一条 DNA 链中，而相关内含子编码的蛋白质则切割相反的链并使用切割的 3' 端作为引物用于插入的内含子 RNA 的逆转录。该机制由 RNP 复合物介导，该复合物包含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，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，使得将移动组 II 内含子开发成高效的细菌基因靶向载体（“targetrons”）成为可能，只需修改内含子即可对其进行重新编程以插入所需的 DNA 靶位点核糖核酸。拟议研究的目的是为非洲爪蟾开发类似的基于 II 组内含子的基因靶向方法。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/或卵中进行基因靶向的方法。 (2) 通过修改常用的爪蟾转基因方案，开发在解压缩的非洲爪蟾精子核中进行 II 类内含子基因靶向的方法。 (3) 通过从注射的 DNA 和/或 RNA 构建体表达 II 组内含子 RNP，开发非洲爪蟾基因靶向方法。 (4)将目标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。我们预计该项目将为爪蟾发育和细胞生物学的遗传研究提供重要的新技术。这也将是朝着我们的最终目标迈出的重要一步，即真核生物基于 II 类内含子的通用基因靶向系统，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。