Group II Intron-Based Gene Targeting Methods for Xenopus

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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.