Targeted forward mutagenesis by transitive RNAi

Petsch, K. A., Chonglie, M., Scanlon, M. J., Jorgensen, R. A. (December 2010) Targeted forward mutagenesis by transitive RNAi. The Plant Journal, 61 (5). pp. 873-882.

URL: http://www.ncbi.nlm.nih.gov/pubmed/20003132
DOI: 10.1111/j.1365-313X.2009.04104.x

Abstract

A novel technique is described that targets specific populations of transcripts for homology-based gene silencing using transitive RNAi. This approach is designed to target a subset of the transcriptome in order to identify genes involved in a particular localized process, such as photosynthesis. As a proof-of-concept approach, mesophyll cells from Arabidopsis thaliana were laser-microdissected from whole leaves to generate a focused cDNA library that was bi-directionally cloned into a transitive RNAi vector that had been designed to induce silencing of homologous, endogenous genes. Approximately 15% of the transformant plants identified from both sense and antisense libraries exhibited visible phenotypes indicative of photosynthetic defects. Amplification from the genome and sequencing of cDNA inserts identified candidate genes underlying the phenotypes. For 10 of 11 such mutants, re-transformation with an RNAi construct corresponding to the candidate gene recapitulated the original mutant phenotype, and reduction of corresponding endogene transcripts was confirmed. In addition, one of the re-transformed transgenes also silenced transcripts of closely related family members, thereby demonstrating the utility of this approach for mutagenesis of redundant gene functions. Preliminary results using tissue-specific transitive RNAi forward mutagenesis of the Arabidopsis vegetative shoot apical meristem demonstrate the broad applicability of this forward mutagenesis technique for a variety of plant cell types. © 2010 Blackwell Publishing Ltd.

Item Type: Paper
Uncontrolled Keywords: Arabidopsis thaliana Forward mutagenesis Laser capture microdissection Mesophyll cell RNAi
Subjects: organism description > plant > Arabidopsis
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > transcription
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > RNAi
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > cDNA
CSHL Authors:
Communities: CSHL Post Doctoral Fellows
CSHL labs > Timmermans lab
Depositing User: CSHL Librarian
Date: 10 December 2010
Date Deposited: 05 Oct 2011 14:00
Last Modified: 03 May 2013 13:39
Related URLs:
URI: http://repository.cshl.edu/id/eprint/15504

Actions (login required)

Administrator's edit/view item Administrator's edit/view item
CSHL HomeAbout CSHLResearchEducationNews & FeaturesCampus & Public EventsCareersGiving