Document Detail


Nuclear gene targeting in Chlamydomonas using engineered zinc-finger nucleases.
MedLine Citation:
PMID:  23137232     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
The unicellular green alga Chlamydomonas reinhardtii is a versatile model for fundamental and biotechnological research. A wide toolset for genetic manipulation has been developed for this alga, but specific modification of nuclear genes is still not routinely possible. Here, we present a nuclear gene targeting strategy for Chlamydomonas that is based on the application of zinc-finger nucleases (ZFNs). Our approach includes 1) design of gene-specific ZFNs using available online tools; 2) evaluation of the designed ZFNs in a Chlamydomonas in situ model system; 3) optimization of ZFN activity by modification of the nuclease domain; and 4) application of the most suitable enzymes for the mutagenesis of an endogenous gene. Initially, we designed a set of ZFNs for targeting the COP3 gene that encodes the light-activated ion channel channelrhodopsin-1. To evaluate the designed ZFNs, we constructed a model strain by inserting a non-functional aminoglycoside 3'-phosphotransferase VIII (aphVIII) selection marker interspaced with a short COP3 target sequence into the nuclear genome. Upon co-transformation of this recipient strain with the engineered ZFNs and an aphVIII DNA template, we were able to restore marker activity and select paromomycin resistant (Pm-R) clones with active nucleases. Of these Pm-R clones, 1% contained a modified COP3 locus as well. In cases where cells were co-transformed with a modified COP3 template, the COP3 locus was specifically modified accordingly by homologous recombination between COP3 and the supplied template DNA. We anticipate that this ZFN technology will be useful for studying the functions of individual genes in Chlamydomonas. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.
Authors:
Irina Sizova; Andre Greiner; Mayanka Awasthi; Suneel Kateriya; Peter Hegemann
Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-11-8
Journal Detail:
Title:  The Plant journal : for cell and molecular biology     Volume:  -     ISSN:  1365-313X     ISO Abbreviation:  Plant J.     Publication Date:  2012 Nov 
Date Detail:
Created Date:  2012-11-9     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9207397     Medline TA:  Plant J     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Copyright Information:
© 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.
Affiliation:
Institute of Biology, Experimental Biophysics, Humboldt-Universität zu Berlin, Berlin, Germany; Division of Radiation Biophysics, Petersburg Nuclear Physics, Institute, Russian Academy of Sciences, Gatchina/St. Petersburg, 188350, Russia.
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