Nuclear-gene Targeting by Using Single-stranded DNA Avoids Illegitimate DNA Integration in Chlamydomonas Reinhardtii

Overview

Journal Eukaryot Cell

Publisher American Society for Microbiology

Specialty Molecular Biology

Date 2005 Jul 9

PMID 16002652

Citations 34

Authors

Boris Zorin

Peter Hegemann

Irina Sizova

Affiliations

Soon will be listed here.

Abstract

Homologous DNA recombination (HR) allows the deletion (knockout), repair (rescuing), and modification of a selected gene, thereby rendering a functional analysis of the gene product possible. However, targeting of nuclear genes has been an inefficient process in most eukaryotes, including algae, plants, and animals, due to the dominance of integration of the applied DNA into nonhomologous regions of the genome. We have shown for the green alga Chlamydomonas reinhardtii by repairing a previously introduced truncated aminoglycoside 3'-phosphotransferase gene, aphVIII, that single-stranded DNA can recombine with a homologous endogenous DNA region of interest. Nonhomologous DNA integration appeared to be more than 100-fold reduced compared with the use of double-stranded DNA, thus allowing isolation of the homologous recombinants. We propose that this method will be applicable to direct targeting of nuclear C. reinhardtii genes.

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