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Nuclear-Gene Targeting by Using Single-Stranded DNA Avoids Illegitimate DNA Integration in Chlamydomonas reinhardtii

Boris Zorin, Peter Hegemann, and Irina Sizova^*

Institut für Biologie, Experimentelle Biophysik, Humboldt-Universität zu Berlin, Invalidenstr.42, 10115 Berlin, Germany

Received 26 March 2005/ Accepted 3 May 2005

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.

This publication is dedicated to Karen Kindle, who convinced P.H. in 1994 that nuclear-gene targeting is possible in Chlamydomonas.

Present address: Division of Molecular and Radiation Biophysics, Petersburg Nuclear Physics Institute, Russian Academy of Sciences, Gatchina/St. Petersburg, 188350 Russia.

Present address: Biological Institute, St. Petersburg State University, Oranienbaumskoye sch, 2, St. Petersburg 198904, Russia.

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