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Open Access Methodology

Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants

David Gonzalez-Ballester12*, Wirulda Pootakham13, Florence Mus14, Wenqiang Yang1, Claudia Catalanotti1, Leonardo Magneschi15, Amaury de Montaigu26, Jose J Higuera2, Matthew Prior1, Aurora Galván2, Emilio Fernandez2 and Arthur R Grossman1

Author Affiliations

1 Department of Plant Biology, The Carnegie Institution for Science, Stanford, CA 94305, USA

2 Departamento de Bioquímica y Biología Molecular, Universidad de Córdoba, Córdoba 14071, Spain

3 National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathumthani, 12120, Thailand

4 Montana State University, Department of Chemical and Biological Engineering, and Department of Microbiology, Bozeman, MT 59171, USA

5 PlantLab, Scuola Superiore Sant'Anna, 56127 Pisa, Italy

6 Max Planck Insitute for Plant Breeding Research, Department of Plant Developmental Biology, D-50829, Köln, Germany

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Plant Methods 2011, 7:24  doi:10.1186/1746-4811-7-24

Published: 27 July 2011

Abstract

A method was developed to identify insertional mutants of Chlamydomonas reinhardtii disrupted for selected target genes. The approach relies on the generation of thousands of transformants followed by PCR-based screenings that allow for identification of strains harboring the introduced marker gene within specific genes of interest. Our results highlight the strengths and limitations of two independent screens that differed in the nature of the marker DNA used (PCR-amplified fragment containing the plasmid-free marker versus entire linearized plasmid with the marker) and in the strategies used to maintain and store transformants.

Keywords:
reverse genetics; insertional mutants; transformation; mutant library; mutant screening; paromomycin resistance; PCR-based screening