1. Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants
- Author
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de Montaigu Amaury, Magneschi Leonardo, Catalanotti Claudia, Yang Wenqiang, Mus Florence, Pootakham Wirulda, Gonzalez-Ballester David, Higuera Jose J, Prior Matthew, Galván Aurora, Fernandez Emilio, and Grossman Arthur R
- Subjects
reverse genetics ,insertional mutants ,transformation ,mutant library ,mutant screening ,paromomycin resistance ,PCR-based screening ,Plant culture ,SB1-1110 ,Biology (General) ,QH301-705.5 - Abstract
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.
- Published
- 2011
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