Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants

衣藻的反向遗传学:分离插入突变体的平台

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作者:David Gonzalez-Ballester, Wirulda Pootakham, Florence Mus, Wenqiang Yang, Claudia Catalanotti, Leonardo Magneschi, Amaury de Montaigu, Jose J Higuera, Matthew Prior, Aurora Galván, Emilio Fernandez, Arthur R Grossman

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.

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