Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants

Plant Methods

Arthur R Grossman²  Emilio Fernandez¹  Aurora Galván¹  Matthew Prior²  Jose J Higuera¹  Amaury de Montaigu⁴  Leonardo Magneschi⁵  Claudia Catalanotti²  Wenqiang Yang²  Florence Mus³  Wirulda Pootakham⁶  David Gonzalez-Ballester¹ 

[1] Departamento de Bioquímica y Biología Molecular, Universidad de Córdoba, Córdoba 14071, Spain;Department of Plant Biology, The Carnegie Institution for Science, Stanford, CA 94305, USA;Montana State University, Department of Chemical and Biological Engineering, and Department of Microbiology, Bozeman, MT 59171, USA;Max Planck Insitute for Plant Breeding Research, Department of Plant Developmental Biology, D-50829, Köln, Germany;PlantLab, Scuola Superiore Sant'Anna, 56127 Pisa, Italy;National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathumthani, 12120, Thailand

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

Others  :  822985
DOI  :  10.1186/1746-4811-7-24

 received in 2011-04-11, accepted in 2011-07-27,  发布年份 2011

【 摘 要 】

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.

【 授权许可】

   
2011 Gonzalez-Ballester et al; licensee BioMed Central Ltd.

【 图 表 】

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