Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants

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Author
Pootakham, Wirulda
Mus, Florence
Yang, Wenqiang
Catalanotti, Claudia
Magneschi, Leonardo
Montaigu, Amaury de
Higuera, Jose J.
Prior, Matthew
Galván Cejudo, Aurora
Fernández Reyes, Emilio
Grossman, Arthur R.
González-Ballester, David
Publisher
BioMed CentralDate
2011Subject
Reverse geneticsInsertional mutants
Transformation
Mutant library
Mutant screening
Paromomyce resistance
PCR-based screening
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Show full item recordAbstract
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.