Developing of a high-throughput insertional mutation-based screening strategy in Chlamydomonas reinhardtii
Wenqiang Yang1, Claudia Catalanotti1, David Dewez1, David Gonzales-Ballester1, Mark Heinnikel1, Wirulda Pootakham1,2, Florence Mus1, and Arthur R. Grossman1
1) Carnegie Institution for Science, Stanford, CA
2) Stanford University, Department of Biology, Stanford, CA
In Chlamydomonas reinhardtii, random insertional mutagenesis, using drug resistance marker genes, has proven to be a very useful tool for generating mutants with specific phenotypes. The disrupted gene in the mutants can then be rapidly identified based on localization of the marker gene in the genome (forward genetics). Alternatively, a PCR-based approach can be used to screen an insertional library for disruptions in specific genes that are associated with biological processes of interest (reverse genetics). We have used this latter screening approach with a population of mutants in which the AphVIII gene (paromomycin resistance) was randomly inserted into the Chlamydomonas genome. Paromomycin-resistant transformants were individually picked, transferred to 96-well microtiter plates and grown for ~5 days. Genomic DNA was then isolated from each pool of 96 transformants (one microtitre plate) and 10 pools of DNA were combined to create a “DNA superpool”. To screen for strains with lesions of interest, PCR reactions using the superpool DNA as template were performed with gene-specific primers (in the gene for which a disruption is sought) and a primer in the AphVIII-sequence (designated RB2 primer). The PCR products generated in these reactions were excised from the gel, purified and sequenced to insure that the specific gene disrupted was the one that was targeted. We then identified the specific microtitre plates that contained the disrupted gene and purified individual mutants by analyzing transformants in the wells within both the columns and rows of the selected plates. This strategy has been used to generate insertions in genes encoding proteins involved in fermentation metabolism, sulfur deprivation responses, and photosynthetic function.
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