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A protocol for screening of autophagy regulatory genes applying cell sorting and next generation sequencing technologies
Author(s) -
Huang WeiPang,
Liu AngYu
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.994.9
Subject(s) - autophagy , vacuole , mutant , saccharomyces cerevisiae , biology , microbiology and biotechnology , gene , genetic screen , cell sorting , vacuolar protein sorting , yeast , cytoplasm , cell , computational biology , genetics , apoptosis
Autophagy, as a conserved pathway in eukaryotic cells, plays a critical role in maintaining cellular homeostasis by delivering cytoplasmic materials to lysosomes/vacuoles for degradation. Abnormal autophagy activities are associated with several human diseases, such as diabetes, neurodegenerative diseases, and cancers. These observation leads to the possibilities of manipulating autophagy for therapeutic applications. To achieve this purpose, detailed information on the molecular mechanisms of autophagy regulation is essential. In this study, we have developed a protocol to screen for more selective autophagy regulatory genes based on cell image analysis, which is compliable to cell sorter application. We constructed a plasmid for expressing peroxisome‐targeted pH‐sensitive green fluorescent proteins in Saccharomyces cerevisiae . The plasmid was introduced into yeast deletion mutant pools and pexophagy‐deficient cells with high fluorescent signals after induction of pexophagy was sorted out. The identities of the mutated genes of those selected cells were determined by next generation sequencing of the gene barcodes. Our results proved it is able to distinguish atg mutants from wild types cells. We are currently characterizing the isolated autophagy mutants from the nonessential yeast gene deletion mutant pool. Grant support: NSC 101–2311‐B‐002–005