Abstract
Sphingolipids are essential components in eukaryotes and have various cellular functions. Recent developments in genome-editing technologies have facilitated gene disruption in various organisms and cell lines. We here show the disruption of various sphingolipid metabolic genes in human cervical carcinoma HeLa cells by using transcription activator-like effector nucleases (TALENs). A TALEN pair targeting the human CERT gene (alternative name COL4A3BP) encoding a ceramide transport protein induced a loss-of-function phenotype in more than 60% of HeLa cells even though the cell line has a pseudo-triploid karyotype. We have isolated several loss-of-function mutant clones for CERT, UGCG (encoding glucosylceramide synthase), and B4GalT5 (encoding the major lactosylceramide synthase), and also a CERT/UGCG double-deficient clone. Characterization of these clones supported previous proposals that CERT primarily contributes to the synthesis of SM but not GlcCer, and that B4GalT5 is the major LacCer synthase. These newly established sphingolipid-deficient HeLa cell mutants together with our previously established stable transfectants provide a 'sphingolipid-modified HeLa cell panel,' which will be useful to elucidate the functions of various sphingolipid species against essentially the same genomic background.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Base Sequence
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Cationic Amino Acid Transporter 1 / genetics*
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Cationic Amino Acid Transporter 1 / metabolism
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Deoxyribonucleases / metabolism*
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Galactosyltransferases / genetics*
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Galactosyltransferases / metabolism
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Glucosyltransferases / genetics*
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Glucosyltransferases / metabolism
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HeLa Cells
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Humans
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Mice
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Molecular Sequence Data
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Mutation / genetics*
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Protein Serine-Threonine Kinases / genetics*
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Protein Serine-Threonine Kinases / metabolism
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RNA, Messenger / genetics
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Real-Time Polymerase Chain Reaction
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Reverse Transcriptase Polymerase Chain Reaction
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Sequence Homology, Nucleic Acid
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Sphingolipids / metabolism*
Substances
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Cationic Amino Acid Transporter 1
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RNA, Messenger
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Sphingolipids
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Galactosyltransferases
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Glucosyltransferases
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UDPgalactose-glucosylceramide galactosyltransferase
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ceramide glucosyltransferase
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CERT1 protein, human
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Protein Serine-Threonine Kinases
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Deoxyribonucleases
Grants and funding
This work was supported by the Japan Society for the Promotion of Science (Grant-in-Aid for Young Scientists (B), No. 23770164 to T.Y., Grant-in-Aid for Scientific Research (B), No. 22370054 to K.H., and Grant-in-Aid for Challenging Exploratory Research, No. 25670045 to K.H.), and the Naito Foundation (the 39th Subsidy for Promotion of Specific Research Projects to T.Y.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.