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Article: One-step multiplex toolkit for efficient generation of conditional gene silencing human cell lines

TitleOne-step multiplex toolkit for efficient generation of conditional gene silencing human cell lines
Authors
Issue Date2021
Citation
Molecular Biology of the Cell, 2021, v. 32, n. 14, p. 1320-1330 How to Cite?
AbstractLoss-of-function analysis is one of the major arsenals we have for understanding gene functions in mammalian cells. For analysis of essential genes, the major challenge is to develop simple methodologies for tight and rapid inducible gene inactivation. One approach involves CRISPR-Cas9-mediated disruption of the endogenous locus in conjunction with the expression of a rescue construct, which can subsequently be turned off to produce a gene inactivation effect. Here we describe the development of a set of Sleeping Beauty transposon-based vectors for expressing auxin-inducible degron (AID)-tagged genes under the regulation of a tetracycline-controlled promoter. The dual transcriptional and degron-mediated post-translational regulation allows rapid and tight silencing of protein expression in mammalian cells. We demonstrated that both non-essential and essential genes could be targeted in human cell lines using a one-step transfection method. Moreover, multiple genes could be simultaneously or sequentially targeted, allowing inducible inactivation of multiple genes. These resources enable highly efficient generation of conditional gene silencing cell lines to facilitate functional studies of essential genes.
Persistent Identifierhttp://hdl.handle.net/10722/307314
ISSN
2023 Impact Factor: 3.1
2023 SCImago Journal Rankings: 1.566
PubMed Central ID
ISI Accession Number ID

 

DC FieldValueLanguage
dc.contributor.authorYeung, Tsz Kwan-
dc.contributor.authorLau, Ho Wai-
dc.contributor.authorMa, Hoi Tang-
dc.contributor.authorPoon, Randy Y.C.-
dc.date.accessioned2021-11-03T06:22:21Z-
dc.date.available2021-11-03T06:22:21Z-
dc.date.issued2021-
dc.identifier.citationMolecular Biology of the Cell, 2021, v. 32, n. 14, p. 1320-1330-
dc.identifier.issn1059-1524-
dc.identifier.urihttp://hdl.handle.net/10722/307314-
dc.description.abstractLoss-of-function analysis is one of the major arsenals we have for understanding gene functions in mammalian cells. For analysis of essential genes, the major challenge is to develop simple methodologies for tight and rapid inducible gene inactivation. One approach involves CRISPR-Cas9-mediated disruption of the endogenous locus in conjunction with the expression of a rescue construct, which can subsequently be turned off to produce a gene inactivation effect. Here we describe the development of a set of Sleeping Beauty transposon-based vectors for expressing auxin-inducible degron (AID)-tagged genes under the regulation of a tetracycline-controlled promoter. The dual transcriptional and degron-mediated post-translational regulation allows rapid and tight silencing of protein expression in mammalian cells. We demonstrated that both non-essential and essential genes could be targeted in human cell lines using a one-step transfection method. Moreover, multiple genes could be simultaneously or sequentially targeted, allowing inducible inactivation of multiple genes. These resources enable highly efficient generation of conditional gene silencing cell lines to facilitate functional studies of essential genes.-
dc.languageeng-
dc.relation.ispartofMolecular Biology of the Cell-
dc.rightsThis work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.-
dc.titleOne-step multiplex toolkit for efficient generation of conditional gene silencing human cell lines-
dc.typeArticle-
dc.description.naturepublished_or_final_version-
dc.identifier.doi10.1091/mbc.E21-02-0051-
dc.identifier.pmid33979199-
dc.identifier.pmcidPMC8351548-
dc.identifier.scopuseid_2-s2.0-85108998266-
dc.identifier.volume32-
dc.identifier.issue14-
dc.identifier.spage1320-
dc.identifier.epage1330-
dc.identifier.eissn1939-4586-
dc.identifier.isiWOS:000667949900006-

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