Cargando…
One-step multiplex toolkit for efficient generation of conditional gene silencing human cell lines
Loss-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 disr...
Autores principales: | , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The American Society for Cell Biology
2021
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8351548/ https://www.ncbi.nlm.nih.gov/pubmed/33979199 http://dx.doi.org/10.1091/mbc.E21-02-0051 |
_version_ | 1783735994722287616 |
---|---|
author | Yeung, Tsz Kwan Lau, Ho Wai Ma, Hoi Tang Poon, Randy Y. C. |
author_facet | Yeung, Tsz Kwan Lau, Ho Wai Ma, Hoi Tang Poon, Randy Y. C. |
author_sort | Yeung, Tsz Kwan |
collection | PubMed |
description | Loss-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. |
format | Online Article Text |
id | pubmed-8351548 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | The American Society for Cell Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-83515482021-09-16 One-step multiplex toolkit for efficient generation of conditional gene silencing human cell lines Yeung, Tsz Kwan Lau, Ho Wai Ma, Hoi Tang Poon, Randy Y. C. Mol Biol Cell Articles Loss-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. The American Society for Cell Biology 2021-07-01 /pmc/articles/PMC8351548/ /pubmed/33979199 http://dx.doi.org/10.1091/mbc.E21-02-0051 Text en © 2021 Yeung et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology. https://creativecommons.org/licenses/by-nc-sa/3.0/This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License. |
spellingShingle | Articles Yeung, Tsz Kwan Lau, Ho Wai Ma, Hoi Tang Poon, Randy Y. C. One-step multiplex toolkit for efficient generation of conditional gene silencing human cell lines |
title | One-step multiplex toolkit for efficient generation of conditional gene silencing human cell lines |
title_full | One-step multiplex toolkit for efficient generation of conditional gene silencing human cell lines |
title_fullStr | One-step multiplex toolkit for efficient generation of conditional gene silencing human cell lines |
title_full_unstemmed | One-step multiplex toolkit for efficient generation of conditional gene silencing human cell lines |
title_short | One-step multiplex toolkit for efficient generation of conditional gene silencing human cell lines |
title_sort | one-step multiplex toolkit for efficient generation of conditional gene silencing human cell lines |
topic | Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8351548/ https://www.ncbi.nlm.nih.gov/pubmed/33979199 http://dx.doi.org/10.1091/mbc.E21-02-0051 |
work_keys_str_mv | AT yeungtszkwan onestepmultiplextoolkitforefficientgenerationofconditionalgenesilencinghumancelllines AT lauhowai onestepmultiplextoolkitforefficientgenerationofconditionalgenesilencinghumancelllines AT mahoitang onestepmultiplextoolkitforefficientgenerationofconditionalgenesilencinghumancelllines AT poonrandyyc onestepmultiplextoolkitforefficientgenerationofconditionalgenesilencinghumancelllines |