One-step multiplex toolkit for efficient generation of conditional gene silencing human cell lines
| Autor: | Hoi Tang Ma, Ho Wai Lau, Tsz Kwan Yeung, Randy Yat Choi Poon |
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| Rok vydání: | 2021 |
| Předmět: |
Genetic Vectors
Gene Expression Transposases Locus (genetics) Computational biology Biology Transfection Cell Line 03 medical and health sciences 0302 clinical medicine Genes Reporter Loss of Function Mutation Animals Humans Gene silencing Multiplex Gene Silencing Promoter Regions Genetic Molecular Biology Gene 030304 developmental biology 0303 health sciences Genes Essential Indoleacetic Acids Articles Cell Biology Sleeping Beauty transposon system Gene Expression Regulation Cell culture 030220 oncology & carcinogenesis CRISPR-Cas Systems Degron Genetic Engineering |
| Zdroj: | Molecular Biology of the Cell |
| ISSN: | 1939-4586 1059-1524 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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