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Genetic Screening of New Genes Responsible for Cellular Adaptation to Hypoxia Using a Genome-Wide shRNA Library
Oxygen is a vital requirement for multi-cellular organisms to generate energy and cells have developed multiple compensatory mechanisms to adapt to stressful hypoxic conditions. Such adaptive mechanisms are intricately interconnected with other signaling pathways that regulate cellular functions suc...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3327663/ https://www.ncbi.nlm.nih.gov/pubmed/22523603 http://dx.doi.org/10.1371/journal.pone.0035590 |
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author | Yoshino, Seiko Hara, Toshiro Weng, Jane S. Takahashi, Yuka Seiki, Motoharu Sakamoto, Takeharu |
author_facet | Yoshino, Seiko Hara, Toshiro Weng, Jane S. Takahashi, Yuka Seiki, Motoharu Sakamoto, Takeharu |
author_sort | Yoshino, Seiko |
collection | PubMed |
description | Oxygen is a vital requirement for multi-cellular organisms to generate energy and cells have developed multiple compensatory mechanisms to adapt to stressful hypoxic conditions. Such adaptive mechanisms are intricately interconnected with other signaling pathways that regulate cellular functions such as cell growth. However, our understanding of the overall system governing the cellular response to the availability of oxygen remains limited. To identify new genes involved in the response to hypoxic stress, we have performed a genome-wide gene knockdown analysis in human lung carcinoma PC8 cells using an shRNA library carried by a lentiviral vector. The knockdown analysis was performed under both normoxic and hypoxic conditions to identify shRNA sequences enriched or lost in the resulting selected cell populations. Consequently, we identified 56 candidate genes that might contribute to the cellular response to hypoxia. Subsequent individual knockdown of each gene demonstrated that 13 of these have a significant effect upon oxygen-sensitive cell growth. The identification of BCL2L1, which encodes a Bcl-2 family protein that plays a role in cell survival by preventing apoptosis, validates the successful design of our screen. The other selected genes have not previously been directly implicated in the cellular response to hypoxia. Interestingly, hypoxia did not directly enhance the expression of any of the identified genes, suggesting that we have identified a new class of genes that have been missed by conventional gene expression analyses to identify hypoxia response genes. Thus, our genetic screening method using a genome-wide shRNA library and the newly-identified genes represent useful tools to analyze the cellular systems that respond to hypoxic stress. |
format | Online Article Text |
id | pubmed-3327663 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-33276632012-04-20 Genetic Screening of New Genes Responsible for Cellular Adaptation to Hypoxia Using a Genome-Wide shRNA Library Yoshino, Seiko Hara, Toshiro Weng, Jane S. Takahashi, Yuka Seiki, Motoharu Sakamoto, Takeharu PLoS One Research Article Oxygen is a vital requirement for multi-cellular organisms to generate energy and cells have developed multiple compensatory mechanisms to adapt to stressful hypoxic conditions. Such adaptive mechanisms are intricately interconnected with other signaling pathways that regulate cellular functions such as cell growth. However, our understanding of the overall system governing the cellular response to the availability of oxygen remains limited. To identify new genes involved in the response to hypoxic stress, we have performed a genome-wide gene knockdown analysis in human lung carcinoma PC8 cells using an shRNA library carried by a lentiviral vector. The knockdown analysis was performed under both normoxic and hypoxic conditions to identify shRNA sequences enriched or lost in the resulting selected cell populations. Consequently, we identified 56 candidate genes that might contribute to the cellular response to hypoxia. Subsequent individual knockdown of each gene demonstrated that 13 of these have a significant effect upon oxygen-sensitive cell growth. The identification of BCL2L1, which encodes a Bcl-2 family protein that plays a role in cell survival by preventing apoptosis, validates the successful design of our screen. The other selected genes have not previously been directly implicated in the cellular response to hypoxia. Interestingly, hypoxia did not directly enhance the expression of any of the identified genes, suggesting that we have identified a new class of genes that have been missed by conventional gene expression analyses to identify hypoxia response genes. Thus, our genetic screening method using a genome-wide shRNA library and the newly-identified genes represent useful tools to analyze the cellular systems that respond to hypoxic stress. Public Library of Science 2012-04-16 /pmc/articles/PMC3327663/ /pubmed/22523603 http://dx.doi.org/10.1371/journal.pone.0035590 Text en Yoshino et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Yoshino, Seiko Hara, Toshiro Weng, Jane S. Takahashi, Yuka Seiki, Motoharu Sakamoto, Takeharu Genetic Screening of New Genes Responsible for Cellular Adaptation to Hypoxia Using a Genome-Wide shRNA Library |
title | Genetic Screening of New Genes Responsible for Cellular Adaptation to Hypoxia Using a Genome-Wide shRNA Library |
title_full | Genetic Screening of New Genes Responsible for Cellular Adaptation to Hypoxia Using a Genome-Wide shRNA Library |
title_fullStr | Genetic Screening of New Genes Responsible for Cellular Adaptation to Hypoxia Using a Genome-Wide shRNA Library |
title_full_unstemmed | Genetic Screening of New Genes Responsible for Cellular Adaptation to Hypoxia Using a Genome-Wide shRNA Library |
title_short | Genetic Screening of New Genes Responsible for Cellular Adaptation to Hypoxia Using a Genome-Wide shRNA Library |
title_sort | genetic screening of new genes responsible for cellular adaptation to hypoxia using a genome-wide shrna library |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3327663/ https://www.ncbi.nlm.nih.gov/pubmed/22523603 http://dx.doi.org/10.1371/journal.pone.0035590 |
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