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Exploiting orthologue diversity for systematic detection of gain-of-function phenotypes
BACKGROUND: Systematic search for genes whose gain-of-function by exogenous expression confers an advantage in cell-based selective screenings is a powerful method for unbiased functional exploration of the genome, and has the potential to disclose new targets for cancer therapy. A major limit of th...
Autores principales: | , , , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2008
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2435555/ https://www.ncbi.nlm.nih.gov/pubmed/18510758 http://dx.doi.org/10.1186/1471-2164-9-254 |
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author | Martelli, Maria Luisa Isella, Claudio Mira, Alessia Fu, Limin Cantarella, Daniela Medico, Enzo |
author_facet | Martelli, Maria Luisa Isella, Claudio Mira, Alessia Fu, Limin Cantarella, Daniela Medico, Enzo |
author_sort | Martelli, Maria Luisa |
collection | PubMed |
description | BACKGROUND: Systematic search for genes whose gain-of-function by exogenous expression confers an advantage in cell-based selective screenings is a powerful method for unbiased functional exploration of the genome, and has the potential to disclose new targets for cancer therapy. A major limit of this approach resides in the labor-intensive cloning of resistant cells, identification of the integrated genes and validation of their ability to confer a selective advantage. Moreover, the selection has to be drastic and genes conferring a limited advantage are typically missed. RESULTS: We developed a new functional screening strategy based on transduction of mammalian cells of a given species with an expression library from another species, followed by one-shot quantitative tracing with DNA microarrays of all library-derived transcripts before and after selection. In this way, exogenous transcripts enriched after selection, and therefore likely to confer resistance, are readily detected. We transduced a retroviral cDNA expression library from mouse testis into human and canine cells, and optimized the use of commercial murine gene expression arrays for species-specific detection of library-derived transcripts. We then conducted a functional screening by growing library-transduced canine MDCK cells in suspension, to enrich for cDNAs conferring anchorage independence. Notably, these cells show partial resistance to loss of anchorage, and the selection can be of limited stringency, compromising approaches based on clonal selection or anyway requiring high stringency. Microarray analysis revealed reproducible enrichment after three weeks of growth on polyhema for seven genes, among which the Hras proto-oncogene and Sox5. When individually transduced into MDCK cells, Sox5 specifically promoted anchorage-independent growth, thereby confirming the validity and specificity of the approach. CONCLUSION: The procedure described here brings substantial advantages to the field of expression cloning, being faster, more systematic and more sensitive. Indeed, this strategy allowed identification and validation of genes promoting anchorage-independent growth of epithelial cells under selection conditions not amenable to conventional expression cloning. |
format | Text |
id | pubmed-2435555 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2008 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-24355552008-06-24 Exploiting orthologue diversity for systematic detection of gain-of-function phenotypes Martelli, Maria Luisa Isella, Claudio Mira, Alessia Fu, Limin Cantarella, Daniela Medico, Enzo BMC Genomics Methodology Article BACKGROUND: Systematic search for genes whose gain-of-function by exogenous expression confers an advantage in cell-based selective screenings is a powerful method for unbiased functional exploration of the genome, and has the potential to disclose new targets for cancer therapy. A major limit of this approach resides in the labor-intensive cloning of resistant cells, identification of the integrated genes and validation of their ability to confer a selective advantage. Moreover, the selection has to be drastic and genes conferring a limited advantage are typically missed. RESULTS: We developed a new functional screening strategy based on transduction of mammalian cells of a given species with an expression library from another species, followed by one-shot quantitative tracing with DNA microarrays of all library-derived transcripts before and after selection. In this way, exogenous transcripts enriched after selection, and therefore likely to confer resistance, are readily detected. We transduced a retroviral cDNA expression library from mouse testis into human and canine cells, and optimized the use of commercial murine gene expression arrays for species-specific detection of library-derived transcripts. We then conducted a functional screening by growing library-transduced canine MDCK cells in suspension, to enrich for cDNAs conferring anchorage independence. Notably, these cells show partial resistance to loss of anchorage, and the selection can be of limited stringency, compromising approaches based on clonal selection or anyway requiring high stringency. Microarray analysis revealed reproducible enrichment after three weeks of growth on polyhema for seven genes, among which the Hras proto-oncogene and Sox5. When individually transduced into MDCK cells, Sox5 specifically promoted anchorage-independent growth, thereby confirming the validity and specificity of the approach. CONCLUSION: The procedure described here brings substantial advantages to the field of expression cloning, being faster, more systematic and more sensitive. Indeed, this strategy allowed identification and validation of genes promoting anchorage-independent growth of epithelial cells under selection conditions not amenable to conventional expression cloning. BioMed Central 2008-05-29 /pmc/articles/PMC2435555/ /pubmed/18510758 http://dx.doi.org/10.1186/1471-2164-9-254 Text en Copyright © 2008 Martelli et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methodology Article Martelli, Maria Luisa Isella, Claudio Mira, Alessia Fu, Limin Cantarella, Daniela Medico, Enzo Exploiting orthologue diversity for systematic detection of gain-of-function phenotypes |
title | Exploiting orthologue diversity for systematic detection of gain-of-function phenotypes |
title_full | Exploiting orthologue diversity for systematic detection of gain-of-function phenotypes |
title_fullStr | Exploiting orthologue diversity for systematic detection of gain-of-function phenotypes |
title_full_unstemmed | Exploiting orthologue diversity for systematic detection of gain-of-function phenotypes |
title_short | Exploiting orthologue diversity for systematic detection of gain-of-function phenotypes |
title_sort | exploiting orthologue diversity for systematic detection of gain-of-function phenotypes |
topic | Methodology Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2435555/ https://www.ncbi.nlm.nih.gov/pubmed/18510758 http://dx.doi.org/10.1186/1471-2164-9-254 |
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