A high-throughput platform for lentiviral overexpression screening of the human ORFeome.
In response to the growing need for functional analysis of the human genome, we have developed a platform for high-throughput functional screening of genes overexpressed from lentiviral vectors. Protein-coding human open reading frames (ORFs) from the Mammalian Gene Collection were transferred into...
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doaj-f824c7d2e31f4b4fb025a4c305a54c2e2020-11-25T02:31:04ZengPublic Library of Science (PLoS)PLoS ONE1932-62032011-01-0165e2005710.1371/journal.pone.0020057A high-throughput platform for lentiviral overexpression screening of the human ORFeome.Dubravka ŠkalameraMax V RanallBenjamin M WilsonPaul LeoAmy S PurdonCarolyn HydeEhsan NourbakhshSean M GrimmondSimon C BarryBrian GabrielliThomas J GondaIn response to the growing need for functional analysis of the human genome, we have developed a platform for high-throughput functional screening of genes overexpressed from lentiviral vectors. Protein-coding human open reading frames (ORFs) from the Mammalian Gene Collection were transferred into lentiviral expression vector using the highly efficient Gateway recombination cloning. Target ORFs were inserted into the vector downstream of a constitutive promoter and upstream of an IRES controlled GFP reporter, so that their transfection, transduction and expression could be monitored by fluorescence. The expression plasmids and viral packaging plasmids were combined and transfected into 293T cells to produce virus, which was then used to transduce the screening cell line. We have optimised the transfection and transduction procedures so that they can be performed using robotic liquid handling systems in arrayed 96-well microplate, one-gene-per-well format, without the need to concentrate the viral supernatant. Since lentiviruses can infect both dividing and non-dividing cells, this system can be used to overexpress human ORFs in a broad spectrum of experimental contexts. We tested the platform in a 1990 gene pilot screen for genes that can increase proliferation of the non-tumorigenic mammary epithelial cell line MCF-10A after removal of growth factors. Transduced cells were labelled with the nucleoside analogue 5-ethynyl-2'-deoxyuridine (EdU) to detect cells progressing through S phase. Hits were identified using high-content imaging and statistical analysis and confirmed with vectors using two different promoters (CMV and EF1α). The screen demonstrates the reliability, versatility and utility of our screening platform, and identifies novel cell cycle/proliferative activities for a number of genes.http://europepmc.org/articles/PMC3101218?pdf=render |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Dubravka Škalamera Max V Ranall Benjamin M Wilson Paul Leo Amy S Purdon Carolyn Hyde Ehsan Nourbakhsh Sean M Grimmond Simon C Barry Brian Gabrielli Thomas J Gonda |
spellingShingle |
Dubravka Škalamera Max V Ranall Benjamin M Wilson Paul Leo Amy S Purdon Carolyn Hyde Ehsan Nourbakhsh Sean M Grimmond Simon C Barry Brian Gabrielli Thomas J Gonda A high-throughput platform for lentiviral overexpression screening of the human ORFeome. PLoS ONE |
author_facet |
Dubravka Škalamera Max V Ranall Benjamin M Wilson Paul Leo Amy S Purdon Carolyn Hyde Ehsan Nourbakhsh Sean M Grimmond Simon C Barry Brian Gabrielli Thomas J Gonda |
author_sort |
Dubravka Škalamera |
title |
A high-throughput platform for lentiviral overexpression screening of the human ORFeome. |
title_short |
A high-throughput platform for lentiviral overexpression screening of the human ORFeome. |
title_full |
A high-throughput platform for lentiviral overexpression screening of the human ORFeome. |
title_fullStr |
A high-throughput platform for lentiviral overexpression screening of the human ORFeome. |
title_full_unstemmed |
A high-throughput platform for lentiviral overexpression screening of the human ORFeome. |
title_sort |
high-throughput platform for lentiviral overexpression screening of the human orfeome. |
publisher |
Public Library of Science (PLoS) |
series |
PLoS ONE |
issn |
1932-6203 |
publishDate |
2011-01-01 |
description |
In response to the growing need for functional analysis of the human genome, we have developed a platform for high-throughput functional screening of genes overexpressed from lentiviral vectors. Protein-coding human open reading frames (ORFs) from the Mammalian Gene Collection were transferred into lentiviral expression vector using the highly efficient Gateway recombination cloning. Target ORFs were inserted into the vector downstream of a constitutive promoter and upstream of an IRES controlled GFP reporter, so that their transfection, transduction and expression could be monitored by fluorescence. The expression plasmids and viral packaging plasmids were combined and transfected into 293T cells to produce virus, which was then used to transduce the screening cell line. We have optimised the transfection and transduction procedures so that they can be performed using robotic liquid handling systems in arrayed 96-well microplate, one-gene-per-well format, without the need to concentrate the viral supernatant. Since lentiviruses can infect both dividing and non-dividing cells, this system can be used to overexpress human ORFs in a broad spectrum of experimental contexts. We tested the platform in a 1990 gene pilot screen for genes that can increase proliferation of the non-tumorigenic mammary epithelial cell line MCF-10A after removal of growth factors. Transduced cells were labelled with the nucleoside analogue 5-ethynyl-2'-deoxyuridine (EdU) to detect cells progressing through S phase. Hits were identified using high-content imaging and statistical analysis and confirmed with vectors using two different promoters (CMV and EF1α). The screen demonstrates the reliability, versatility and utility of our screening platform, and identifies novel cell cycle/proliferative activities for a number of genes. |
url |
http://europepmc.org/articles/PMC3101218?pdf=render |
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