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https://hdl.handle.net/2440/70924
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Type: | Journal article |
Title: | A high-throughput platform for lentiviral overexpression screening of the human ORFeome |
Author: | Skalamera, D. Ranall, M. Wilson, B. Leo, P. Purdon, A. Hyde, C. Nourbakhsh, E. Grimmond, S. Barry, S. Gabrielli, B. Gonda, T. |
Citation: | PLoS One, 2011; 6(5):e20057-1-e20057-14 |
Publisher: | Public Library of Science |
Issue Date: | 2011 |
ISSN: | 1932-6203 1932-6203 |
Editor: | Jothi, R. |
Statement of Responsibility: | 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 and Thomas J. Gonda |
Abstract: | In response to the growing need for functional analysis of the human genome, we have developed a platform for highthroughput 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-29-deoxyuridine (EdU) to detect cells progressing through S phase. Hits were identified using highcontent imaging and statistical analysis and confirmed with vectors using two different promoters (CMV and EF1a). The screen demonstrates the reliability, versatility and utility of our screening platform, and identifies novel cell cycle/proliferative activities for a number of genes. |
Keywords: | Cell Line Humans Lentivirus Blotting, Western Cell Cycle Cell Proliferation Open Reading Frames Plasmids Models, Genetic |
Description: | Extent: 14p. |
Rights: | Copyright © 2011 Škalamera et al. 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 credited. |
DOI: | 10.1371/journal.pone.0020057 |
Grant ID: | http://purl.org/au-research/grants/arc/LE0668241 |
Published version: | http://dx.doi.org/10.1371/journal.pone.0020057 |
Appears in Collections: | Aurora harvest Paediatrics publications |
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hdl_70924.pdf | Published version | 1.64 MB | Adobe PDF | View/Open |
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