Abstract
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.
Highlights
Elucidating gene function is a pressing challenge for human biology and medicine
This is in part due to the fact that gene silencing can be achieved with readily synthetised oligonucleotides while overexpression requires cloning full length open reading frames (ORFs) into expression plasmids [4,5]
The ORF is inserted upstream of the IRES-controlled hrGFP, so that transgene expression can be monitored by GFP fluorescence
Summary
Elucidating gene function is a pressing challenge for human biology and medicine. Given that the human genome consists of up to 25000 protein-coding genes [1], this task requires highthroughput approaches. Only a few studies have investigated the effect of ectopic cDNA expression on a genomic scale using individually arrayed expression clones This is in part due to the fact that gene silencing can be achieved with readily synthetised oligonucleotides while overexpression requires cloning full length open reading frames (ORFs) into expression plasmids [4,5]. Another difficulty is that foreign plasmids can be transfected into only a limited number of human cell types, so that the existing reports have focused on highly transfectable cell lines such as HEK293T [6,7,8], U2OS2 [9], HCT116 [10] and SMC1772 [11]. We describe a high-throughput platform for overexpression screening of the human genome in an arrayed one gene per well format that circumvents these difficulties by using Gateway cloning and lentiviral expression vectors
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