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A high-throughput platform for lentiviral overexpression screening of the human ORFeome.

Škalamera D, Ranall MV, Wilson BM, Leo P, Purdon AS, Hyde C, Nourbakhsh E, Grimmond SM, Barry SC, Gabrielli B, Gonda TJ - PLoS ONE (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: University of Queensland Diamantina Institute, Princess Alexandra Hospital, Brisbane, Queensland, Australia. d.skalamera@uq.edu.au

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.

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Summary measurements for hits showing increased total cell proliferation.CMV, EF1α indicate values for ORFs expressed under control of the CMV and EF1α promoters, respectively. A – ratio of total objects counted on day 4 over day 2 of the assay (Figure 2); B – ratio of the proportion of EdU positive cells in GFP positive over that in GFP negative objects on day 4; C-percentage of GFP positive objects on day4. All bars represent a mean of three wells, error bars are standard deviation. Genes represented here were significantly different (p≤0.05) in A and B from the control (in this case vectors encoding a truncated peptide originating from CPNE3, which had no effect on proliferation in primary and validation screens). Data for all genes tested is in Table S3.
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pone-0020057-g004: Summary measurements for hits showing increased total cell proliferation.CMV, EF1α indicate values for ORFs expressed under control of the CMV and EF1α promoters, respectively. A – ratio of total objects counted on day 4 over day 2 of the assay (Figure 2); B – ratio of the proportion of EdU positive cells in GFP positive over that in GFP negative objects on day 4; C-percentage of GFP positive objects on day4. All bars represent a mean of three wells, error bars are standard deviation. Genes represented here were significantly different (p≤0.05) in A and B from the control (in this case vectors encoding a truncated peptide originating from CPNE3, which had no effect on proliferation in primary and validation screens). Data for all genes tested is in Table S3.

Mentions: From the multiple rounds of screening we identified 47 genes that satisfy criteria for increased nucleotide incorporation rate, suggesting that they may be increasing cell proliferation after EGF removal. We took advantage of the flexibility of our platform to further analyse the effect of hit ORFs on cell proliferation rate and cell cycle profile. These hit ORFs were also subcloned into alternative lentiviral vector plv411G, which is identical to plv101G except that it utilises the EF1α promoter in place of the CMV promoter. The EF1α promoter presented advantages over the CMV promoter when we generated stable cell lines overexpressing transgenes. In the MCF-10A cell line background we detected silencing of the CMV promoter, while the EF1α promoter maintained stable expression through multiple cell passages and at least one freeze/thaw cycle (data not shown). With these two sets of expression clones we performed a time course experiment similar to the assay described in Figure 2, except that triplicate plates were EdU labelled and harvested on days 2 and 4 (i.e. 1 and 3 days respectively after transduction and EGF withdrawal). This allowed us to measure the effect of transgene overexpression on the increase in cell number, as well as to observe the changes in cell cycle induced by transduction and EGF withdrawal. As a negative control we used a clone expressing a 24 amino acid truncated version of CPNE3 which produced neutral z-scores in screens described here, as well as in previous preliminary experiments. The results for all 47 genes are presented in Tables S3 and S4, while the data for the 11 genes that significantly (P≤0.05) increased both cell number and EdU incorporation rate compared to control (see below) are shown in Figure 4.


A high-throughput platform for lentiviral overexpression screening of the human ORFeome.

Škalamera D, Ranall MV, Wilson BM, Leo P, Purdon AS, Hyde C, Nourbakhsh E, Grimmond SM, Barry SC, Gabrielli B, Gonda TJ - PLoS ONE (2011)

Summary measurements for hits showing increased total cell proliferation.CMV, EF1α indicate values for ORFs expressed under control of the CMV and EF1α promoters, respectively. A – ratio of total objects counted on day 4 over day 2 of the assay (Figure 2); B – ratio of the proportion of EdU positive cells in GFP positive over that in GFP negative objects on day 4; C-percentage of GFP positive objects on day4. All bars represent a mean of three wells, error bars are standard deviation. Genes represented here were significantly different (p≤0.05) in A and B from the control (in this case vectors encoding a truncated peptide originating from CPNE3, which had no effect on proliferation in primary and validation screens). Data for all genes tested is in Table S3.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3101218&req=5

pone-0020057-g004: Summary measurements for hits showing increased total cell proliferation.CMV, EF1α indicate values for ORFs expressed under control of the CMV and EF1α promoters, respectively. A – ratio of total objects counted on day 4 over day 2 of the assay (Figure 2); B – ratio of the proportion of EdU positive cells in GFP positive over that in GFP negative objects on day 4; C-percentage of GFP positive objects on day4. All bars represent a mean of three wells, error bars are standard deviation. Genes represented here were significantly different (p≤0.05) in A and B from the control (in this case vectors encoding a truncated peptide originating from CPNE3, which had no effect on proliferation in primary and validation screens). Data for all genes tested is in Table S3.
Mentions: From the multiple rounds of screening we identified 47 genes that satisfy criteria for increased nucleotide incorporation rate, suggesting that they may be increasing cell proliferation after EGF removal. We took advantage of the flexibility of our platform to further analyse the effect of hit ORFs on cell proliferation rate and cell cycle profile. These hit ORFs were also subcloned into alternative lentiviral vector plv411G, which is identical to plv101G except that it utilises the EF1α promoter in place of the CMV promoter. The EF1α promoter presented advantages over the CMV promoter when we generated stable cell lines overexpressing transgenes. In the MCF-10A cell line background we detected silencing of the CMV promoter, while the EF1α promoter maintained stable expression through multiple cell passages and at least one freeze/thaw cycle (data not shown). With these two sets of expression clones we performed a time course experiment similar to the assay described in Figure 2, except that triplicate plates were EdU labelled and harvested on days 2 and 4 (i.e. 1 and 3 days respectively after transduction and EGF withdrawal). This allowed us to measure the effect of transgene overexpression on the increase in cell number, as well as to observe the changes in cell cycle induced by transduction and EGF withdrawal. As a negative control we used a clone expressing a 24 amino acid truncated version of CPNE3 which produced neutral z-scores in screens described here, as well as in previous preliminary experiments. The results for all 47 genes are presented in Tables S3 and S4, while the data for the 11 genes that significantly (P≤0.05) increased both cell number and EdU incorporation rate compared to control (see below) are shown in Figure 4.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: University of Queensland Diamantina Institute, Princess Alexandra Hospital, Brisbane, Queensland, Australia. d.skalamera@uq.edu.au

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.

Show MeSH
Related in: MedlinePlus