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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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Related in: MedlinePlus

Cell cycle analysis of proliferation-inducing hits.A- DAPI intensity (x-axis) histograms (y-axis = number of objects) obtained by CellCycle v3 application in the Cellomics ArrayScan Software, representing typical profiles observed on Day 0, Day 2 and Day 4 of the assay (Figure 2), depending on growth conditions and viral transduction. Profiles and data shown are derived from a representative single well from each of the categories: not transduced (no virus), transduced with empty plv101 vector, or with the vector expressing NEK6, analysed using cells grown in complete or restrictive medium as indicated. The proportion of cells in each cell cycle phase is indicated (based on DNA content: 2N (%G1), between 2N and 4N (%S), and 4N (%G2)). B, C – Graphs representing the proportion of cells in G2 on day 2 (B) and day 4 (C) for proliferation-inducing hits (Bars = mean of 3 wells; error bars = standard deviation). CMV, EF1α - values for clones expressed under control of the CMV and EF1α promoters respectively. N.T-not transduced, control – truncated CPNE3. Values for all analysed genes are in Table S4, for all transduced wells, values represent GFP positive cell population only.
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pone-0020057-g006: Cell cycle analysis of proliferation-inducing hits.A- DAPI intensity (x-axis) histograms (y-axis = number of objects) obtained by CellCycle v3 application in the Cellomics ArrayScan Software, representing typical profiles observed on Day 0, Day 2 and Day 4 of the assay (Figure 2), depending on growth conditions and viral transduction. Profiles and data shown are derived from a representative single well from each of the categories: not transduced (no virus), transduced with empty plv101 vector, or with the vector expressing NEK6, analysed using cells grown in complete or restrictive medium as indicated. The proportion of cells in each cell cycle phase is indicated (based on DNA content: 2N (%G1), between 2N and 4N (%S), and 4N (%G2)). B, C – Graphs representing the proportion of cells in G2 on day 2 (B) and day 4 (C) for proliferation-inducing hits (Bars = mean of 3 wells; error bars = standard deviation). CMV, EF1α - values for clones expressed under control of the CMV and EF1α promoters respectively. N.T-not transduced, control – truncated CPNE3. Values for all analysed genes are in Table S4, for all transduced wells, values represent GFP positive cell population only.

Mentions: Cells in our screen were exposed to two antiproliferative conditions: EGF withdrawal which causes accumulation of cells in G1 phase of the cell cycle [21] and lentiviral infection which has been shown to promote G2 phase accumulation [34], [35]. To determine which of these antiproliferative conditions the hit transgenes were overcoming, we examined cell cycle progression in transduced cells by DNA content analysis. Using the DAPI intensity histograms obtained by high-content imaging, nuclei were assigned cell cycle phases based on their deduced DNA content (G1 = 2N, G2 = 4N, and S = (2N–4N)), by modelling on the histograms derived from the reference untransduced wells (Figure 6A). Three hours after plating, cells that had not been exposed to viral supernatants and were grown in complete media had a mean G1/G2 ratio of 1.6±0.13 (n = 12) (Figure 6A). On day 2 (Figure 2) of the assay, after a medium change to 1% serum without EGF, cells in untransduced wells started accumulating in G1 (G1/G2 = 2.1–2.4). In contrast, most wells exposed to viral supernatant had an increased proportion of cells in G2 phase compared to cells in untransduced wells (Figure 6A, B; Table S4). Fewer cells were observed in the transduced wells at this time, indicating that the G2 accumulation was due to G2 block rather than faster progression through G1/S. This effect was observed with both CMV and EF1α promoters, and was detected in both GFP positive and negative cells in transduced wells, as well as in cells exposed to mock supernatant (from cells transfected only with the packaging vectors), suggesting that it may be caused by both empty viral particles as well as the particles containing the ORF expressing RNA. Irrespective of the promoter used, the G2 phase accumulation was less pronounced in cells overexpressing hit genes CADM1, CDC20, CDK2, CELA2B, CLK2, KRT19, NEK6, PACSIN, or RIT1 compared to control and other hits (Figure 6, Table S4). On Day 4 of the assay (Figure 6A, and C), untransduced cells growing in EGF-free media were arresting in G1 phase, evident from the reduced proportion of cells in S phase and an increased proportion in G1. This EGF withdrawal-induced G1 arrest was more pronounced and happened at lower cell densities compared to the G1 accumulation observed in untransduced cells in complete media that almost reached confluence (Figure 6A). In contrast, cells transduced with the truncated CPNE3 control still had a significantly higher proportion of cells in G2 phase compared to those in untransduced wells, although not as high as on day 2. Cells transduced with hit genes had G2 proportion values between those of the transduced control and untransduced wells in EGF withdrawal (Figure 6A, C, Table S4), indicat ingthat at least in some cases transgene overexpression compensated for the transduction induced G2 phase arrest observed on Day2. For most hit genes, the proportion of cells in G1 phase was lower compared to the untransduced controls subjected to EGF withdrawal (Table S4), indicating that these genes may also compensate for growth factor removal.


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)

Cell cycle analysis of proliferation-inducing hits.A- DAPI intensity (x-axis) histograms (y-axis = number of objects) obtained by CellCycle v3 application in the Cellomics ArrayScan Software, representing typical profiles observed on Day 0, Day 2 and Day 4 of the assay (Figure 2), depending on growth conditions and viral transduction. Profiles and data shown are derived from a representative single well from each of the categories: not transduced (no virus), transduced with empty plv101 vector, or with the vector expressing NEK6, analysed using cells grown in complete or restrictive medium as indicated. The proportion of cells in each cell cycle phase is indicated (based on DNA content: 2N (%G1), between 2N and 4N (%S), and 4N (%G2)). B, C – Graphs representing the proportion of cells in G2 on day 2 (B) and day 4 (C) for proliferation-inducing hits (Bars = mean of 3 wells; error bars = standard deviation). CMV, EF1α - values for clones expressed under control of the CMV and EF1α promoters respectively. N.T-not transduced, control – truncated CPNE3. Values for all analysed genes are in Table S4, for all transduced wells, values represent GFP positive cell population only.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020057-g006: Cell cycle analysis of proliferation-inducing hits.A- DAPI intensity (x-axis) histograms (y-axis = number of objects) obtained by CellCycle v3 application in the Cellomics ArrayScan Software, representing typical profiles observed on Day 0, Day 2 and Day 4 of the assay (Figure 2), depending on growth conditions and viral transduction. Profiles and data shown are derived from a representative single well from each of the categories: not transduced (no virus), transduced with empty plv101 vector, or with the vector expressing NEK6, analysed using cells grown in complete or restrictive medium as indicated. The proportion of cells in each cell cycle phase is indicated (based on DNA content: 2N (%G1), between 2N and 4N (%S), and 4N (%G2)). B, C – Graphs representing the proportion of cells in G2 on day 2 (B) and day 4 (C) for proliferation-inducing hits (Bars = mean of 3 wells; error bars = standard deviation). CMV, EF1α - values for clones expressed under control of the CMV and EF1α promoters respectively. N.T-not transduced, control – truncated CPNE3. Values for all analysed genes are in Table S4, for all transduced wells, values represent GFP positive cell population only.
Mentions: Cells in our screen were exposed to two antiproliferative conditions: EGF withdrawal which causes accumulation of cells in G1 phase of the cell cycle [21] and lentiviral infection which has been shown to promote G2 phase accumulation [34], [35]. To determine which of these antiproliferative conditions the hit transgenes were overcoming, we examined cell cycle progression in transduced cells by DNA content analysis. Using the DAPI intensity histograms obtained by high-content imaging, nuclei were assigned cell cycle phases based on their deduced DNA content (G1 = 2N, G2 = 4N, and S = (2N–4N)), by modelling on the histograms derived from the reference untransduced wells (Figure 6A). Three hours after plating, cells that had not been exposed to viral supernatants and were grown in complete media had a mean G1/G2 ratio of 1.6±0.13 (n = 12) (Figure 6A). On day 2 (Figure 2) of the assay, after a medium change to 1% serum without EGF, cells in untransduced wells started accumulating in G1 (G1/G2 = 2.1–2.4). In contrast, most wells exposed to viral supernatant had an increased proportion of cells in G2 phase compared to cells in untransduced wells (Figure 6A, B; Table S4). Fewer cells were observed in the transduced wells at this time, indicating that the G2 accumulation was due to G2 block rather than faster progression through G1/S. This effect was observed with both CMV and EF1α promoters, and was detected in both GFP positive and negative cells in transduced wells, as well as in cells exposed to mock supernatant (from cells transfected only with the packaging vectors), suggesting that it may be caused by both empty viral particles as well as the particles containing the ORF expressing RNA. Irrespective of the promoter used, the G2 phase accumulation was less pronounced in cells overexpressing hit genes CADM1, CDC20, CDK2, CELA2B, CLK2, KRT19, NEK6, PACSIN, or RIT1 compared to control and other hits (Figure 6, Table S4). On Day 4 of the assay (Figure 6A, and C), untransduced cells growing in EGF-free media were arresting in G1 phase, evident from the reduced proportion of cells in S phase and an increased proportion in G1. This EGF withdrawal-induced G1 arrest was more pronounced and happened at lower cell densities compared to the G1 accumulation observed in untransduced cells in complete media that almost reached confluence (Figure 6A). In contrast, cells transduced with the truncated CPNE3 control still had a significantly higher proportion of cells in G2 phase compared to those in untransduced wells, although not as high as on day 2. Cells transduced with hit genes had G2 proportion values between those of the transduced control and untransduced wells in EGF withdrawal (Figure 6A, C, Table S4), indicat ingthat at least in some cases transgene overexpression compensated for the transduction induced G2 phase arrest observed on Day2. For most hit genes, the proportion of cells in G1 phase was lower compared to the untransduced controls subjected to EGF withdrawal (Table S4), indicating that these genes may also compensate for growth factor removal.

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