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Polymerase II promoter strength determines efficacy of microRNA adapted shRNAs.

Lebbink RJ, Lowe M, Chan T, Khine H, Wang X, McManus MT - PLoS ONE (2011)

Bottom Line: One of these systems makes use of endogenous microRNA pri-cursors that are expressed from polymerase II promoters where the mature microRNA sequence is replaced by gene specific duplexes that guide RNAi (shRNA-miRs).Differences in RNAi from the shRNA-miRs expressed from the various promoters were particularly pronounced in immune cells.Our findings have direct implications for the design of shRNA-directed RNAi experiments and the preferred RNAi system to use for each cell type.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Diabetes Center, University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT
Since the discovery of RNAi and microRNAs more than 10 years ago, much research has focused on the development of systems that usurp microRNA pathways to downregulate gene expression in mammalian cells. One of these systems makes use of endogenous microRNA pri-cursors that are expressed from polymerase II promoters where the mature microRNA sequence is replaced by gene specific duplexes that guide RNAi (shRNA-miRs). Although shRNA-miRs are effective in directing target mRNA knockdown and hence reducing protein expression in many cell types, variability of RNAi efficacy in cell lines has been an issue. Here we show that the choice of the polymerase II promoter used to drive shRNA expression is of critical importance to allow effective mRNA target knockdown. We tested the abundance of shRNA-miRs expressed from five different polymerase II promoters in 6 human cell lines and measured their ability to drive target knockdown. We observed a clear positive correlation between promoter strength, siRNA expression levels, and protein target knockdown. Differences in RNAi from the shRNA-miRs expressed from the various promoters were particularly pronounced in immune cells. Our findings have direct implications for the design of shRNA-directed RNAi experiments and the preferred RNAi system to use for each cell type.

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Efficacy of shRNA-miRs-guided target knockdown in various cell lines is dependent on the type of polymerase II promoter used.EGFP-expressing human immune cell types (Raji B cells, Jurkat T cells, and THP-1 monocytic cells) and adherent cell lines (293T, HeLa, and HT29 cells) were infected at an MOI of <0.2 with anti-EGFP shRNAs expressed from a miR-30 backbone driven by various indicated polymerase-II promoter. Cells were allowed to grow for the indicated number of days and the expression of EGFP (to monitor knockdown) and mCherry (as marker for infected cells) was assessed by flow cytometry. The presented percentage of EGFP knockdown is calculated by ((Geo-mean of uninfected cells minus Geo-mean of infected cells)/Geo-mean of uninfected cells)*100. The presented data is a representative experiment of 2 experiments performed in triplicate. For all data points, the standard deviation was below 5%.
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pone-0026213-g003: Efficacy of shRNA-miRs-guided target knockdown in various cell lines is dependent on the type of polymerase II promoter used.EGFP-expressing human immune cell types (Raji B cells, Jurkat T cells, and THP-1 monocytic cells) and adherent cell lines (293T, HeLa, and HT29 cells) were infected at an MOI of <0.2 with anti-EGFP shRNAs expressed from a miR-30 backbone driven by various indicated polymerase-II promoter. Cells were allowed to grow for the indicated number of days and the expression of EGFP (to monitor knockdown) and mCherry (as marker for infected cells) was assessed by flow cytometry. The presented percentage of EGFP knockdown is calculated by ((Geo-mean of uninfected cells minus Geo-mean of infected cells)/Geo-mean of uninfected cells)*100. The presented data is a representative experiment of 2 experiments performed in triplicate. For all data points, the standard deviation was below 5%.

Mentions: RNAi is often performed in cells that are relatively easy to grow and transfect with commercially available lipids. We asked whether any of the broadly used cell lines would display differing RNAi effectiveness using our panel of promoters expressing the same shRNA. To address this question we chose to monitor three commonly used adherent cell lines (293T embryonic kidney cells, HeLa cervical carcinoma cells, and HT29 adenocarcinoma cells) and three commonly used non-adherent immune cell lines (Jurkat T cell lymphoblast cells, Raji Burkitt lymphoma B cells, and THP-1 acute monocytic leukemia cells). Stable EGFP-expressing cells were generated for all six lines, and infected at a low MOI with the various viruses. EGFP expression was then monitored at 2, 3, 4, 6, and 8 days post infection by flow cytometry (See Figure 3). In all cases, the EF1A-anti-EGFP construct was most efficient in driving EGFP knockdown as compared to the other four polymerase II-promoters. This observation was most prominent in the non-adherent immune cell types (Raji, Jurkat, and THP1 cells, See Figure 3 left panels). In general EGFP-knockdown was more variable using the different polymerase-II promoters in immune cells as compared to the adherent lines. The EF1A, CAGGS and CMV promoters were equally potent in 293T and HeLa cells, whereas the PGK and UbiC promoters displayed much-reduced EGFP-target knockdown in these lines. Optimal EGFP-knockdown was observed 6 to 8 days post infection for all promoters in all lines. In Table 1 we present a summarizing overview of the RNAi efficiencies of the tested promoters in the different cell lines.


Polymerase II promoter strength determines efficacy of microRNA adapted shRNAs.

Lebbink RJ, Lowe M, Chan T, Khine H, Wang X, McManus MT - PLoS ONE (2011)

Efficacy of shRNA-miRs-guided target knockdown in various cell lines is dependent on the type of polymerase II promoter used.EGFP-expressing human immune cell types (Raji B cells, Jurkat T cells, and THP-1 monocytic cells) and adherent cell lines (293T, HeLa, and HT29 cells) were infected at an MOI of <0.2 with anti-EGFP shRNAs expressed from a miR-30 backbone driven by various indicated polymerase-II promoter. Cells were allowed to grow for the indicated number of days and the expression of EGFP (to monitor knockdown) and mCherry (as marker for infected cells) was assessed by flow cytometry. The presented percentage of EGFP knockdown is calculated by ((Geo-mean of uninfected cells minus Geo-mean of infected cells)/Geo-mean of uninfected cells)*100. The presented data is a representative experiment of 2 experiments performed in triplicate. For all data points, the standard deviation was below 5%.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198731&req=5

pone-0026213-g003: Efficacy of shRNA-miRs-guided target knockdown in various cell lines is dependent on the type of polymerase II promoter used.EGFP-expressing human immune cell types (Raji B cells, Jurkat T cells, and THP-1 monocytic cells) and adherent cell lines (293T, HeLa, and HT29 cells) were infected at an MOI of <0.2 with anti-EGFP shRNAs expressed from a miR-30 backbone driven by various indicated polymerase-II promoter. Cells were allowed to grow for the indicated number of days and the expression of EGFP (to monitor knockdown) and mCherry (as marker for infected cells) was assessed by flow cytometry. The presented percentage of EGFP knockdown is calculated by ((Geo-mean of uninfected cells minus Geo-mean of infected cells)/Geo-mean of uninfected cells)*100. The presented data is a representative experiment of 2 experiments performed in triplicate. For all data points, the standard deviation was below 5%.
Mentions: RNAi is often performed in cells that are relatively easy to grow and transfect with commercially available lipids. We asked whether any of the broadly used cell lines would display differing RNAi effectiveness using our panel of promoters expressing the same shRNA. To address this question we chose to monitor three commonly used adherent cell lines (293T embryonic kidney cells, HeLa cervical carcinoma cells, and HT29 adenocarcinoma cells) and three commonly used non-adherent immune cell lines (Jurkat T cell lymphoblast cells, Raji Burkitt lymphoma B cells, and THP-1 acute monocytic leukemia cells). Stable EGFP-expressing cells were generated for all six lines, and infected at a low MOI with the various viruses. EGFP expression was then monitored at 2, 3, 4, 6, and 8 days post infection by flow cytometry (See Figure 3). In all cases, the EF1A-anti-EGFP construct was most efficient in driving EGFP knockdown as compared to the other four polymerase II-promoters. This observation was most prominent in the non-adherent immune cell types (Raji, Jurkat, and THP1 cells, See Figure 3 left panels). In general EGFP-knockdown was more variable using the different polymerase-II promoters in immune cells as compared to the adherent lines. The EF1A, CAGGS and CMV promoters were equally potent in 293T and HeLa cells, whereas the PGK and UbiC promoters displayed much-reduced EGFP-target knockdown in these lines. Optimal EGFP-knockdown was observed 6 to 8 days post infection for all promoters in all lines. In Table 1 we present a summarizing overview of the RNAi efficiencies of the tested promoters in the different cell lines.

Bottom Line: One of these systems makes use of endogenous microRNA pri-cursors that are expressed from polymerase II promoters where the mature microRNA sequence is replaced by gene specific duplexes that guide RNAi (shRNA-miRs).Differences in RNAi from the shRNA-miRs expressed from the various promoters were particularly pronounced in immune cells.Our findings have direct implications for the design of shRNA-directed RNAi experiments and the preferred RNAi system to use for each cell type.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Diabetes Center, University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT
Since the discovery of RNAi and microRNAs more than 10 years ago, much research has focused on the development of systems that usurp microRNA pathways to downregulate gene expression in mammalian cells. One of these systems makes use of endogenous microRNA pri-cursors that are expressed from polymerase II promoters where the mature microRNA sequence is replaced by gene specific duplexes that guide RNAi (shRNA-miRs). Although shRNA-miRs are effective in directing target mRNA knockdown and hence reducing protein expression in many cell types, variability of RNAi efficacy in cell lines has been an issue. Here we show that the choice of the polymerase II promoter used to drive shRNA expression is of critical importance to allow effective mRNA target knockdown. We tested the abundance of shRNA-miRs expressed from five different polymerase II promoters in 6 human cell lines and measured their ability to drive target knockdown. We observed a clear positive correlation between promoter strength, siRNA expression levels, and protein target knockdown. Differences in RNAi from the shRNA-miRs expressed from the various promoters were particularly pronounced in immune cells. Our findings have direct implications for the design of shRNA-directed RNAi experiments and the preferred RNAi system to use for each cell type.

Show MeSH