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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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Promoter strength determines potency of shRNA-miRs.EGFP-expressing human immune cell types (Raji B, Jurkat T, 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 (left panels) expressed from a miR-30 backbone driven by various polymerase-II promoter. Cells were allowed to grow for 8 days and the expression of EGFP (to monitor knockdown) was assessed by flow cytometry. Relative promoter strength of the polymerase II promoters (mCherry Geo Mean) was measured from control vectors that lacked the miR30-backbone, since processing of shRNAs out of the miR30 backbone destabilized the transcript. Relative promoter strength is plotted against% EGFP knockdown in the indicated cellines. Trendlines and R2 values of fitted curves are indicated.
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pone-0026213-g005: Promoter strength determines potency of shRNA-miRs.EGFP-expressing human immune cell types (Raji B, Jurkat T, 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 (left panels) expressed from a miR-30 backbone driven by various polymerase-II promoter. Cells were allowed to grow for 8 days and the expression of EGFP (to monitor knockdown) was assessed by flow cytometry. Relative promoter strength of the polymerase II promoters (mCherry Geo Mean) was measured from control vectors that lacked the miR30-backbone, since processing of shRNAs out of the miR30 backbone destabilized the transcript. Relative promoter strength is plotted against% EGFP knockdown in the indicated cellines. Trendlines and R2 values of fitted curves are indicated.

Mentions: To this end, we measured the relative strength of the promoters by assessing the mCherry fluorescence of infected cells 8 days post infection and plotted this to the percentage of target knockdown at this same time point (see Figure 5). For all lines, there was a clear correlation between promoter strength (as determined by mCherry expression) and EGFP-knockdown by the miR30-backbone anti-EGFP shRNA, suggesting that the strength of the promoter is a major determinant for shRNA-miR potency.


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)

Promoter strength determines potency of shRNA-miRs.EGFP-expressing human immune cell types (Raji B, Jurkat T, 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 (left panels) expressed from a miR-30 backbone driven by various polymerase-II promoter. Cells were allowed to grow for 8 days and the expression of EGFP (to monitor knockdown) was assessed by flow cytometry. Relative promoter strength of the polymerase II promoters (mCherry Geo Mean) was measured from control vectors that lacked the miR30-backbone, since processing of shRNAs out of the miR30 backbone destabilized the transcript. Relative promoter strength is plotted against% EGFP knockdown in the indicated cellines. Trendlines and R2 values of fitted curves are indicated.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026213-g005: Promoter strength determines potency of shRNA-miRs.EGFP-expressing human immune cell types (Raji B, Jurkat T, 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 (left panels) expressed from a miR-30 backbone driven by various polymerase-II promoter. Cells were allowed to grow for 8 days and the expression of EGFP (to monitor knockdown) was assessed by flow cytometry. Relative promoter strength of the polymerase II promoters (mCherry Geo Mean) was measured from control vectors that lacked the miR30-backbone, since processing of shRNAs out of the miR30 backbone destabilized the transcript. Relative promoter strength is plotted against% EGFP knockdown in the indicated cellines. Trendlines and R2 values of fitted curves are indicated.
Mentions: To this end, we measured the relative strength of the promoters by assessing the mCherry fluorescence of infected cells 8 days post infection and plotted this to the percentage of target knockdown at this same time point (see Figure 5). For all lines, there was a clear correlation between promoter strength (as determined by mCherry expression) and EGFP-knockdown by the miR30-backbone anti-EGFP shRNA, suggesting that the strength of the promoter is a major determinant for shRNA-miR potency.

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