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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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anti-EGFP shRNAs expressed from a mouse U6 promoter is more potent as compared to anti-EGFP shRNA-miRs expressed from an EF1A-promoter.A) 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 expressed from a mouse U6 promoter (blue histograms) or with anti-EGFP shRNA-miRs expressed from the human EF1A promoter (red histograms). Eight days post infection, infected cells were monitored for EGFP expression by flow cytometry. The presented percentage of EGFP knockdown of the U6-driven shRNAs relative to the EF1A-driven shRNAs is calculated by ((Geo-mean of EF1A infected cells minus Geo-mean of U6 infected cells)/Geo-mean of EF1A infected cells)*100. B) mCherry expression is reduced upon cloning of an shRNA-containing miR-30 cassette in the 3′UTR of the fluorescent protein. Two examples are shown where either the CAGGS or EF1A promoter drives expression of mCherry with/without a functional miR cassette in it's 3′UTR. The percentage of reduced relative mCherry expression caused by the miR-cassette containing UTR is indicated. The presented data are representative experiments of 2 experiments performed in triplicate.
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pone-0026213-g004: anti-EGFP shRNAs expressed from a mouse U6 promoter is more potent as compared to anti-EGFP shRNA-miRs expressed from an EF1A-promoter.A) 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 expressed from a mouse U6 promoter (blue histograms) or with anti-EGFP shRNA-miRs expressed from the human EF1A promoter (red histograms). Eight days post infection, infected cells were monitored for EGFP expression by flow cytometry. The presented percentage of EGFP knockdown of the U6-driven shRNAs relative to the EF1A-driven shRNAs is calculated by ((Geo-mean of EF1A infected cells minus Geo-mean of U6 infected cells)/Geo-mean of EF1A infected cells)*100. B) mCherry expression is reduced upon cloning of an shRNA-containing miR-30 cassette in the 3′UTR of the fluorescent protein. Two examples are shown where either the CAGGS or EF1A promoter drives expression of mCherry with/without a functional miR cassette in it's 3′UTR. The percentage of reduced relative mCherry expression caused by the miR-cassette containing UTR is indicated. The presented data are representative experiments of 2 experiments performed in triplicate.

Mentions: Although some groups reported that miR-backbone expressed shRNAs are more potent as polymerase III U6-promoter expressed shRNAs [19], [21], [24], [25], we did not observe this result. In contrast, the U6 promoter expressed shRNAs were more potent in directing target knockdown as compared to the most effective EF1A promoter in all 6 cell lines (See Figure 3 and Figure 4A). The U6 promoter caused an additional target knockdown of 22.5%, 10.4%, and 30.7% in 293T, HeLas, and HT29 cells as compared to the EF1A promoter (see Figure 4A). This enhanced target knockdown was even more pronounced in the immune lines Raji, Jurkat and THP1 in which an additional 47.7%, 26.5%, and 50.3% of target knockdown was achieved over the EF1A promoter (Figure 4A). We conclude that shRNA-miRs efficacy is largely dependent on the polymerase II used to drive expression of the shRNA, and that this differential target knockdown is dependent on the cell type used.


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)

anti-EGFP shRNAs expressed from a mouse U6 promoter is more potent as compared to anti-EGFP shRNA-miRs expressed from an EF1A-promoter.A) 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 expressed from a mouse U6 promoter (blue histograms) or with anti-EGFP shRNA-miRs expressed from the human EF1A promoter (red histograms). Eight days post infection, infected cells were monitored for EGFP expression by flow cytometry. The presented percentage of EGFP knockdown of the U6-driven shRNAs relative to the EF1A-driven shRNAs is calculated by ((Geo-mean of EF1A infected cells minus Geo-mean of U6 infected cells)/Geo-mean of EF1A infected cells)*100. B) mCherry expression is reduced upon cloning of an shRNA-containing miR-30 cassette in the 3′UTR of the fluorescent protein. Two examples are shown where either the CAGGS or EF1A promoter drives expression of mCherry with/without a functional miR cassette in it's 3′UTR. The percentage of reduced relative mCherry expression caused by the miR-cassette containing UTR is indicated. The presented data are representative experiments of 2 experiments performed in triplicate.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198731&req=5

pone-0026213-g004: anti-EGFP shRNAs expressed from a mouse U6 promoter is more potent as compared to anti-EGFP shRNA-miRs expressed from an EF1A-promoter.A) 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 expressed from a mouse U6 promoter (blue histograms) or with anti-EGFP shRNA-miRs expressed from the human EF1A promoter (red histograms). Eight days post infection, infected cells were monitored for EGFP expression by flow cytometry. The presented percentage of EGFP knockdown of the U6-driven shRNAs relative to the EF1A-driven shRNAs is calculated by ((Geo-mean of EF1A infected cells minus Geo-mean of U6 infected cells)/Geo-mean of EF1A infected cells)*100. B) mCherry expression is reduced upon cloning of an shRNA-containing miR-30 cassette in the 3′UTR of the fluorescent protein. Two examples are shown where either the CAGGS or EF1A promoter drives expression of mCherry with/without a functional miR cassette in it's 3′UTR. The percentage of reduced relative mCherry expression caused by the miR-cassette containing UTR is indicated. The presented data are representative experiments of 2 experiments performed in triplicate.
Mentions: Although some groups reported that miR-backbone expressed shRNAs are more potent as polymerase III U6-promoter expressed shRNAs [19], [21], [24], [25], we did not observe this result. In contrast, the U6 promoter expressed shRNAs were more potent in directing target knockdown as compared to the most effective EF1A promoter in all 6 cell lines (See Figure 3 and Figure 4A). The U6 promoter caused an additional target knockdown of 22.5%, 10.4%, and 30.7% in 293T, HeLas, and HT29 cells as compared to the EF1A promoter (see Figure 4A). This enhanced target knockdown was even more pronounced in the immune lines Raji, Jurkat and THP1 in which an additional 47.7%, 26.5%, and 50.3% of target knockdown was achieved over the EF1A promoter (Figure 4A). We conclude that shRNA-miRs efficacy is largely dependent on the polymerase II used to drive expression of the shRNA, and that this differential target knockdown is dependent on the cell type used.

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
Related in: MedlinePlus