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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 miRNA backbone are less efficient in target-knockdown in Jurkat T cells than in 293T cells.A) schematic representation of the miR-context anti-EGFP shRNA construct cloned in the 3′UTR of mCherry which is expressed under the control of the polymerase II CMV promoter (lower panel) and the control anti-EGFP shRNA expressed from a mouse polymerase III U6 promoter (top panel). B) EGFP-expressing Jurkat T cells (top panels) or 293T cells (lower panels) were infected at an MOI of <0.2 with anti-EGFP shRNAs expressed from a mouse U6 promoter (U6 anti-EGFP shRNA) or from a miR-30 backbone expressed from a CMV promoter (miR-context anti-EGFP shRNA) or the relevant control viruses that lack shRNA inserts. Cells were allowed to grow for 8 days and the expression of EGFP (to monitor knockdown) and mCherry (as marker for infected cells) were assessed by flow cytometry. The indicated 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.
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pone-0026213-g001: anti-EGFP shRNAs expressed from a miRNA backbone are less efficient in target-knockdown in Jurkat T cells than in 293T cells.A) schematic representation of the miR-context anti-EGFP shRNA construct cloned in the 3′UTR of mCherry which is expressed under the control of the polymerase II CMV promoter (lower panel) and the control anti-EGFP shRNA expressed from a mouse polymerase III U6 promoter (top panel). B) EGFP-expressing Jurkat T cells (top panels) or 293T cells (lower panels) were infected at an MOI of <0.2 with anti-EGFP shRNAs expressed from a mouse U6 promoter (U6 anti-EGFP shRNA) or from a miR-30 backbone expressed from a CMV promoter (miR-context anti-EGFP shRNA) or the relevant control viruses that lack shRNA inserts. Cells were allowed to grow for 8 days and the expression of EGFP (to monitor knockdown) and mCherry (as marker for infected cells) were assessed by flow cytometry. The indicated 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.

Mentions: In the process of generating stable EGFP knockdown cell lines in human Jurkat T cells by using an anti-EGFP shRNA expressed from CMV-driven miR30-backbone expression vectors (shRNA-miRs, see Figure 1A for design of the constructs), we noted a much reduced target knockdown (52.7%) as compared to identical shRNAs expressed from the mouse polymerase III U6 promoter (90.2%, see Figure 1B, top panels). These same lentiviral shRNA vectors were equally potent in driving EGFP knockdown in human 293T cells (88.0% and 92.5% target knockdown for shRNA-miR and U6-driven shRNAs respectively, see Figure 1B lower panels), suggesting that shRNA-miRs might be less potent in Jurkat T cells as in 293T cells. We set out to explore the nature of this discrepancy, and assessed whether the potency of the polymerase II promoter used to express the miR30-backbone anti-EGFP shRNAs could impact the shRNA-directed silencing in Jurkat T cells. To this end, we cloned five different polymerase II promoters upstream the miR-30 anti-EGFP shRNA cassette that is located in the 3′UTR of the fluorescent protein mCherry to mark cells that have been infected with the lentiviral construct. We selected the ubiquitin C (UbiC) promoter, the phosphoglycerate kinase (PGK) promoter, the cytomegalovirus (CMV) promoter, a beta-actin promoter with an upstream CMV IV enhancer (CAGGS), and the elongation factor 1 alpha promoter (EF1A) since these promoters are among the most commonly used polymerase II promoters to drive mRNA expression. We analyzed the potency of these promoters in driving anti-EGFP shRNA expression by measuring EGFP protein knockdown in EGFP-expressing cells via flow cytometry (See Figure 2). shRNAs expressed from these promoters displayed various potency in target-knockdown in Jurkat cells. The ubiquitin C (UbiC) promoter, the phosphoglycerate kinase (PGK) promoter, and the cytomegalovirus (CMV) promoter were equally potent in driving EGFP-target knockdown (52.2%, 52.9%, and 52.7% respectively), whereas the beta-actin promoter with an upstream CMV IV enhancer (CAGGS, 70.7%), and the elongation factor 1 alpha promoter (EF1A, 86.3%) were much more effective (See Figure 2). These data reveal a significant impact of the choice of polymerase II promoter used in shRNA-directed target knockdown in human Jurkat T cells.


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 miRNA backbone are less efficient in target-knockdown in Jurkat T cells than in 293T cells.A) schematic representation of the miR-context anti-EGFP shRNA construct cloned in the 3′UTR of mCherry which is expressed under the control of the polymerase II CMV promoter (lower panel) and the control anti-EGFP shRNA expressed from a mouse polymerase III U6 promoter (top panel). B) EGFP-expressing Jurkat T cells (top panels) or 293T cells (lower panels) were infected at an MOI of <0.2 with anti-EGFP shRNAs expressed from a mouse U6 promoter (U6 anti-EGFP shRNA) or from a miR-30 backbone expressed from a CMV promoter (miR-context anti-EGFP shRNA) or the relevant control viruses that lack shRNA inserts. Cells were allowed to grow for 8 days and the expression of EGFP (to monitor knockdown) and mCherry (as marker for infected cells) were assessed by flow cytometry. The indicated 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198731&req=5

pone-0026213-g001: anti-EGFP shRNAs expressed from a miRNA backbone are less efficient in target-knockdown in Jurkat T cells than in 293T cells.A) schematic representation of the miR-context anti-EGFP shRNA construct cloned in the 3′UTR of mCherry which is expressed under the control of the polymerase II CMV promoter (lower panel) and the control anti-EGFP shRNA expressed from a mouse polymerase III U6 promoter (top panel). B) EGFP-expressing Jurkat T cells (top panels) or 293T cells (lower panels) were infected at an MOI of <0.2 with anti-EGFP shRNAs expressed from a mouse U6 promoter (U6 anti-EGFP shRNA) or from a miR-30 backbone expressed from a CMV promoter (miR-context anti-EGFP shRNA) or the relevant control viruses that lack shRNA inserts. Cells were allowed to grow for 8 days and the expression of EGFP (to monitor knockdown) and mCherry (as marker for infected cells) were assessed by flow cytometry. The indicated 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.
Mentions: In the process of generating stable EGFP knockdown cell lines in human Jurkat T cells by using an anti-EGFP shRNA expressed from CMV-driven miR30-backbone expression vectors (shRNA-miRs, see Figure 1A for design of the constructs), we noted a much reduced target knockdown (52.7%) as compared to identical shRNAs expressed from the mouse polymerase III U6 promoter (90.2%, see Figure 1B, top panels). These same lentiviral shRNA vectors were equally potent in driving EGFP knockdown in human 293T cells (88.0% and 92.5% target knockdown for shRNA-miR and U6-driven shRNAs respectively, see Figure 1B lower panels), suggesting that shRNA-miRs might be less potent in Jurkat T cells as in 293T cells. We set out to explore the nature of this discrepancy, and assessed whether the potency of the polymerase II promoter used to express the miR30-backbone anti-EGFP shRNAs could impact the shRNA-directed silencing in Jurkat T cells. To this end, we cloned five different polymerase II promoters upstream the miR-30 anti-EGFP shRNA cassette that is located in the 3′UTR of the fluorescent protein mCherry to mark cells that have been infected with the lentiviral construct. We selected the ubiquitin C (UbiC) promoter, the phosphoglycerate kinase (PGK) promoter, the cytomegalovirus (CMV) promoter, a beta-actin promoter with an upstream CMV IV enhancer (CAGGS), and the elongation factor 1 alpha promoter (EF1A) since these promoters are among the most commonly used polymerase II promoters to drive mRNA expression. We analyzed the potency of these promoters in driving anti-EGFP shRNA expression by measuring EGFP protein knockdown in EGFP-expressing cells via flow cytometry (See Figure 2). shRNAs expressed from these promoters displayed various potency in target-knockdown in Jurkat cells. The ubiquitin C (UbiC) promoter, the phosphoglycerate kinase (PGK) promoter, and the cytomegalovirus (CMV) promoter were equally potent in driving EGFP-target knockdown (52.2%, 52.9%, and 52.7% respectively), whereas the beta-actin promoter with an upstream CMV IV enhancer (CAGGS, 70.7%), and the elongation factor 1 alpha promoter (EF1A, 86.3%) were much more effective (See Figure 2). These data reveal a significant impact of the choice of polymerase II promoter used in shRNA-directed target knockdown in human Jurkat T cells.

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