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An RNA polymerase II construct synthesizes short-hairpin RNA with a quantitative indicator and mediates highly efficient RNAi.

Zhou H, Xia XG, Xu Z - Nucleic Acids Res. (2005)

Bottom Line: Previous efforts in developing a Pol II system have been sparse and the results were conflicting, and the usefulness of those Pol II vectors has been limited due to low efficacy.In addition, this system synthesizes a marker protein under control of the same promoter as the shRNA, thus providing an unequivocal indicator, not only to the cells that express the shRNA, but also to the levels of the shRNA expression.This system may be adapted for in vivo shRNA expression and gene silencing.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School 364 Plantation Street, Worcester, MA 01605, USA. zuoshang.xu@umassmed.edu

ABSTRACT
RNA interference (RNAi) mediates gene silencing in many eukaryotes and has been widely used to investigate gene functions. A common method to induce sustained RNAi is introducing plasmids that synthesize short hairpin RNAs (shRNAs) using Pol III promoters. While these promoters synthesize shRNAs and elicit RNAi efficiently, they lack cell specificity. Monitoring shRNA expression levels in individual cells by Pol III promoters is also difficult. An alternative way to deliver RNAi is to use Pol II-directed synthesis of shRNA. Previous efforts in developing a Pol II system have been sparse and the results were conflicting, and the usefulness of those Pol II vectors has been limited due to low efficacy. Here we demonstrate a new Pol II system that directs efficient shRNA synthesis and mediates strong RNAi at levels that are comparable with the commonly used Pol III systems. In addition, this system synthesizes a marker protein under control of the same promoter as the shRNA, thus providing an unequivocal indicator, not only to the cells that express the shRNA, but also to the levels of the shRNA expression. This system may be adapted for in vivo shRNA expression and gene silencing.

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Comparisons in the levels of shRNA expression and in silencing efficacy. (A) Northern analysis of SOD1-shRNA expression under control of either Pol-II or Pol-III promoters. Each lane was loaded with equal amount of total RNA (20 μg/lane) extracted from HEK 293 cells transfected with indicated shRNA constructs. The equal loading was verified by staining the gel with ethidium bromide before transferring the RNA to the membrane (data not shown). Lane numbers correspond to the construct numbers shown in Figure 1. (B) Silencing activity of each construct. HEK 293 cells were transfected with Rr-luc-sod1 and Pp-luc plasmids together with various shRNA constructs against human SOD1 mRNA, located at 3′-UTR of firefly luciferase (Pp-luc) gene. Renilla luciferase (Rr-luc) plasmid served as transfection control. All luciferase activities were normalized to the value measured in the lysate from cells transfected with the empty vector pcDNA3 (carrier). The values are means with SD (n = 6). The construct numbers match with those in Figure 1.
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fig2: Comparisons in the levels of shRNA expression and in silencing efficacy. (A) Northern analysis of SOD1-shRNA expression under control of either Pol-II or Pol-III promoters. Each lane was loaded with equal amount of total RNA (20 μg/lane) extracted from HEK 293 cells transfected with indicated shRNA constructs. The equal loading was verified by staining the gel with ethidium bromide before transferring the RNA to the membrane (data not shown). Lane numbers correspond to the construct numbers shown in Figure 1. (B) Silencing activity of each construct. HEK 293 cells were transfected with Rr-luc-sod1 and Pp-luc plasmids together with various shRNA constructs against human SOD1 mRNA, located at 3′-UTR of firefly luciferase (Pp-luc) gene. Renilla luciferase (Rr-luc) plasmid served as transfection control. All luciferase activities were normalized to the value measured in the lysate from cells transfected with the empty vector pcDNA3 (carrier). The values are means with SD (n = 6). The construct numbers match with those in Figure 1.

Mentions: To find the optimal construct for Pol II-directed synthesis of shRNA, we made a series of variants of human miR-30 hairpin structure (Figure 1). We chose miR-30 as a model hairpin structure because its stem sequence could be substituted with other sequences that match different targets (20,29). Because the previous studies suggested that simplifying miR-30 structure by eliminating the mismatch, the bulge and most of the base sequence does not significantly alter the RNAi efficiency (20,29), we first constructed a similar hairpin and a series of its variations (Figure 1, constructs 1–7). These hairpins targeted the human SOD1 mRNA and were placed downstream from the CMV promoter in an mRNA transcript. This construct design was similar to several previously published Pol II-shRNA synthesizing constructs (20,29). In cotransfections with a report luciferase construct that contains the target sequence in its 3′-UTR, most of these hairpins (1–6 and 9) were expressed (Figure 2A) and mediated RNAi against the target (Figure 2B), albeit all at lowered efficiency compared with the construct that carried the unmodified miR-30 hairpin structure (Figure 1, construct 8; and Figure 2). The expression of construct 7 was not detected, nor was its RNAi efficacy (Figures 1 and 2). This lack of expression of shRNA in construct 7 is unlikely to be caused by RNA degradation as shown by ethidium bromide staining of this gel before transferring (data not shown). Rather, it might be caused by a lack of processing due to the relatively drastic change from the authentic miR-30 structure in this construct.


An RNA polymerase II construct synthesizes short-hairpin RNA with a quantitative indicator and mediates highly efficient RNAi.

Zhou H, Xia XG, Xu Z - Nucleic Acids Res. (2005)

Comparisons in the levels of shRNA expression and in silencing efficacy. (A) Northern analysis of SOD1-shRNA expression under control of either Pol-II or Pol-III promoters. Each lane was loaded with equal amount of total RNA (20 μg/lane) extracted from HEK 293 cells transfected with indicated shRNA constructs. The equal loading was verified by staining the gel with ethidium bromide before transferring the RNA to the membrane (data not shown). Lane numbers correspond to the construct numbers shown in Figure 1. (B) Silencing activity of each construct. HEK 293 cells were transfected with Rr-luc-sod1 and Pp-luc plasmids together with various shRNA constructs against human SOD1 mRNA, located at 3′-UTR of firefly luciferase (Pp-luc) gene. Renilla luciferase (Rr-luc) plasmid served as transfection control. All luciferase activities were normalized to the value measured in the lysate from cells transfected with the empty vector pcDNA3 (carrier). The values are means with SD (n = 6). The construct numbers match with those in Figure 1.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1074311&req=5

fig2: Comparisons in the levels of shRNA expression and in silencing efficacy. (A) Northern analysis of SOD1-shRNA expression under control of either Pol-II or Pol-III promoters. Each lane was loaded with equal amount of total RNA (20 μg/lane) extracted from HEK 293 cells transfected with indicated shRNA constructs. The equal loading was verified by staining the gel with ethidium bromide before transferring the RNA to the membrane (data not shown). Lane numbers correspond to the construct numbers shown in Figure 1. (B) Silencing activity of each construct. HEK 293 cells were transfected with Rr-luc-sod1 and Pp-luc plasmids together with various shRNA constructs against human SOD1 mRNA, located at 3′-UTR of firefly luciferase (Pp-luc) gene. Renilla luciferase (Rr-luc) plasmid served as transfection control. All luciferase activities were normalized to the value measured in the lysate from cells transfected with the empty vector pcDNA3 (carrier). The values are means with SD (n = 6). The construct numbers match with those in Figure 1.
Mentions: To find the optimal construct for Pol II-directed synthesis of shRNA, we made a series of variants of human miR-30 hairpin structure (Figure 1). We chose miR-30 as a model hairpin structure because its stem sequence could be substituted with other sequences that match different targets (20,29). Because the previous studies suggested that simplifying miR-30 structure by eliminating the mismatch, the bulge and most of the base sequence does not significantly alter the RNAi efficiency (20,29), we first constructed a similar hairpin and a series of its variations (Figure 1, constructs 1–7). These hairpins targeted the human SOD1 mRNA and were placed downstream from the CMV promoter in an mRNA transcript. This construct design was similar to several previously published Pol II-shRNA synthesizing constructs (20,29). In cotransfections with a report luciferase construct that contains the target sequence in its 3′-UTR, most of these hairpins (1–6 and 9) were expressed (Figure 2A) and mediated RNAi against the target (Figure 2B), albeit all at lowered efficiency compared with the construct that carried the unmodified miR-30 hairpin structure (Figure 1, construct 8; and Figure 2). The expression of construct 7 was not detected, nor was its RNAi efficacy (Figures 1 and 2). This lack of expression of shRNA in construct 7 is unlikely to be caused by RNA degradation as shown by ethidium bromide staining of this gel before transferring (data not shown). Rather, it might be caused by a lack of processing due to the relatively drastic change from the authentic miR-30 structure in this construct.

Bottom Line: Previous efforts in developing a Pol II system have been sparse and the results were conflicting, and the usefulness of those Pol II vectors has been limited due to low efficacy.In addition, this system synthesizes a marker protein under control of the same promoter as the shRNA, thus providing an unequivocal indicator, not only to the cells that express the shRNA, but also to the levels of the shRNA expression.This system may be adapted for in vivo shRNA expression and gene silencing.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School 364 Plantation Street, Worcester, MA 01605, USA. zuoshang.xu@umassmed.edu

ABSTRACT
RNA interference (RNAi) mediates gene silencing in many eukaryotes and has been widely used to investigate gene functions. A common method to induce sustained RNAi is introducing plasmids that synthesize short hairpin RNAs (shRNAs) using Pol III promoters. While these promoters synthesize shRNAs and elicit RNAi efficiently, they lack cell specificity. Monitoring shRNA expression levels in individual cells by Pol III promoters is also difficult. An alternative way to deliver RNAi is to use Pol II-directed synthesis of shRNA. Previous efforts in developing a Pol II system have been sparse and the results were conflicting, and the usefulness of those Pol II vectors has been limited due to low efficacy. Here we demonstrate a new Pol II system that directs efficient shRNA synthesis and mediates strong RNAi at levels that are comparable with the commonly used Pol III systems. In addition, this system synthesizes a marker protein under control of the same promoter as the shRNA, thus providing an unequivocal indicator, not only to the cells that express the shRNA, but also to the levels of the shRNA expression. This system may be adapted for in vivo shRNA expression and gene silencing.

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