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An inducible system for expression and validation of the specificity of short hairpin RNA in mammalian cells.

Ma HT, On KF, Tsang YH, Poon RY - Nucleic Acids Res. (2007)

Bottom Line: Conversely, the rescue protein can be activated after the endogenous protein is completely repressed.This approach is particularly suitable when prolonged expression of either the shRNA or the compensatory cDNA is detrimental to cell growth.This system allows a convenient one-step validation of shRNA and generation of stable shRNA-expressing cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong.

ABSTRACT
RNA interference (RNAi) by means of short hairpin RNA (shRNA) has developed into a powerful tool for loss-of-function analysis in mammalian cells. The principal problem in RNAi experiments is off-target effects, and the most vigorous demonstration of the specificity of shRNA is the rescue of the RNAi effects with a shRNA-resistant target gene. This presents its own problems, including the unpredictable relative expression of shRNA and rescue cDNA in individual cells, and the difficulty in generating stable cell lines. In this report, we evaluated the plausibility of combining the expression of shRNA and rescue cDNA in the same vector. In addition to facilitate the validation of shRNA specificity, this system also considerably simplifies the generation of shRNA-expressing cell lines. Since the compensatory cDNA is under the control of an inducible promoter, stable shRNA-expressing cells can be generated before the knockdown phenotypes are studied by conditionally turning off the rescue protein. Conversely, the rescue protein can be activated after the endogenous protein is completely repressed. This approach is particularly suitable when prolonged expression of either the shRNA or the compensatory cDNA is detrimental to cell growth. This system allows a convenient one-step validation of shRNA and generation of stable shRNA-expressing cells.

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A vector for co-expression of shRNA and shRNA-resistant cDNA. (A) Schematic diagram of pKAR1. The various elements are shown to scale: TRE, tetracycline response element; PminCMV, minimal immediate early cytomegalovirus (CMV) promoter; FLAG-3C-MCS, multiple cloning sites; SV40 pA, SV40 polyadenylation signal; PU6, mouse U6 RNA promoter; Ampr, ampicillin-resistant gene; ColE1 ori, ColE1 replication origin. The shRNA is first inserted between Bbs I-Xba I exactly as described for mU6pro (5), and the shRNA-resistant cDNA is cloned into the multiple cloning sites (see panel B). (B) The cDNA cloning region of pKAR1. The protein sequence of the N-terminal tag is shown. After the cloning of the shRNA, the cDNA can be inserted in frame into the unique Apa I, Nco I, Xho I, EcoR I and BamH I sites. Un-tagged version can be cloned using the Nhe I site. The FLAG-tag is highlighted, the 3C protease recognition sequence is underlined, and the arrow indicates the site of cleavage.
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Figure 1: A vector for co-expression of shRNA and shRNA-resistant cDNA. (A) Schematic diagram of pKAR1. The various elements are shown to scale: TRE, tetracycline response element; PminCMV, minimal immediate early cytomegalovirus (CMV) promoter; FLAG-3C-MCS, multiple cloning sites; SV40 pA, SV40 polyadenylation signal; PU6, mouse U6 RNA promoter; Ampr, ampicillin-resistant gene; ColE1 ori, ColE1 replication origin. The shRNA is first inserted between Bbs I-Xba I exactly as described for mU6pro (5), and the shRNA-resistant cDNA is cloned into the multiple cloning sites (see panel B). (B) The cDNA cloning region of pKAR1. The protein sequence of the N-terminal tag is shown. After the cloning of the shRNA, the cDNA can be inserted in frame into the unique Apa I, Nco I, Xho I, EcoR I and BamH I sites. Un-tagged version can be cloned using the Nhe I site. The FLAG-tag is highlighted, the 3C protease recognition sequence is underlined, and the arrow indicates the site of cleavage.

Mentions: We constructed the pKAR (Knockdown And Rescue) plasmids based on an inducible system designed by Hermann Bujard's group (14), and a shRNA-expressing system originated from David Turner's group (5) (Figure 1A). The shRNA was expressed from a mouse U6 RNA promoter, and the rescue cDNA was expressed under the control of doxycycline. The cDNA expressed from pKAR1 was engineered to fuse at the N-terminus with a FLAG-tag and a 3C protease cleavage site (Figure 1B). Due to the slight increase in size conferred by the epitope tag, both the endogenous protein to be silenced and the ectopically expressed version can be detected simultaneously. The tag also allowed the recombinant protein to be specifically detected or immunoprecipitated. Furthermore, the epitope tag can be removed using 3C proteases.Figure 1.


An inducible system for expression and validation of the specificity of short hairpin RNA in mammalian cells.

Ma HT, On KF, Tsang YH, Poon RY - Nucleic Acids Res. (2007)

A vector for co-expression of shRNA and shRNA-resistant cDNA. (A) Schematic diagram of pKAR1. The various elements are shown to scale: TRE, tetracycline response element; PminCMV, minimal immediate early cytomegalovirus (CMV) promoter; FLAG-3C-MCS, multiple cloning sites; SV40 pA, SV40 polyadenylation signal; PU6, mouse U6 RNA promoter; Ampr, ampicillin-resistant gene; ColE1 ori, ColE1 replication origin. The shRNA is first inserted between Bbs I-Xba I exactly as described for mU6pro (5), and the shRNA-resistant cDNA is cloned into the multiple cloning sites (see panel B). (B) The cDNA cloning region of pKAR1. The protein sequence of the N-terminal tag is shown. After the cloning of the shRNA, the cDNA can be inserted in frame into the unique Apa I, Nco I, Xho I, EcoR I and BamH I sites. Un-tagged version can be cloned using the Nhe I site. The FLAG-tag is highlighted, the 3C protease recognition sequence is underlined, and the arrow indicates the site of cleavage.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851631&req=5

Figure 1: A vector for co-expression of shRNA and shRNA-resistant cDNA. (A) Schematic diagram of pKAR1. The various elements are shown to scale: TRE, tetracycline response element; PminCMV, minimal immediate early cytomegalovirus (CMV) promoter; FLAG-3C-MCS, multiple cloning sites; SV40 pA, SV40 polyadenylation signal; PU6, mouse U6 RNA promoter; Ampr, ampicillin-resistant gene; ColE1 ori, ColE1 replication origin. The shRNA is first inserted between Bbs I-Xba I exactly as described for mU6pro (5), and the shRNA-resistant cDNA is cloned into the multiple cloning sites (see panel B). (B) The cDNA cloning region of pKAR1. The protein sequence of the N-terminal tag is shown. After the cloning of the shRNA, the cDNA can be inserted in frame into the unique Apa I, Nco I, Xho I, EcoR I and BamH I sites. Un-tagged version can be cloned using the Nhe I site. The FLAG-tag is highlighted, the 3C protease recognition sequence is underlined, and the arrow indicates the site of cleavage.
Mentions: We constructed the pKAR (Knockdown And Rescue) plasmids based on an inducible system designed by Hermann Bujard's group (14), and a shRNA-expressing system originated from David Turner's group (5) (Figure 1A). The shRNA was expressed from a mouse U6 RNA promoter, and the rescue cDNA was expressed under the control of doxycycline. The cDNA expressed from pKAR1 was engineered to fuse at the N-terminus with a FLAG-tag and a 3C protease cleavage site (Figure 1B). Due to the slight increase in size conferred by the epitope tag, both the endogenous protein to be silenced and the ectopically expressed version can be detected simultaneously. The tag also allowed the recombinant protein to be specifically detected or immunoprecipitated. Furthermore, the epitope tag can be removed using 3C proteases.Figure 1.

Bottom Line: Conversely, the rescue protein can be activated after the endogenous protein is completely repressed.This approach is particularly suitable when prolonged expression of either the shRNA or the compensatory cDNA is detrimental to cell growth.This system allows a convenient one-step validation of shRNA and generation of stable shRNA-expressing cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong.

ABSTRACT
RNA interference (RNAi) by means of short hairpin RNA (shRNA) has developed into a powerful tool for loss-of-function analysis in mammalian cells. The principal problem in RNAi experiments is off-target effects, and the most vigorous demonstration of the specificity of shRNA is the rescue of the RNAi effects with a shRNA-resistant target gene. This presents its own problems, including the unpredictable relative expression of shRNA and rescue cDNA in individual cells, and the difficulty in generating stable cell lines. In this report, we evaluated the plausibility of combining the expression of shRNA and rescue cDNA in the same vector. In addition to facilitate the validation of shRNA specificity, this system also considerably simplifies the generation of shRNA-expressing cell lines. Since the compensatory cDNA is under the control of an inducible promoter, stable shRNA-expressing cells can be generated before the knockdown phenotypes are studied by conditionally turning off the rescue protein. Conversely, the rescue protein can be activated after the endogenous protein is completely repressed. This approach is particularly suitable when prolonged expression of either the shRNA or the compensatory cDNA is detrimental to cell growth. This system allows a convenient one-step validation of shRNA and generation of stable shRNA-expressing cells.

Show MeSH