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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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Knockdown and rescue by transient transfection of pKAR1 constructs. (A) Knockdown of endogenous cyclin A and expression of recombinant cyclin A by transient transfection. HtTA1 cells were transfected with either control vectors or cyclin A/shRNA-expressing plasmids. After enriching the transfected cells (Materials and Methods), over 90% of cells expressed a cotransfected GFP-tagged histone H2B (data not shown). Cells expressing cyclin A and shRNA were either mock-treated or treated with doxycycline for 48 h as indicated. Cell-free extracts were prepared and subjected to immunoblotting for cyclin A and FLAG. Uniform loading of lysates was confirmed by immunoblotting for CDC2. (B) Suppression of recombinant cyclin A expression by doxycycline. Cells were transfected with cyclin A/shRNA in pKAR1 as described in panel A. After doxycycline was applied, cell-free extracts were prepared at the indicated time points and subjected to immunoblotting for cyclin A. Lysates from control cells were loaded in lane 1. The lower band in lane 2 probably is a degradative product of FLAG-cyclin A. (C) Knockdown and rescue of MAD2. HtTA1 cells were transfected with either control vectors (lane 1) or MAD2/shRNA-expressing plasmids in the presence of doxycycline. After enriching the transfected cells, the MAD2- and shRNA-expressing cells were washed and grown in doxycycline-free medium. At the indicated time points, cell-free extracts were prepared and subjected to immunoblotting for MAD2.
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Figure 3: Knockdown and rescue by transient transfection of pKAR1 constructs. (A) Knockdown of endogenous cyclin A and expression of recombinant cyclin A by transient transfection. HtTA1 cells were transfected with either control vectors or cyclin A/shRNA-expressing plasmids. After enriching the transfected cells (Materials and Methods), over 90% of cells expressed a cotransfected GFP-tagged histone H2B (data not shown). Cells expressing cyclin A and shRNA were either mock-treated or treated with doxycycline for 48 h as indicated. Cell-free extracts were prepared and subjected to immunoblotting for cyclin A and FLAG. Uniform loading of lysates was confirmed by immunoblotting for CDC2. (B) Suppression of recombinant cyclin A expression by doxycycline. Cells were transfected with cyclin A/shRNA in pKAR1 as described in panel A. After doxycycline was applied, cell-free extracts were prepared at the indicated time points and subjected to immunoblotting for cyclin A. Lysates from control cells were loaded in lane 1. The lower band in lane 2 probably is a degradative product of FLAG-cyclin A. (C) Knockdown and rescue of MAD2. HtTA1 cells were transfected with either control vectors (lane 1) or MAD2/shRNA-expressing plasmids in the presence of doxycycline. After enriching the transfected cells, the MAD2- and shRNA-expressing cells were washed and grown in doxycycline-free medium. At the indicated time points, cell-free extracts were prepared and subjected to immunoblotting for MAD2.

Mentions: To determine if the endogenous cyclin A could be downregulated by the cyclin A/shRNA construct, cells were transfected with either control vectors or the cyclin A/shRNA construct. Figure 3A shows that the expression of cyclin A was effectively attenuated by the shRNA (lanes 1 and 2). As expected, FLAG-cyclin A (which exhibited a slightly slower gel mobility than the endogenous cyclin A) was expressed in the absence, but not the presence of doxycycline. The expression of the recombinant FLAG-cyclin A was also confirmed by immunoblotting for FLAG. Figure 3B shows that FLAG-cyclin A was suppressed by doxycycline progressively over the time course of the experiment. These results indicate that while the endogenous cyclin A could be silenced by the shRNA, the co-expressed recombinant cyclin A was refractory to the knockdown.Figure 3.


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)

Knockdown and rescue by transient transfection of pKAR1 constructs. (A) Knockdown of endogenous cyclin A and expression of recombinant cyclin A by transient transfection. HtTA1 cells were transfected with either control vectors or cyclin A/shRNA-expressing plasmids. After enriching the transfected cells (Materials and Methods), over 90% of cells expressed a cotransfected GFP-tagged histone H2B (data not shown). Cells expressing cyclin A and shRNA were either mock-treated or treated with doxycycline for 48 h as indicated. Cell-free extracts were prepared and subjected to immunoblotting for cyclin A and FLAG. Uniform loading of lysates was confirmed by immunoblotting for CDC2. (B) Suppression of recombinant cyclin A expression by doxycycline. Cells were transfected with cyclin A/shRNA in pKAR1 as described in panel A. After doxycycline was applied, cell-free extracts were prepared at the indicated time points and subjected to immunoblotting for cyclin A. Lysates from control cells were loaded in lane 1. The lower band in lane 2 probably is a degradative product of FLAG-cyclin A. (C) Knockdown and rescue of MAD2. HtTA1 cells were transfected with either control vectors (lane 1) or MAD2/shRNA-expressing plasmids in the presence of doxycycline. After enriching the transfected cells, the MAD2- and shRNA-expressing cells were washed and grown in doxycycline-free medium. At the indicated time points, cell-free extracts were prepared and subjected to immunoblotting for MAD2.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Figure 3: Knockdown and rescue by transient transfection of pKAR1 constructs. (A) Knockdown of endogenous cyclin A and expression of recombinant cyclin A by transient transfection. HtTA1 cells were transfected with either control vectors or cyclin A/shRNA-expressing plasmids. After enriching the transfected cells (Materials and Methods), over 90% of cells expressed a cotransfected GFP-tagged histone H2B (data not shown). Cells expressing cyclin A and shRNA were either mock-treated or treated with doxycycline for 48 h as indicated. Cell-free extracts were prepared and subjected to immunoblotting for cyclin A and FLAG. Uniform loading of lysates was confirmed by immunoblotting for CDC2. (B) Suppression of recombinant cyclin A expression by doxycycline. Cells were transfected with cyclin A/shRNA in pKAR1 as described in panel A. After doxycycline was applied, cell-free extracts were prepared at the indicated time points and subjected to immunoblotting for cyclin A. Lysates from control cells were loaded in lane 1. The lower band in lane 2 probably is a degradative product of FLAG-cyclin A. (C) Knockdown and rescue of MAD2. HtTA1 cells were transfected with either control vectors (lane 1) or MAD2/shRNA-expressing plasmids in the presence of doxycycline. After enriching the transfected cells, the MAD2- and shRNA-expressing cells were washed and grown in doxycycline-free medium. At the indicated time points, cell-free extracts were prepared and subjected to immunoblotting for MAD2.
Mentions: To determine if the endogenous cyclin A could be downregulated by the cyclin A/shRNA construct, cells were transfected with either control vectors or the cyclin A/shRNA construct. Figure 3A shows that the expression of cyclin A was effectively attenuated by the shRNA (lanes 1 and 2). As expected, FLAG-cyclin A (which exhibited a slightly slower gel mobility than the endogenous cyclin A) was expressed in the absence, but not the presence of doxycycline. The expression of the recombinant FLAG-cyclin A was also confirmed by immunoblotting for FLAG. Figure 3B shows that FLAG-cyclin A was suppressed by doxycycline progressively over the time course of the experiment. These results indicate that while the endogenous cyclin A could be silenced by the shRNA, the co-expressed recombinant cyclin A was refractory to the knockdown.Figure 3.

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
Related in: MedlinePlus