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Comparison of chicken 7SK and U6 RNA polymerase III promoters for short hairpin RNA expression.

Bannister SC, Wise TG, Cahill DM, Doran TJ - BMC Biotechnol. (2007)

Bottom Line: We transfected chicken DF-1 cells with these constructs and found that anti-EGFP-shRNAs (shEGFP) expressed from the ch7SK promoter could induce efficient knockdown of EGFP expression.We further compared the efficiency of ch7SK-directed knockdown to that of chicken U6 (cU6) promoters and found that the efficiency of the ch7SK promoter was not greater than, but comparable to the efficiency of cU6 promoters.This aside, our results clearly indicate that the ch7SK promoter is an efficient alternative to U6-based shRNA expression systems for inducing efficient RNAi activity in chicken cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: CSIRO Livestock Industries, Australian Animal Health Laboratory, Geelong 3220, Australia. stephanie.bannister@csiro.au

ABSTRACT

Background: RNA polymerase III (pol III) type 3 promoters such as U6 or 7SK are commonly used to express short-hairpin RNA (shRNA) effectors for RNA interference (RNAi). To extend the use of RNAi for studies of development using the chicken as a model system, we have developed a system for expressing shRNAs using the chicken 7SK (ch7SK) promoter.

Results: We identified and characterised the ch7SK promoter sequence upstream of the full-length 7SK small nuclear RNA (snRNA) sequence in the chicken genome and used this to construct vectors to express shRNAs targeting enhanced green fluorescent protein (EGFP). We transfected chicken DF-1 cells with these constructs and found that anti-EGFP-shRNAs (shEGFP) expressed from the ch7SK promoter could induce efficient knockdown of EGFP expression. We further compared the efficiency of ch7SK-directed knockdown to that of chicken U6 (cU6) promoters and found that the efficiency of the ch7SK promoter was not greater than, but comparable to the efficiency of cU6 promoters.

Conclusion: In this study we have demonstrated that the ch7SK promoter can express shRNAs capable of mediating efficient RNAi in a chicken cell line. However, our finding that RNAi driven by the ch7SK promoter is not more efficient than cU6 promoters contrasts previous comparisons of mammalian U6 and 7SK promoters. Since the ch7SK promoter is the first non-mammalian vertebrate 7SK promoter to be characterised, this finding may be helpful in understanding the divergence of pol III promoter activities between mammalian and non-mammalian vertebrates. This aside, our results clearly indicate that the ch7SK promoter is an efficient alternative to U6-based shRNA expression systems for inducing efficient RNAi activity in chicken cells.

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EGFP knockdown conferred by chicken 7SK and U6 promoters in DF-1 cells. (a) Fluorescence microscopy images of DF-1 cells transfected with pEGFP-N1 only, or co-transfected with pEGFP-N1 and various shEGFP expression plasmids as indicated for each image. TA is transfection reagent-only control (no-plasmid DNA). Images presented are representative of results from three independent experiments at 60 hours post-transfection (Magnification 50×). (b) Flow cytometry results for EGFP knockdown assays in co-transfected DF-1 cells. shEGFP expression constructs co-transfected with pEGFP-N1 are indicated on the x axis. EGFP knockdown was measured as a percent mean fluorescence intensity (MFI %), normalised to the average MFI of the negative control pch7SK-shIrr cells (100%). Error bars represent the standard error of the mean (SEM) calculated from at three independent experiments. Where no bars or error bars are visible the MFI and or SEM is less than 1%.
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Figure 3: EGFP knockdown conferred by chicken 7SK and U6 promoters in DF-1 cells. (a) Fluorescence microscopy images of DF-1 cells transfected with pEGFP-N1 only, or co-transfected with pEGFP-N1 and various shEGFP expression plasmids as indicated for each image. TA is transfection reagent-only control (no-plasmid DNA). Images presented are representative of results from three independent experiments at 60 hours post-transfection (Magnification 50×). (b) Flow cytometry results for EGFP knockdown assays in co-transfected DF-1 cells. shEGFP expression constructs co-transfected with pEGFP-N1 are indicated on the x axis. EGFP knockdown was measured as a percent mean fluorescence intensity (MFI %), normalised to the average MFI of the negative control pch7SK-shIrr cells (100%). Error bars represent the standard error of the mean (SEM) calculated from at three independent experiments. Where no bars or error bars are visible the MFI and or SEM is less than 1%.

Mentions: To verify that the shEGFP expressed by the ch7SK promoter could direct RNAi-mediated knockdown of an EGFP reporter gene, we conducted EGFP knockdown assays by co-transfecting chicken DF-1 cells with the pch7SK-shEGFP, pch7SK-MCS-shEGFP or positive control, pmU6-shEGFP and pEZ-b7SK-shEGFP [14] constructs, with an EGFP expression vector (pEGFP-N1) (Fig. 3). Given that co-transfection of reporter and shRNA expression plasmids is considered to be 100% efficient for validation of specific RNAi activity [24], we considered any reduction in EGFP fluorescence intensity to reflect RNAi-mediated EGFP knockdown. EGFP knockdown was assessed for each co-transfection condition in duplicate using fluorescence microscopy (Fig. 3a) and quantified using flow cytometry by sampling the mean fluorescence intensity (MFI) from triplicate co-transfections for each condition (See Materials & Methods) (Fig. 3b).


Comparison of chicken 7SK and U6 RNA polymerase III promoters for short hairpin RNA expression.

Bannister SC, Wise TG, Cahill DM, Doran TJ - BMC Biotechnol. (2007)

EGFP knockdown conferred by chicken 7SK and U6 promoters in DF-1 cells. (a) Fluorescence microscopy images of DF-1 cells transfected with pEGFP-N1 only, or co-transfected with pEGFP-N1 and various shEGFP expression plasmids as indicated for each image. TA is transfection reagent-only control (no-plasmid DNA). Images presented are representative of results from three independent experiments at 60 hours post-transfection (Magnification 50×). (b) Flow cytometry results for EGFP knockdown assays in co-transfected DF-1 cells. shEGFP expression constructs co-transfected with pEGFP-N1 are indicated on the x axis. EGFP knockdown was measured as a percent mean fluorescence intensity (MFI %), normalised to the average MFI of the negative control pch7SK-shIrr cells (100%). Error bars represent the standard error of the mean (SEM) calculated from at three independent experiments. Where no bars or error bars are visible the MFI and or SEM is less than 1%.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2235858&req=5

Figure 3: EGFP knockdown conferred by chicken 7SK and U6 promoters in DF-1 cells. (a) Fluorescence microscopy images of DF-1 cells transfected with pEGFP-N1 only, or co-transfected with pEGFP-N1 and various shEGFP expression plasmids as indicated for each image. TA is transfection reagent-only control (no-plasmid DNA). Images presented are representative of results from three independent experiments at 60 hours post-transfection (Magnification 50×). (b) Flow cytometry results for EGFP knockdown assays in co-transfected DF-1 cells. shEGFP expression constructs co-transfected with pEGFP-N1 are indicated on the x axis. EGFP knockdown was measured as a percent mean fluorescence intensity (MFI %), normalised to the average MFI of the negative control pch7SK-shIrr cells (100%). Error bars represent the standard error of the mean (SEM) calculated from at three independent experiments. Where no bars or error bars are visible the MFI and or SEM is less than 1%.
Mentions: To verify that the shEGFP expressed by the ch7SK promoter could direct RNAi-mediated knockdown of an EGFP reporter gene, we conducted EGFP knockdown assays by co-transfecting chicken DF-1 cells with the pch7SK-shEGFP, pch7SK-MCS-shEGFP or positive control, pmU6-shEGFP and pEZ-b7SK-shEGFP [14] constructs, with an EGFP expression vector (pEGFP-N1) (Fig. 3). Given that co-transfection of reporter and shRNA expression plasmids is considered to be 100% efficient for validation of specific RNAi activity [24], we considered any reduction in EGFP fluorescence intensity to reflect RNAi-mediated EGFP knockdown. EGFP knockdown was assessed for each co-transfection condition in duplicate using fluorescence microscopy (Fig. 3a) and quantified using flow cytometry by sampling the mean fluorescence intensity (MFI) from triplicate co-transfections for each condition (See Materials & Methods) (Fig. 3b).

Bottom Line: We transfected chicken DF-1 cells with these constructs and found that anti-EGFP-shRNAs (shEGFP) expressed from the ch7SK promoter could induce efficient knockdown of EGFP expression.We further compared the efficiency of ch7SK-directed knockdown to that of chicken U6 (cU6) promoters and found that the efficiency of the ch7SK promoter was not greater than, but comparable to the efficiency of cU6 promoters.This aside, our results clearly indicate that the ch7SK promoter is an efficient alternative to U6-based shRNA expression systems for inducing efficient RNAi activity in chicken cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: CSIRO Livestock Industries, Australian Animal Health Laboratory, Geelong 3220, Australia. stephanie.bannister@csiro.au

ABSTRACT

Background: RNA polymerase III (pol III) type 3 promoters such as U6 or 7SK are commonly used to express short-hairpin RNA (shRNA) effectors for RNA interference (RNAi). To extend the use of RNAi for studies of development using the chicken as a model system, we have developed a system for expressing shRNAs using the chicken 7SK (ch7SK) promoter.

Results: We identified and characterised the ch7SK promoter sequence upstream of the full-length 7SK small nuclear RNA (snRNA) sequence in the chicken genome and used this to construct vectors to express shRNAs targeting enhanced green fluorescent protein (EGFP). We transfected chicken DF-1 cells with these constructs and found that anti-EGFP-shRNAs (shEGFP) expressed from the ch7SK promoter could induce efficient knockdown of EGFP expression. We further compared the efficiency of ch7SK-directed knockdown to that of chicken U6 (cU6) promoters and found that the efficiency of the ch7SK promoter was not greater than, but comparable to the efficiency of cU6 promoters.

Conclusion: In this study we have demonstrated that the ch7SK promoter can express shRNAs capable of mediating efficient RNAi in a chicken cell line. However, our finding that RNAi driven by the ch7SK promoter is not more efficient than cU6 promoters contrasts previous comparisons of mammalian U6 and 7SK promoters. Since the ch7SK promoter is the first non-mammalian vertebrate 7SK promoter to be characterised, this finding may be helpful in understanding the divergence of pol III promoter activities between mammalian and non-mammalian vertebrates. This aside, our results clearly indicate that the ch7SK promoter is an efficient alternative to U6-based shRNA expression systems for inducing efficient RNAi activity in chicken cells.

Show MeSH
Related in: MedlinePlus