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Human RNAi U1 Promoter Works Effectively in Mouse NIH-3T3 Cells

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Abstract

We tested the GeneClip™ U1 Hairpin Cloning System (Cat.# C8790) for activity in a mouse NIH/3T3 cell line. Here we show that the human U1 promoter can achieve an inhibition of a stably expressed human Renilla luciferase gene (hRluc) in NIH/3T3 cells.

Tom Yeager

Promega Corporation
Publication Date: 2005

Introduction

The ability to target certain gene products for knockdown was a very difficult process until recently. The discovery of RNA Interference (RNAi) as a means to achieve reproducible knockdown was one of the most exciting breakthroughs in cell biology. RNA interference is a naturally occurring phenomenon in eukaryotic cells where a double-stranded RNA (dsRNA) can specifically target the cleavage and degradation of a homologous mRNA and reduce the expression of a specific target (1) . The production of a foldback, stem-loop structure or short hairpin RNA (shRNA) from a plasmid-based promoter can make a functional dsRNA that can activate the RNAi pathway (2) . A number of different promoters have been shown to have the correct termination for the insertion into the RNAi pathway including the U1 promoter (3) (4) .The purpose for this experiment was to determine if a human U1 promoter could produce a functional RNAi transcript in a mouse cell line. We have previously shown that this promoter is effective in human cell lines (HeLa, 293T, etc.) as well as a hamster cell line (CHO; (5) ). It would be very useful to have a promoter that would be effective in a variety of cell types.

Methods and Results

To test our system we have developed a NIH/3T3 cell line that stably expressed the humanized Renilla luciferase (hRluc) gene (phRL-SV40 vector). This cell line was cotransfected with a plasmid that contained the neomycin resistance gene and was selected for G418 resistance as well as high-level, stable hRluc expression. For the assay, 3,000 NIH/3T3 cells/well were plated in a 96-well tissue culture plate, white walled with a clear bottom. The following day cells were transfected with 0.1μg/well DNA and either 0.9μl/well or 1.2μl/well Mirus LT1 transfection reagent. The DNA used was the GeneClip™ U1 GFP vector containing either a sequence against hRluc or a nonspecific sequence. Each transfection was performed in eight replicate wells. After 48 hours, cells were visualized for GFP transfection efficiency, which was determined to be 60%. The cells were then assayed for hRluc expression using the ViviRen™ Live Cell Substrate (Cat.# E6492). To correct for live-cell number per well, the plate was assayed with the CellTiter-Glo® Luminescent Cell Viability Assay (Cat.# G7570). The hRluc expression was divided by the CellTiter-Glo® number to give hRluc to cell ratio. The specific target sequence ratio was then divided by the nonspecific ratio to determine inhibition. This inhibition was then corrected for the transfection efficiency as seen with the GFP levels to give a final inhibition in the transfected cells. The results of the experiment are shown in Figure 1. For the GeneClip™ U1 promoter, a Renilla luciferase inhibition of 60% was detected using 0.9μl/well transfection reagent, but this number fell to 51% with the higher level of transfection reagent. These results show that the U1 promoter can be used to direct RNAi in NIH/3T3 cells, but experiments will require optimization for best results.

Inhibition of <i>Renilla</i> luciferase expression in NIH/3T3 cells using a U1 promoter. Figure 1. Inhibition of Renilla luciferase expression in NIH/3T3 cells using a U1 promoter.

Using the GeneClip™ U1 Hairpin Cloning System, an shRNA sequence was cloned into the vectors against either hRluc or a nonspecific sequence. These plasmids were transfected into NIH/3T3 cells using 0.1μg/well DNA and either 0.9μl/well or 1.2μl/well Mirus LT1 transfection reagent. Forty-eight hours after transfection, the cells were assayed for GFP expression for transfection efficiency, Renilla luciferase expression, and assayed with the CellTiter-Glo® Luminescent Cell Viability Assay for cell number. Inhibition levels shown were corrected for transfection efficiency. The inhibition level was calculated by comparing luciferase activity in cells transfected with the shRNA specific for hRluc and in cells transfected with the shRNA with the nonspecific sequence after normalizing to cell number in each well.

Conclusions

These data show that human U1 promoter can function to make shRNAs in the mouse NIH/3T3 cell line to inhibit a stably expressed gene of interest. These findings support earlier data that showed this promoter could function in human (HeLa, 293T, etc.) as well as hamster (CHO) cell lines (5) .

References

  1. Fire, A. et al. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–11.
  2. Sui, G. et al. (2002) A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc. Natl. Acad. Sci. USA 99, 5515–20.
  3. Denti, M.A. et al. (2004) A new vector, based on the PolII promoter of the U1 snRNA gene, for the expression of siRNAs in mammalian cells. Mol. Ther. 10, 191–9.
  4. Paddison, P.J. et al. (2002) Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev. 16, 948–58.
  5. Yeager, T. et al. (2005) GeneClip™ U1 Hairpin Cloning Systems for expression of short hairpin RNAs in vivo Promega Notes 89, 21–4.

How to Cite This Article

Yeager, T. Human RNAi Promoters Work Effectively in Mouse NIH-3T3 Cells. [Internet] 2005. [cited: year, month, date]. Available from: http://www.promega.com.br/resources/pubhub/enotes/human-rnai-promoters-work-effectively-in-mouse-nih3t3-cells/

Yeager, T. Human RNAi Promoters Work Effectively in Mouse NIH-3T3 Cells. Promega Corporation Web site. http://www.promega.com.br/resources/pubhub/enotes/human-rnai-promoters-work-effectively-in-mouse-nih3t3-cells/ Updated 2005. Accessed Month Day, Year.

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Figures

Inhibition of <i>Renilla</i> luciferase expression in NIH/3T3 cells using a U1 promoter. Figure 1. Inhibition of Renilla luciferase expression in NIH/3T3 cells using a U1 promoter.

Using the GeneClip™ U1 Hairpin Cloning System, an shRNA sequence was cloned into the vectors against either hRluc or a nonspecific sequence. These plasmids were transfected into NIH/3T3 cells using 0.1μg/well DNA and either 0.9μl/well or 1.2μl/well Mirus LT1 transfection reagent. Forty-eight hours after transfection, the cells were assayed for GFP expression for transfection efficiency, Renilla luciferase expression, and assayed with the CellTiter-Glo® Luminescent Cell Viability Assay for cell number. Inhibition levels shown were corrected for transfection efficiency. The inhibition level was calculated by comparing luciferase activity in cells transfected with the shRNA specific for hRluc and in cells transfected with the shRNA with the nonspecific sequence after normalizing to cell number in each well.

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