Part:BBa_K4917011
shRNA against DWV virus ver10
Starting position for shRNA ver10 in DWV is 2104
Usage and Biology
| Name | Backbone/Plasmids used for GG assembly | Content | Description |
| p207 | pRS304 | pGAL1_EGFP*siRNAv10_tCYC1 | Plasmid containing GFP sensor fused to siRNA target |
| p222 | Golden Gate assembly | shRNA_v10 | shRNA expression vectors |
| p224 | Golden Gate assembly | pPGK1-AGO1-tPGK1 + pTEF1-DCR1-tPGK1 | Plasmid containing Ago1 and Dcr1 transcriptional units |
Yeast strains used in the study
| Strain name | Genotype | Description |
| DOM90 | w303 MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG}[phi+] |
Background strain |
| I63 | DOM90 Leu2::Ago1+Dcr1 | Strain expressing Ago1 and Dcr1. It was used to transform with vectors expressing shRNA and target sequences |
| I73 | DOM90 Leu2::Ago1+Dcr1 Trp1::GFP-Target_v10 | Strain expressing Ago1, Dcr1, and GFP_V10 target |
| I83 | DOM90 Leu2::Ago1+Dcr1 Trp1::GFP-Target_v10 URA3::shRNA V10 | Strain expressing of shRNA_V10 and its GFP_V10 target |
TEST the Effect of siRNA on GFP Expression
To assess the efficiency of the siRNA we designed a sensor that consisted of GFP fused with the viral target sequence for the siRNA. If the siRNA is active and efficient, the mRNA will be degraded leading to no or decreased GFP fluorescence signal compared to cells without siRNA treatment.
Flow cytometry reveals suppression of EGFP expression by siRNA induction in yeast
Flow cytometry offers a means for efficient and precise evaluation of GFP expression at the individual cell level. In our experimental setup, we cultivated genetically modified yeast strains under tightly regulated environmental conditions. Subsequently, upon initiation of siRNA and GFP production, we subjected each yeast cell culture to flow cytometry. This method enables accurate measurement of any changes in GFP signal. A reduction in GFP fluorescence signifies the efficacy of the siRNA. We used flow cytometry to measure the GFP fluorescence intensities in yeast cultures 24h after inducing the expression of shRNA and the GFP-target sequence reporter. In the absence of shRNA expression, the GFP-reporter-containing cultures showed at least 3 times higher GFP fluorescence signal, confirming sufficient expression of the reporter protein (Fig. 1A). Additionally, we observed variations in fluorescence intensities among different GFP reporter constructs, suggesting that the viral sequence introduced into the 3'-UTR of the transcript may influence mRNA stability. Reduced mRNA stability, in turn, leads to impaired translation and decreased GFP fluorescence. For this reason, to compare the impact of the shRNA on GFP reporter expression, we normalized the GFP fluorescence data for each strain to the data obtained for its parent strain without shRNA expression (Fig. 1B). Interestingly, the anti-DWV shRNA V10 caused a drop in the GFP reporter fluorescence signal to background level (Fig. 1B).
Learn
These experiments lead to three important conclusions. Firstly, the presented work confirms the previously published results, demonstrating that introducing Dicer and Argonaute proteins to S. cerevisiae is sufficient to reconstitute RNAi response. Secondly, the experiments show that when viral sequences are introduced into the 3’-UTR of a GFP-coding transcript, they can be targeted by shRNA in yeast, offering a straightforward means to assess the efficacy of various shRNAs. Thirdly, we observed complete suppression on GFP-reporter expression with anti-DWV shRNA v10, making this strong candidate to move further into studies in bees to investigate possible off-target effects. We have also observed that the presence of the viral target sequence can influence GFP reporter expression, even in the absence of shRNA. As so far all the tested reporters still showed sufficiently high GFP expression to allow measuring of RNAi, further optimization of the length of the viral sequence introduced and the exact positioning of that sequence should be investigated. In the current experimental setup, both shRNA and the GFP reporter are expressed from pGAL1 promoter, as was done in Drinnenberg et al. 2009. However, it would be better to use different inducible promoters for shRNA and GFP to enable the flexibility of expressing either one within the same strain. The current single-promoter design necessitates the use of different strains to capture the reporter fluorescence signal in the absence and presence of shRNA. Utilizing distinct promoters would enable the use of a single strain in which reporter expression can be independently induced from shRNA expression, simplifying the process of measuring uninterrupted reporter expression and streamlining the methodology. ?While the first round of experiments confirmed efficient RNAi by the tested shRNA v10, we were not able to detect quantitative differences in shRNA efficiencies, This limitation arises because all shRNAs led to maximal suppression of the GFP reporter(Fig. 8B, fluorescence dropping to GFP negative background level).? In these strains shRNA v10 is expressed from a high copy number 2μ-plasmid, ensuring extremely high expression levels. In future experiments, we could consider expressing shRNA from a CEN plasmid, which exists in lower numbers within yeast cells, thereby resulting in reduced shRNA expression. Such an adjustment could potentially enhance the method's sensitivity to quantitative differences in shRNA efficiency.
References:
Drinnenberg, I. A., Weinberg, D. E., Xie, K. T., Mower, J. P., Wolfe, K. H., Fink, G. R., & Bartel, D. P. (2009). RNAi in Budding Yeast. Science, 326(5952), 544–550. <a href="https://doi.org/10.1126/science.1176945">https://doi.org/10.1126/science.1176945</a>
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1
Illegal BsaI.rc site found at 66
| None |