Results

Assessment of shRNA knockdown

shRNA Design

We chose an shRNA sequenced published on Addgene to knockdown our eGFP, and we compared the shRNA sequence to the eGFP DNA sequence to make sure the knockdown will theoretically occur. To construct the plasmid, we assembled the shRNA-encoding DNA from two segments that made up the sequence because the natural hairpin structure in the shRNA made the full sequence difficult to synthesize in one piece. Because our project design relies on the shRNA to inhibit gene expression (ultimately oncogene expression) and shRNA is not extremely stable (but not unstable), the bacteria must produce large quantities of shRNA for this gene therapy approach to be effective. Thus, we chose to put the gene in a pUC plasmid, which is a very high-copy plasmid (500-600 copies per cell). To increase the production of shRNA further, we placed the shRNA production under the control of a T7 promoter, which causes very strong gene expression compared to other promoters. Our final shRNA plasmid design is the following:

Figure 1: The SilenshR plasmid. This is an IPTG-inducible circuit with a T7 promoter that produces an shRNA that knocks down commercialized eGFP.

Transformation of Plasmid into BL21 (DE3) E.coli

We transformed this plasmid into BL21 (DE3) E. coli cells and got three colonies. To confirm that cloning was successful, we first performed colony PCR to determine if the insert was successfully cloned into the backbone. As can be seen, the colony PCR results show that the insert was successfully cloned in each of the three colonies:

Figure 2: Lane 1 is the Quick-Load® 2-Log DNA Ladder(0.1-10.0 kb) from NEB, lane 2 is the negative control, and lane 3 through 5 are from the three transformed colonies. The bands in lanes 3 through 5 are about 500bp, which is what we expect, and they are different in size from the negative control. Thus, this shows that cloning was successful.

Sequencing our Results

We then performed Sanger sequencing on each colony to ensure that the shRNA sequence in the plasmid is correct, and analysis of the results showed that it is correct.

Figure 3: Sanger sequencing results for the insert for the 2 colonies

GFP Expression Results

We then produced the shRNA in vitro using the New England Biolabs T7 expression kit to use in the lipofectamine assay. After the in vitro shRNA is successful, we reran the lipofectamine assay with shRNA that was produced in vivo using IPTG induction. The shRNA produced in vivo was isolated using the miRNeasy kit. We extracted a significant quantity of shRNA using both in vitro expression and in vivo expression. In in vitro expression, the concentration of shRNA was about 2230 ng/uL. In in vivo expression, concentrations ranged from 850 ng/uL to 1070 ng/uL. The lipofectamine assay would test whether the shRNA successfully knocks down the eGFP in mammalian cells. In this assay, the shRNA would be introduced into liposomes, and then the liposomes would be introduced into the mammalian cell line. The liposomes would fuse with the cell membrane of the mammalian cells and then the shRNA would enter the cells. Upon entry, the shRNA would knock down the expression of eGFP. The effectiveness of the knockdown of eGFP from the shRNA is measured using a flow cytometer. The flow cytometer measures the GFP florescence in every cell and gives a statistical distribution of the florescence. The results from the synthesized shRNA is compared to a negative control, where none of the shRNA is introduced to the mammalian cells during the lipofectamine assay. The results will also be compared to a positive control, where we used siRNA designed and proven to knock down eGFP from Thermo Fisher. We analyzed the decrease in florescence to determine the effectiveness of the shRNA-mediated knockdown of GFP. (For shRNA synthesized to knockdown oncogenes, we plan to use RT-qPCR to detect knockdown of the target gene. For the RT-qPCR, we will use the reverse transcriptase to transcribe the mRNA produced by the bacteria into cDNA. Then in qPCR, we can determine the amount of mRNA remaining in the mammalian cells from the rate at which qPCR proceeds.)

Figure 4: The x-axis shows the negative control (PBS), the positive controls (siRNA), and the experimental shRNA designed to inhibit eGFP (shRNA). The y-axis shows the percentage of mammalian cells that were fluorescing eGFP (defined as eGFP positive). The error bars are 2 standard error, or a 95% confidence interval. As can be seen, the confidence intervals for the negative control and the experimental shRNA do not overlap, which shows that this difference is statistically significant. This indicates that our shRNA successfully knocks down eGFP expression in HeLa cells. Hard data used to generate this graph can be found here.

These results show that our proof-of-concept works successfully, where shRNA successfully knocks down expression of the targeted gene.