Team:ColumbiaNYC/Improve
Future Considerations
Further avenues of exploration on this project include the following:
Gentamicin Assay
The next step to proving the therapeutic efficacy of the presented mechanism is to ensure that the bacteria are actually entering their target cells and delivering the shRNA payload to them. The way in which to do this is a gentamicin assay. A gentamicin assay allows us to see whether the bacteria have entered mammalian cells. Essentially how this is done is that we leave the cells exposed to the bacteria for a given amount of time, then apply gentamicin to the cells, killing off any external bacteria. Then we allow for incubation and infection and observe the progression of the invasion using microscopy over the course of 24 hours. This will indicate whether the bacteria have truly invaded the cells.
Mouse Models
After the gentamicin assay, another important step in this project will be mouse models. We need mouse models to test the efficacy of this system in vivo, assess toxicity adequately, and ensure that the bacteria can reach their target cancer sites specifically. A study of the delivery of our shRNA containing bacteria to tumors in live mice would be the final step in ensuring the therapetic relevance of this mechanism.
Lipofectamine
eGFP HeLa cells were grown in Dulbecco’s Modified Eagle Media until they had reached 80% confluence. They were then split, and aliquotted into 12 individual wells. These cells were then incubated overnight. In the mean time, bacteria containing the eGFP shRNA were induced to produce this shRNA, which was then isolated with an miRNA mini prep kit. After this, concentrations of shRNA were determined via nanodrop, and a gel was run to ensure that our desired shRNA were present. The shRNAs were then allowed to complex with lipofectamine 2000, producing complexes containing the desired shRNA. The HeLa cells were then exposed to these complexes. Three wells were not exposed to anything, another three were exposed to just liposomes, the next three were exposed to liposomes with control eGFP siRNA, and the final three wells were exposed to liposomes containing eGFP shRNA. The cells were allowed to incubate for 24 hours, after which they were viewed under a microscope and subjected to flow-cytometry.
Lipofectamine results: The results showed that eGFP siRNA knockdown of eGFP expression in HeLa cells was less than that of shRNA knockdown of eGFP but greater than the control indicating that the method of gene knockdown is in-fact a viable method.
