From my gene of interest, Sox9, when cut into my recipient plasmid, pEZT-BM, this may include two WPRE regions and CMV promoter. This would take place if I were to cut with KpnI and NotI. As such, I have decided to use PCR cloning where I design primers either side of the GOI.
In my paper, I need to include research/evidence that shows why two WRPE downstream and two CMV promoters is a bad idea; however, I cannot seem to find anything in the research -- just my supervisor's recommendations.
Thanks heaps!
A single CMV promoter is pretty strong by itself and having two would be redundant or might provide for a marginal increase in transcription efficiency at best. Same goes for the WPRE regions as well, although having two does improve turnover.
Article Effect of posttranscriptional regulatory elements on transge...
As for an argument against having a copy: Viruses are some of the most efficient organisms on the planet. And these sequences evolved in viruses and they usually carry only a single copy. There are viruses with multiple promoters or bidirectional ones, but they are usually under different inducers/regulators. It is rare to find two of the same promoters for a single gene in nature (unless those promoters are separated by a large stretch of DNA and require a spatial criteria for their activation).
From what I understood, you have subcloned your gene from one plasmid to another. It is common practice to only PCR amplify your GOI and flank it with RE sites for your destination vector. You dont need to attest to that on paper. Ideally, it's best if you amplify the GOI from the source genome to avoid point mutations that may have taken place during cloning or maintenance in the bacteria.
- Badges
- Science topic