I understand that shRNAs can be cloned in either specific commercially available vectors or even introduced through lentiviral mediated transduction process. However, if I have to opt the first option then I have a set of queries:
1.Is it necessary to use one of the commercially available vectors only? Is it not possible to clone the shRNA coding gene into any of the stable plasmids? If so what are the features the plasmid needs to possess in order to clone the shRNA into it? (Copy number, promoter, etc)
2.Why do most commercially available vectors possess pol III promoters instead of pol II promoters?
3.Suppose I clone my shRNA into the vector, will the promoter regions also not be transcribed along with? If so will the extra regions thus transcribed not interfere with the silencing mechanism?
shRNA cloning usually aims several goals - strong silencing effect, specifity of expression, large concentration in a target cell, low off-target. Thus, 1) you may use commercially avalable vectors with a proven activity. However if you need to reduce off-target you may use gene specific promoter (commercial or self-made). 2) Pol II and pol III recognize different structures of shRNA. The choice of promotor depends on your shRNA design. 3) shRNA transcript undergoes simple processing, that removes promoter elements. Most commerciall shRNAs contain specific sequences, that facilitate this process.
Lentivirus or other retroviruses viruses are used because they efficiently integrate into the cell's genome, thus expressing the shRNA permanently. Plasmids integrate at a much lower level and often disrupt the gene inserted, whereas viruses integrate with reproducibility using specific sequences like LTRs. There are no real stable plasmids, unless you are talking about episomal plasmids with replication origins.
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