How many RNAi expression cassettes can be subcloned into a single vector?
We haven’t determined the absolute maximum number of expression cassettes that can be subcloned into a single vector. Typically a plasmid vector ranging from 2.0 kb to 4.0 kb in size can uptake an insert over 5 kb or more in length, which is about the size of 12 RNAi expression cassettes (~ 400 bp each). Notably, RNAi is a biological process and saturable in the presence of excessive amount of double-stranded RNAs. Normally 3 – 5 genes can be targeted simultaneously without significant reduction in the gene silencing capacity of each cassette. Therefore, it is the effectiveness of RNAi machinery in the cells, not the size of the plasmid, that becomes the limiting factor when you want to silence multiple gene targets from a single vector.
