isoform specific rnai

i’m working witha gene that has many isoforms and would like to do isoform specific rnai by generating double t7 clones to the various unique regions of each isoform. do people do this? i can’t seem to find a paper where this has been done. any experiences or a reference would be great.



I would imagine this might be problematic in C.elegans because of RNA-Directed-RNA-Polymerase (RDRP) activity. See the 2001 Sijen et al. paper, and see the more recent work profiled in this minireview by David Baucolme (PDF).

I don’t know about t7, however knockdown of isoforms is possible:

Murray P, Clegg RA, Rees HH, Fisher MJ.
siRNA-mediated knockdown of a splice variant of the PK-A catalytic subunit gene causes adult-onset paralysis in C. elegans.
Gene. 2008 Jan 31;408(1-2):157-63.

In the Sijen et al. 2001 paper, you will see that the “spreading” of RNAi effects to nearby regions is distance-dependent. The RDRP activity will extend the siRNA toward the 5’-end of your gene, but its processivity appears weak. If the exons you want to leave untouched are far enough away (i.e., there are one or more large intervening introns) , you may be able to get isoform-specific knockdown.

This is an interesting topic. Does anyone have any knowledge of RNAi on a transcript produced by an operon? Does the interference spread to include all the genes on the transcript?

See Bosher et al. Genetics. 1999 Nov;153(3):1245, for example.

RNAi of an upstream gene in the operon can induce the loss of function of the downstream gene. Such as RNAi lin-15b can induce ~95% Muv of eri-1(-) animals, which indicates the loss of functions of both lin-15b/lin-15a genes.