User:Jonathan Dewerd/Notebook/TeamAllergy/2010/06/16

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Gene Silencing Vector

  1. Wesley SV, Helliwell CA, Smith NA, Wang MB, Rouse DT, Liu Q, Gooding PS, Singh SP, Abbott D, Stoutjesdijk PA, Robinson SP, Gleave AP, Green AG, and Waterhouse PM. . pmid:11576441. PubMed HubMed [Wesley-TPJ-2001]

Deals with the relative efficacy of various post-translational gene silencing (PTGS) techniques. Summary:

  • co-suppression (sRNA) Simply over-express a certain RNA and the cell's regulatory mechanisms will reduce the expression. 30% effective at suppressing GUS.
  • Adj-hp RNA (Adjacent to HairPin RNA): place the target RNA sequence next to a random hairpin and attach a single strand of the target RNA to one of the tails of the hairpin. 25% effective at suppressing GUS
  • hpRNA (HairPin RNA): let one of the hairpin tails be the target sequence. 55% effective.
  • ihpRNA (Intron on HairPin RNA): put part of an intron in the hairpin loop. 90% effective. Overhang makes it slightly less effective.

GUS is a standard reporting gene. When activated, things turn blue.

PCR

The primers arrived today so we were able to PCR the cDNA we made yesterday. We used the Fraa1_{Antisense,Sense}_{FR} promoters. "Sense" and "Antisense" refer to the final product: Sense_F and Sense_R go together. We used the protocol on the protocol page with the Phusion Polymerase. We also poured the gel which we'll run tomorrow.


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