Generation of transgenic drosophila expressing shRNAs in the miR-1 backbone

Abstract

In Drosophila, long-term effects of RNA interference (RNAi) must be achieved by integrating into the genomea template fromwhich anRNAi trigger is transcribed by cellularRNApolymerases, generallyRNA polymerase II or III. With encoded triggers, not only can essentially permanent silencing be achieved, but control can also be exerted over the level of trigger expression, with a resulting variation in the degree to which the target is silenced. Knockdown can also be controlled in a temporal and cell-type-dependent fashionthroughtheuseofwell-establishedtransgenicmethodologiesandwell-testedpromoters.The forms of encoded triggers vary. Long double-stranded RNAs can be expressed as extended inverted repeats. The nearest equivalent of a small interfering RNA is an artificial microRNA (miRNA) or short hairpin RNA (shRNA),whereanaturalmiRNAbackbone(alsocalledascaffold) isremodeledtoproduceadifferent small RNA or a small inverted repeat (<30 nucleotides) is simply expressed. This protocol describes creation of transgenic Drosophila carrying shRNAinserts in a remodeled endogenousmiRNAbackbone.The protocol applies to the use of miRNA-based shRNAs, butmost of the vectors, principles of experimental design, and methods are also applicable to long inverted repeat transgenes