Friday, March 18, 2016

Persistence of RNAi in mosaic studies

Another report regarding in vivo RNAi that fly folks using the technique should be aware of (see this post for another, and check out other posts tagged "in vivo RNAi" or "troubleshooting").

Bosch JA, Sumabat TM, Hariharan IK. Persistence of RNAi-Mediated Knockdown in Drosophila Complicates Mosaic Analysis yet Enables Highly Sensitive Lineage Tracing. Genetics. 2016 Mar 16. PMID: 26984059.

From the abstract: "... By expressing synthetic short hairpin RNAs (shRNAs) using the Gal4/UAS system, knockdown is efficiently achieved in specific tissues or in clones of marked cells. ... knockdown by shRNAs is so potent and persistent that even transient exposure of cells to shRNAs can reduce gene function in their descendants. When using the FLP-out Gal4 method, in some instances we observed unmarked "shadow RNAi" clones adjacent to Gal4-expressing clones, which may have resulted from brief Gal4 expression following recombination but prior to cell division. Similarly, Gal4 driver lines with dynamic expression patterns can generate shadow RNAi cells after their activity has ceased ... Importantly, these effects can lead to erroneous conclusions regarding the cell autonomy of knockdown phenotypes. We have investigated the basis of this phenomenon and suggested experimental designs for eliminating ambiguities in interpretation. We have also exploited the persistence of shRNA-mediated knockdown to design a sensitive lineage-tracing method, i-TRACE, which is capable of detecting even low levels of past reporter expression. ..."

No comments: