2 Department of Infectious Diseases, Microbiology and Parasitology, Faculty of Veterinary Medicine, Grochowska 272, 03-849 Warsaw, Poland
3 || Present address: Veterinary Consultancy, Conrada 01-922 Warsaw, Poland
RNA-interference (Fire et al. 1998) is a popular ‘reverse-genetics’ screening strategy. In particular ingested variant of RNAi (Timmons et al. 1999) gained popularity for genome-wide screens, where bacterially expressed dsRNA is administrated per os utilizing modified plasmids and E. coli as a feeding vector (Timmons et al. 2001). Genome-wide RNAi screens are presently carried using RNAi feeding libraries.
Two types of genome-wide RNAi ‘feeding libraries’ entailing PCR-amplified target regions are presently available: library of predicted gene-overlapping segments (Kamath et al. 2003) and amplified cDNA library (Rual et al. 2004). However, available genome-wide PCR-based recombinant RNAi libraries – resources consisting of dsRNA producing plasmids – depend heavily on gene predictions, hence the bias toward certain exon rich gene regions or certain cDNAs. Here, I report on a complementary resource facilitating an approach to RNAi screen relying on unbiased ‘forward-genetics’ strategy.
The experiments based on this approach started with the construction of the library of genomic segments incorporated into convergent T7 polymerase binding sites plasmid (PBS) vector (L4440 plasmid
Based on the above experiment, I think the forward RNA interference screening is useful and feasible, with strong expectation the presented screening mode will complement and extend on the existing, currently available, genome-wide RNAi resources.
*Other variants of the experiment explored elsewhere, involve the fragmented DNA ligated with other enzymes and/or linkers.
Kamath RS, Fraser AG, Dong Y, Poulin G, Durbin R, Gotta M, Kanapin A, Le Bot N, Moreno S, Sohrmann M, Welchman DP, Zipperlen P, Ahringer J. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature. 2003 Jan 16;421(6920):231-7.
Kapulkin WJ, Hiester BG, Link CD. Compensatory regulation among ER chaperones in C. elegans. FEBS Lett. 2005 Jun 6;579(14):3063-8. Erratum in: FEBS Lett. 2007 Dec 22;581(30):5952. Kapulkin, Vadim [corrected to Kapulkin, Wadim J].
Rual JF, Ceron J, Koreth J, Hao T, Nicot AS, Hirozane-Kishikawa T, Vandenhaute J, Orkin SH, Hill DE, van den Heuvel S, Vidal M. Toward improving Caenorhabditis elegans phenome mapping with an ORFeome-based RNAi library. Genome Res. 2004 Oct;14(10B):2162-8.