The TRiP generated a series of vectors referred to as the "VALIUM series" (Vermilion-AttB-Loxp-Intron-UAS-MCS) (Ni et al., 2008; Ni et al., 2009). Here we describe VALIUM20 and VALIUM22 - the second generation of TRiP vectors. These vectors were designed to deliver siRNAs using the endogenous microRNA pathway.
Our use of the microRNA cassette is based on the shRNA design of Haley et al. (2008, 2010) with modifications (Ni et al., 2010).
VALIUM20 contains vermillion as a selectable marker; an attB sequence to allow phiC31-targeted integration at genomic attP landing sites; two gypsy sequences to enhance hairpin DNA transcription; two pentamers of UAS, one of which can be excised using the Cre/loxP system to generate a 5XUAS derivative; the hsp70 basal promoter; a multiple cloning site (MCS) for cloning shmiRNAs in a miR1 scaffold, and a ftz 3'UTR intron followed by a SV40 3'UTR as a source for a polyA signal sequence.
Our data shows that VALIUM20 gives a stronger knockdown than VALIUM10 in the soma, and works well in the germline (Ni et al., 2010).
VALIUM22 contains vermillion as a selectable marker; an attB sequence to allow phiC31-targeted integration at genomic attP landing sites; two gypsy sequences to enhance hairpin DNA transcription; two pentamers of UAS, one of which can be excised using the Cre/loxP system to generate a 5XUAS derivative; the P-transposase core promoter; a multiple cloning site (MCS) for cloning shmiRNAs in a miR1 scaffold, and a ftz 3'UTR intron followed by a K10 polyA.
VALIUM21, a varient of VALIUM22, differs only in that it lacks the ftz intron and gypsy sequences found in VALIUM22. Only 95 TRiP stocks have been generated in the VALIUM21 vector.
VALIUM22 and VALIUM21 trigger effective RNAi knock-down and corresponding phenotypes in the female germline. The P-element transposase promoter is less effective than the hsp70 basal promoter to drive expression in somatic cells.
Hairpin Design using Short Hairpin microRNA
To design an shRNA for any given gene, the sequences for all exons or portions of exons common to all transcripts are obtained. These sequences are reverse-complemented and all possible 21-bp long subsequences are determined. Any subsequence with matches to other genes 16 bp-long or longer are removed from consideration. Each subsequence has a score calculated based on the formula given by Vert et al. (2006). The top scoring subsequences are selected. A top strand oligo is designed by concatenating "ctagcagt", the sense-strand oligo, "tagttatattcaagcata", the anti-sense oligo, and "gcg". A bottom strand oligo is designed by concatenating "aattcgc", the sense-strand oligo,"tatgcttgaatataacta", the anti-sense oligo, and "actg".
This process is automated by a Perl program developed internally at the TRiP. Primer design can also be done at the DSIR website (http://biodev.extra.cea.fr/DSIR/DSIR.html) using the 21nt siRNA option at default setting. The only difference is that the reverse complement has 2nt offset. We would suggest to only use the guide strand of DSIR output and then reverse complement it without the 2nt offset.
Selected References
Ni JQ, Zhou R, Czech B, Liu LP, Holderbaum L, Yang-Zhou D, Shim HS, Handler D, Karpowicz P, Binari R, Booker M, Brennecke J, Perkins L, Hannon GJ, Perrimon N. (2010) A genome-scale shRNA resource for transgenic RNAi in Drosophila. Nature Methods. Published online 3 April 2011; doi:10.1038/nmeth.1592.
Haley B, Foys B, Levine M. (2010) Vectors and parameters that enhance the efficacy of RNAi-mediated gene disruption in transgenic Drosophila. Proc Natl Acad Sci U S A. 2010 Jun 22;107(25):11435-40. Epub 2010 Jun 4.
Ni JQ, Liu LP, Binari R, Hardy R, Shim HS, Cavallaro A, Booker M, Pfeiffer B, Markstein M, Wang H, Villalta C, Laverty T, Perkins L, Perrimon N. (2009) A Drosophila Resource of Transgenic RNAi Lines for Neurogenetics. Genetics. 182(4):1089-100. Epub 2009 Jun 1.
Ni, J-Q., Markstein, M., Binari, R., Pfeiffer,
B., Liu, L-P., Villalta, C., Booker, M., Perkins, L. A., and
Perrimon, N. (2008) Vector and Parameters for Targeted
Transgenic RNAi in Drosophila melanogaster. Nature Methods 5,
49-51.
Haley B, Hendrix D, Trang V, Levine M. A simplified miRNA-based gene silencing method for Drosophila melanogaster. (2008)
Dev Biol. 15;321(2):482-90. Epub 2008 Jun 19.
