The Transgenic RNAi Project, Harvard Medical School, is producing a second-generation collection of TRiP lines that target gene expression during oogenesis. Two new vectors, pVALIUM20 and pVALIUM22, have been designed to deliver small interfering RNAs using the endogenous microRNA pathway. Short hairpin RNA transgenes are created by inserting synthetic oligonucleotides into pVALIUM20 or pVALIUM22. VALIUM20 transgenes give excellent knockdown in the soma and work well in the germline. VALIUM22 transgenes give excellent knockdown in the germline and have little or no effect in the soma.
VALIUM20 contains vermillion (v+t1.8) 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. Short hairpin RNAs in VALIUM20 give stronger knockdown than VALIUM10 long hairpin RNAs in the soma and they work well in the germline.
VALIUM22 contains vermillion (v+t1.8) 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. VALIUM22's P-element transposase promoter works well in the female germline and directs little or no 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".
shRNA constructs in VALIUM20 or VALIUM22 are inserted into flies at one of the genomic phiC31 landing sites P{CaryP}attP2 (chromosome 3) or P{CaryP}attP40 (chromosome 2) in a y1 sc* v1 background. TRiP line numbers with the format HMS##### (HMS for Harvard Medical Short hairpin) carry VALIUM20 shRNA constructs. TRiP line numbers with the format GL##### (GL for germline) carry VALIUM22 shRNA constructs. TRiP lines are contributed to the Bloomington Drosophila Stock Center where they are available for distribution.