Primers for the TRiP hairpins are designed using the DRSC's amplicon design tool "Snapdragon". The PCR product from the hairpin is designed to be 400 - 600bp long. Portions of gene transcript sequences are used that are common to all splice-forms of the gene of interest. Ideally regions that are free of 19bp matches to other genes are used. If there are no such regions of sufficient length, then regions with less than five 21bp matches to other genes are used. Primer3 (Rozen and Skaletsky, 2000) is used to cull the primers once an appropriate sequence is chosen. Any primer pairs whose reverse primer has the sequence CCAC at the 5' end are rejected. An extension of CACC is added to the 5' end of the forward primer.

The targeted method to generate hairpin lines has many practical advantages over P-element based methods: 1. the frequency of recovering transformants using the integrase method, either following co-injection of integrase mRNA or by injecting into the nanos-integrase strain, is almost five-fold higher than in conventional P-element transformation; 2. establishment of the lines is greatly facilitated as no mapping of the transformants to a specific chromosome is needed; and 3. unlike P-element-based methods, insertions into the attP landing site are homozygous viable.

Further, since the efficacy of the transgenic RNAi technology depends upon the level of expression of the UAS-driven constructs, the VALIUM constructs with their modular number of UAS copies allow the generation of a phenotypic series. From the original 10XUAS construct, a 5XUAS derivative can be recovered, and because the attP-containing chromosome can be homozygosed, it is also possible to generate both 15XUAS and 20XUAS combinations (Ni et al., 2008). The ability to generate a phenotypic series from 5XUAS to 20XUAS may prove useful, in particular, when variation of the gene expression dosage is important for phenotypic studies of pleiotropic genes. To generate different levels of RNAi knockdown, the features described above can be used together with other means to vary expression (different Gal4 lines of different strength, temperature) or processing (with coexpression of UAS-Dcr2, Dietzl et al., 2007) of the hairpin construct.

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.

Markstein, M., Pitsouli, C., Villalta, C., Celniker, S. and Perrimon, N. (2008) Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes. Nature Genetics. 135, 1439-1449.

Dietzl, G. et al. (2007) A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature 448, 151-156.

Groth, A.C., Fish, M., Nusse, R. & Calos, M.P. (2004) Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31. Genetics 166, 1775-1782.

Rozen, S. & Skaletsky, H. (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132, 365-86.