No abstract

Conditional expression of hairpin constructs in Drosophila is a powerful method to disrupt the activity of single genes with a spatial and temporal resolution that is impossible, or exceedingly difficult, using classical genetic methods. We previously described a method (Ni et al. 2008) whereby RNAi constructs are targeted into the genome by the phiC31-mediated integration approach using Vermilion-AttB-Loxp-Intron-UAS-MCS (VALIUM), a vector that contains vermilion as a selectable marker, an attB sequence to allow for phiC31-targeted integration at genomic attP landing sites, two pentamers of UAS, the hsp70 core promoter, a multiple cloning site, and two introns. As the level of gene activity knockdown associated with transgenic RNAi depends on the level of expression of the hairpin constructs, we generated a number of derivatives of our initial vector, called the ''VALIUM'' series, to improve the efficiency of the method. Here, we report the results from the systematic analysis of these derivatives and characterize VALIUM10 as the most optimal vector of this series. A critical feature of VALIUM10 is the presence of gypsy insulator sequences that boost dramatically the level of knockdown. We document the efficacy of VALIUM as a vector to analyze the phenotype of genes expressed in the nervous system and have generated a library of 2282 constructs targeting 2043 genes that will be particularly useful for studies of the nervous system as they target, in particular, transcription factors, ion channels, and transporters.

The conditional expression of hairpin constructs in Drosophila melanogaster has emerged in recent years as a method of choice in functional genomic studies. To date, upstream activating site-driven RNA interference constructs have been inserted into the genome randomly using Pelement-mediated transformation, which can result in false negatives due to variable expression. To avoid this problem, we have developed a transgenic RNA interference vector based on the phiC31 site-specific integration method.Transgenic RNA interference (RNAi) has emerged as an important method for analyzing gene function in D. melanogaster and has joined the already rich arsenal of tools available for functional genomic studies in this organism1. The method relies on the Gal4-upstream activating site (UAS) system2 to control the expression of a gene fragment that is dimerized to produce a double-stranded RNA (dsRNA) hairpin structure, which then triggers a sequence-specific post-transcriptional silencing and RNAi response. Tissue-or cell-specific expression of the transgenic RNAi constructs is achieved after a cross between UAS-hairpin and Gal4 driver lines. The main advantage of the method, in addition to its relatively simple design and fast execution time, is that it allows spatial and temporal control of the knockdown construct, which is essential for characterizing genes with pleiotropic functions.A problem with current methodology is the variability in the level of hairpin expression due to the random integration in the genome of the P-element-based UAS-hairpin constructs. For example, one recent report in which two random insertions per construct were tested showed that in 40% of cases the two behaved differently, with one insertion showing lethality and the other viability when tested with the ubiquitously expressed actin5C-Gal4 driver1. To avoid the high incidence of false negatives resulting from random integration, we decided to develop a vector for transgenic RNAi based on the phiC31 targeted integration method3. Targeting RNAi hairpin constructs to a specific region of the genome

The maternal Dorsal regulatory gradient initiates the differentiation of several tissues in the early Drosophila embryo. Whole-genome microarray assays identified as many as 40 new Dorsal target genes, which encode a broad spectrum of cell signaling proteins and transcription factors. Evidence is presented that a tissue-specific form of the NF-Y transcription complex is essential for the activation of gene expression in the mesoderm. Tissue-specific enhancers were identified for new Dorsal target genes, and bioinformatics methods identified conserved cis-regulatory elements for coordinately regulated genes that respond to similar thresholds of the Dorsal gradient. The new Dorsal target genes and enhancers represent one of the most extensive gene networks known for any developmental process.