Efficient and heritable functional knock-out of an adult phenotype in Drosophila using a GAL4-driven hairpin RNA incorporating a heterologous spacer

Abstract

We have developed a modified RNA interference (RNAi) method for generating gene knock-outs in Drosophila melanogaster. We used the sequence of the yellow (y) locus to construct an inverted repeat that will form a double-stranded hairpin structure (y-IR) that is under the control of the upstream activating sequence (UAS) of the yeast transcriptional activator GAL4. Hairpins are extremely difficult to manipulate in Escherichia coli, so our method makes use of a heterologous 330 bp spacer encoding sequences from green fluorescent protein to facilitate the cloning steps. When the UAS-y-IR hairpin is expressed under the control of different promoter-GAL4 fusions, a high frequency of y pigment phenocopies is obtained in adults. Consequently this method for producing gene knock-outs has several advantages over previous methods in that it is applicable to any gene within the fly genome, greatly facilitates cloning of the hairpin, can be used if required with GAL4 drivers to avoid lethality or to induce RNAi in a specific developmental stage and/or tissue, is useful for generating knock-outs of adult phenotypes as reported here and, finally, the system can be manipulated to investigate the trans-acting factors that are involved in the RNAi mechanism.

Post-transcriptional Silencing and Functional Characterization of theDrosophila melanogasterHomolog of HumanSurf1

“…This tool has previously been proven to induce a very efficient and highly specific attenuation in the expression of a target gene (Piccin et al 2001). The GAL4-UAS binary system (Brand and Perrimon 1993) was exploited to drive the knockdown of the target gene transcript under the control of two alternative promoters, namely those of Actin5C, an early-expressed housekeeping gene, or of elav, an early expressed neuron-specific gene.…”

“…Plasmid construction: The procedure adopted is as in Piccin et al (2001). In brief, an 858-bp fragment from the coding sequence of the Drosophila Surf1 gene (CG9943, Surf1) was amplified using the pair of primers 59-ATACA GACTGTATTCCGAAAC-39 and 59-GTCAAGAGGATACCCTT CTGA-39 and cloned in the T vector pDK101 to yield pDKSurf1.…”

“…Chromosomes harboring the UAS-Surf1-IR insert were balanced and later made homozygous. In situ hybridization was used to map the position and detect the number of inserts in each line as described in Piccin et al (2001).…”

On the origin and functions of RNA-mediated silencing: from protists to man

“…Indeed, Dcl1 only includes the RNaseIII motifs and a C-terminal DSRM. In the incomplete genome of Phytophthora sojae the only recognizable Dicer-like sequence consists of a poorly conserved helicase domain, the DUF283 motif, and the two RNaseIII Lee et al (1993) and Wienholds and Plasterk (2005) Anopheles gambiae Hoa et al (2003) and Keene et al (2004) Drosophila melanogaster Aravin et al (2003) and Lagos-Quintana et al (2001) Kennerdell and Carthew (1998) and Piccin et al (2001) Direct b Pal-Bhadra et al (2004) and Kavi et al (2005) Gesellchen and Boutros (2004) and Nakahara et al…”

Illuminating the role of caspases during Drosophila oogenesis

“…The inclusion of an intron in the genomic piece allowed for easier cloning with traditional molecular biology techniques, but it was assumed to be processed out in the final mRNA transcript. 38 Creation of transgenic flies Drosophila w 1118 embryos were collected 20 min after egg laying, dechorionated by rolling on double-stick tape, and microinjected under mineral oil as described in Spradling 39 with 0.65 mg/ml of UASp-CIETDY plasmid or UASp-dronc RNAi plasmid mixed with 0.4 mg/ml transposasecontaining plasmid RIO pActNPII. 40 w þ transformants were screened by eye color and crossed to w 1118 ;Cyo/T(2;3)ap Xa to balance and map.…”