Heritable and inducible RNAi knockdown system in Drosophila

Heritable and inducible RNAi knockdown system in Drosophila

Previously, more than 168 human glycan-related transferases (GRTs), including glycosyltransferases and sulfotransferases, have been cloned and their activities identified. Using these human GRT sequences, we performed a Basic Local Alignment Search Tool (BLAST) search of the Drosophila genome database and identified 91 putative GRTs (Nishihara S. 2008). RNAi knockdown of these GRTs can clarify the functions of glycans.

A schema of the heritable and inducible RNAi knockdown system is shown in Fig. 1 (Takemae H. et al. 2003, Nishihara S. et al. 2004, Nishihara S. 2008). We used 2 transgenic fly lines—the GAL4 driver and UAS-inverted repeat (UAS-IR). The GAL4 driver fly has a transgene that contains the yeast transcription factor GAL4; GAL4 expression is controlled by a tissue-specific promoter. The UAS-IR fly has a transgene containing an IR of the target gene that is ligated to the UAS, a target of GAL4. In the F1 generation of these flies, the double-stranded RNA (dsRNA) expression of the target gene is controlled by a tissue-specific promoter, resulting in gene silencing.

We have used this system for 4 glycosyltransferases—protein O-mannosyltransferase 1; protein O-mannosyltransferase 2 (Ichimiya T. et al. 2004); proteoglycan β-1,4-galactosyltransferase I (Takemae H. et al. 2003); and proteoglycan β-1,3-galactosyltransferase II (Ueyama M. et al. 2008)—and for two 3’-phosphoadenosine 5’-phosphosulfate (PAPS) transporters—sll/dPAPST1(Kamiyama et al. 2003) and dPAPST2 (Goda E. et al. 2006). The results clearly indicate that RNAi specifically reduces the expression of target genes in this system and that this reduction induces the development of abnormal phenotypes in flies.

Figure & Legends

Fig. 1. Schema of the heritable and inducible RNAi system

Two transgenic fly lines—GAL4 driver and UAS-IR—are used in this heritable and inducible RNAi system. The GAL4 driver fly has a transgene containing the yeast transcriptional factor GAL4; GAL4 expression is controlled by a tissue-specific promoter. The UAS-IR fly has a transgene containing an inverted repeat (IR) of the target gene that is ligated to the UAS, a target of GAL4. In the F1 generation of these flies, the dsRNA of the target gene is expressed in a tissue-specific manner and induces gene silencing.

This figure was obtained and modified from "Experimental Glycoscience -Glycobiology" edited by Taniguchi N. et al. Springer Japan KK. 2008, pp.285-289 (Nishihara S. Part2: Section XIV).

Fig. 2. pUAST-R57 vector construction and cloning strategy

pUAST-R57 is the modified pUAST vector used for cloning the IRs of the target gene sequence. A fragment comprising exons 5–7 of the fly Ret oncogene is placed between the insertion sites of the IRs. (a) PCR primer set and cloning strategy, (b) pUAST-R57 vector construction.

This figure was obtained and modified from National Institute of Genetics Fly Stock Center (website: http://www.shigen.nig.ac.jp/fly/nigfly/about/aboutRnai.jsp), Method "Vector construction and cloning strategies (Fig.1)". 

Fig. 3. Mating scheme for establishment of UAS-IR transgenic fly lines

This figure was obtained and modified from National Institute of Genetics Fly Stock Center (website: http://www.shigen.nig.ac.jp/fly/nigfly/about/aboutRnai.jsp), Method "Injection and line establishment (Fig. 2)".