, 2004 and Bischof et al., 2007). These libraries are still being constructed ( Dietzl et al., 2007 and Ni et al., 2009). Another advantage of the ΦC31 system is that RNAi parameters can directly be compared to each
other and therefore be optimized ( Ni et al., 2008 and Ni et al., 2009). These studies also illustrated that short hairpin RNAs (shRNA) modeled on an endogenous microRNA are an effective alternative for classical dsRNA mediated RNAi in the generation of genome-wide RNAi libraries ( Ni et al., 2011). shRNA-mediated RNAi can be directed toward Tenofovir price alternative exons and allowed studying the function of alternative splice variants ( Shi et al., 2007 and Yu et al., 2009b). RNAi experiments can result in unwanted phenotypes due to off-target knockdown. RNAi rescue strategies provide a solution to this problem: one exploits genome-wide libraries of a related species, Drosophila pseudoobscura ( Kondo et al., 2009, Ejsmont et al., 2009 and Langer
et al., 2010), since genes and their regulatory regions of Drosophila pseudoobscura are similar enough to rescue genes of Drosophila melanogaster, but divergent enough to resist the RNAi machinery. Another strategy uses GAL4 to express a UAS rescue construct with altered codon usage that resists the RNAi degradation ( Schulz et al., 2009). In summary, advantages of RNAi experiments are that they can be performed in a tissue-specific fashion using the GAL4-UAS system. Disadvantages Selleck Vorinostat are that off-target effects are not uncommon and knockdowns are almost always incomplete. It is difficult to compare the efficiency of different screening strategies. An RNAi screen to identify novel players in the Notch pathway (Mummery-Widmer et al., 2009) did not identify any of the genes that have been isolated using Flp/FRT screens with EMS mutagenesis ( Jafar-Nejad et al.,
2005, Acar et al., 2008 and Tien et al., 2008) with one exception PRKACG ( Rajan et al., 2009). Homologous recombination or gene targeting can be used to generate modifications or mutations in specific genes in their normal chromosomal context. Gene targeting in Drosophila is performed using one of two methods: ends-in gene targeting and ends-out gene targeting ( Wesolowska and Rong, 2010). The result of ends-in gene targeting is a local duplication at the targeting site, due to the integration of the entire targeting vector ( Rong and Golic, 2000 and Rong and Golic, 2001). This duplication can be resolved during a second round of homologous recombination catalyzed by the meganuclease I-CreI ( Rong et al., 2002), resulting in precisely engineered alleles of several genes required in the nervous system that include point mutations, deletions, gene swaps, protein tags, GAL4 insertion, or splice form reduction ( Demir and Dickson, 2005, Stockinger et al., 2005, Brankatschk and Dickson, 2006, Hattori et al., 2007, Hattori et al., 2009 and Spitzweck et al., 2010).