![]() ![]() In addition, a UAS-human-homologue cDNA of the GOI permits humanization of the flies and assessment of human variants ( Bellen and Yamamoto, 2015 Kanca et al., 2017 Şentürk and Bellen, 2018 Chao et al., 2017 Yoon et al., 2017). This provides a means for rigorous quality assessment of the genetic reagent and, when combined with mutant and/or truncated forms of the UAS-GOI cDNA, facilitates structure-function analysis. ![]() Alternatively, a UAS-GOI cDNA can be used to test for rescue of the loss of function phenotype induced by the insertion cassette. The resulting GAL4 can then be used to drive a UAS-nuclear localization signal (NLS)::mCherry to determine which cells express the gene or a UAS-membrane (CD8)::mCherry to outline the cell projections ( Brand and Perrimon, 1993 Shaner et al., 2004). This insert typically creates a severe loss of function allele and generates a GAL4 protein that is expressed in the target gene’s spatial and temporal expression pattern ( Diao et al., 2015 Gnerer et al., 2015 Lee et al., 2018a). The CRIMIC variety of SIC currently used by the GDP is an artificial exon consisting of attP-FRT-SA-T2A-GAL4-polyA-3XP3-EGFP-FRT-attP inserted in a coding intron (intron flanked by two coding exons) of the GOI ( Lee et al., 2018a). A SIC is typically flanked with attP sites and can be replaced using Recombinase Mediated Cassette Exchange (RMCE) ( Bateman et al., 2006 Venken et al., 2011). These elegant and precise manipulations are made possible by the integration of a Swappable Integration Cassette (SIC) in the gene of interest (GOI) using transposon mediated integration ( Minos-mediated Integration Cassette Venken et al., 2011 Nagarkar-Jaiswal et al., 2015a) or homologous recombination mediated by CRISPR, a technique we named CRIMIC ). This involves assessment of the loss of function phenotype, identification of the cells that express the gene, determination of the subcellular protein localization, selective removal of the transcript or protein in any tissue, the ability to perform immunoprecipitation of the protein and its interacting proteins or DNA, rescue of the induced fly mutant phenotypes with fly or human cDNAs and assessment of the consequences of amino acid variants in vivo. A main goal of the Drosophila Gene Disruption Project (GDP) is to create genetic tools that facilitate an integrated approach to analyze the function of each gene in detail. ![]()
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