RNA interference screening in Drosophila primary cells for genes involved in muscle assembly and maintenance
Department of Neurobiology
Medical Subject Headings
Animals; Animals, Genetically Modified; Base Sequence; Cells, Cultured; DNA Primers; Drosophila; Drosophila Proteins; Gene Expression Regulation, Developmental; *Genes, Insect; Humans; Muscle Development; Muscular Diseases; Phenotype; Plasma Membrane Calcium-Transporting ATPases; RNA Interference
Neuroscience and Neurobiology
To facilitate the genetic analysis of muscle assembly and maintenance, we have developed a method for efficient RNA interference (RNAi) in Drosophila primary cells using double-stranded RNAs (dsRNAs). First, using molecular markers, we confirm and extend the observation that myogenesis in primary cultures derived from Drosophila embryonic cells follows the same developmental course as that seen in vivo. Second, we apply this approach to analyze 28 Drosophila homologs of human muscle disease genes and find that 19 of them, when disrupted, lead to abnormal muscle phenotypes in primary culture. Third, from an RNAi screen of 1140 genes chosen at random, we identify 49 involved in late muscle differentiation. We validate our approach with the in vivo analyses of three genes. We find that Fermitin 1 and Fermitin 2, which are involved in integrin-containing adhesion structures, act in a partially redundant manner to maintain muscle integrity. In addition, we characterize CG2165, which encodes a plasma membrane Ca2+-ATPase, and show that it plays an important role in maintaining muscle integrity. Finally, we discuss how Drosophila primary cells can be manipulated to develop cell-based assays to model human diseases for RNAi and small-molecule screens.
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Citation: Development. 2008 Apr;135(8):1439-49. Link to article on publisher's site