Targeted mutagenesis of the <it>Sap47 </it>gene of <it>Drosophila</it>: Flies lacking the synapse associated protein of 47 kDa are viable and fertile

• Main Authors: Huber Saskia, Becker Sonja, Funk Natalja, Brunner Marion, Buchner Erich
• Format: Article
• Language: English
• Published: BMC 2004-04-01
• Series: BMC Neuroscience
• Online Access: http://www.biomedcentral.com/1471-2202/5/16

<p>Abstract</p> <p>Background</p> <p>Conserved proteins preferentially expressed in synaptic terminals of the nervous system are likely to play a significant role in brain function. We have previously identified and molecularly characterized the <it>Sap47 </it>gene which codes for a novel synapse associated protein of 47 kDa in <it>Drosophila</it>. Sequence comparison identifies homologous proteins in numerous species including <it>C. elegans</it>, fish, mouse and human. First hints as to the function of this novel protein family can be obtained by generating mutants for the <it>Sap47 </it>gene in <it>Drosophila</it>.</p> <p>Results</p> <p>Attempts to eliminate the <it>Sap47 </it>gene through targeted mutagenesis by homologous recombination were unsuccessful. However, several mutants were generated by transposon remobilization after an appropriate insertion line had become available from the <it>Drosophila </it>P-element screen of the Bellen/Hoskins/Rubin/Spradling labs. Characterization of various deletions in the <it>Sap47 </it>gene due to imprecise excision of the P-element identified three null mutants and three hypomorphic mutants. Null mutants are viable and fertile and show no gross structural or obvious behavioural deficits. For cell-specific over-expression and "rescue" of the knock-out flies a transgenic line was generated which expresses the most abundant transcript under the control of the yeast enhancer UAS. In addition, knock-down of the <it>Sap47 </it>gene was achieved by generating 31 transgenic lines expressing <it>Sap47 </it>RNAi constructs, again under UAS control. When driven by a ubiquitously expressed yeast transcription factor (GAL4), <it>Sap47 </it>gene suppression in several of these lines is highly efficient resulting in residual SAP47 protein concentrations in heads as low as 6% of wild type levels.</p> <p>Conclusion</p> <p>The conserved synaptic protein SAP47 of <it>Drosophila </it>is not essential for basic synaptic function. The Sap47 gene region may be refractory to targeted mutagenesis by homologous recombination. RNAi using a construct linking genomic DNA to anti-sense cDNA in our hands is not more effective than using a cDNA-anti-sense cDNA construct. The tools developed in this study will now allow a detailed analysis of the molecular, cellular and systemic function of the SAP47 protein in <it>Drosophila</it>.</p>