Identification Of Drosophila Heart Candidate Gene CG3295 In Knockout Stocks

Our laboratory has cloned a heart related candidate gene named CG3295 using bioinformatics combined with molecular biology techniques.Bioinformatics analysis showed that:the fruit fly genes-CG3295 is located in the 57B9-57B9 segments of the second chromosomes, and has only one exon with a transcript, the full-length of cDNA is 1860bp and the length of open reading frame (ORF) is 1298bp and encode a total of 432 amino acids. Our laboratory has used these techniques to determine the function of CG3295 in heart development, such as: observation of phenotype in mutant heart tube, a whole mount in situ hybridization techniques, the analysis of heart rate in adult levels and embryonic antibody staining technique.In order to study the biological function of CG3295, we use the unique P transposon knockout technology in Drosophila and successfully obtain the knockout stocks of CG3295 and then got 20 knock-out stocks which is fatal to development by group unique P transposon.We identify the knockout stocks in three aspects, such as:First,we detect the knockout strains by western blot analysis and then use PCR technique to identify knockout strains,at last observe the phenotypes of knockout strains to detect the knockout strains.First, we establish the green fluorescent strain expressing in 20 whole-body of CG3295 knockout stocks, and then pick out the homozygous of the knockout stocks by the green fluorescent strain unexpressing and then extract protein from the homozygous Drosophila for western blot protein detection. We selected 5 stocks which CG3295 is completely knocked out according to experimental results, and the results show that the protein expression in 7,8,9,12,16 strains is not seen, indicating successful CG3295 gene knocked out. Then use PCR to identify the knockout stocks.First we design primers according to the sites of P-element located in CG3295, and then extract genome from the heterozygous Drosophila used as template in PCR, the product of CG3295 knockout strains would have two bands compared to wild-type Drosophila, this experiment further verify 7,8,9,12,16 knockout strains. Finally we establish green fluorescence expressing in whole body and heart strains of CG3295 knockout strains, and then observe the phenotype of homozygous CG3295,the results show that the pericardia cells are disordered and partly missing. Selecting CG3295 knockout strains may lay the foundation for the study of the role of the CG3295 gene in heart development.