Phenotypes resulting from RNAi knockdown

Both the human and the fly ADCK3 gene will be codon optimized by gene script so that the RNAi gene does not recognize them as a target. Each gene will be inserted into a pUASTattB plasmid which can then be microinjection into drosophila yellow eyed embryos where the C31-based integration system will insert the plasmid into one of the drosophilas chromosomes. Selecting for transformants will be done by observing eye color as the pUASTattB plasmid encodes a gene required for white eye pigmentation so when white eyes are seen it is due to a successful transformation.
 
The quantity of each product is then analyzed using qRT-PCR and the brains characterized to see if adding these genes has rescued the function of ADCK3 in the RNAi fly lineshow more contentTo check the level of non-specific binding of the anti-FLAG antibody the same procedure will be carried out on wild-type flies which do not contain the FLAG tag.
 
Determine if the mutant genes can rescue the phenotypes resulting from RNAi knockdown
 
Three mutants will be generated by site directed mutagenesis of WT CG32649 and tested to see the effect each has on ADCK3 function within the brain. Of these mutants one contains p.Leu271Pro another has c.1506 1G>A and the third is a double mutant containing both these mutations. Testing each of these mutations separately allows a better understanding of each ADCK3 mutation as it will show whether either one alone makes any difference or if only the combination of both mutations is enough to change ADCK3 expression or function.
 
 
To test these mutations transgenic fly lines containing the mutants must first be made and then recombined into the RNAi line so that the normal expression of ADCK3 in the flies is knocked down and the effect of the mutation can be seen. This experiment therefore is determined on the products of the previous experiments as it cannot be carried out without a successful RNAi knockdown fly line and will not work if the wildtype rescue experiment did not work. The mutants will be generated in the same manner as those used in the rescue experiment. They will be fully characterized using the same methods that were used to characterize a normal flies brain and the RNAi rescue experiments.
 
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