Overview of methods used to study GUS and VUS in Drosophila. Multiple complementary techniques can be used to study the function of candidate genes and variants identified as a novel disease candidate in patients. (A) Loss-of-function (LOF) studies look at the consequences of complete loss or reduction of the fly ortholog's function. This can happen through (1) mutant alleles that limit/eliminate protein function, (2) T2A-GAL4 cassettes inserted into the first intron, causing only a short portion of the protein to be made that is likely LOF, or Kozak-GAL4 cassettes that replace the coding sequence of the gene and either knock down or knock out the gene, and (3) transgenic RNAi lines to knock down the endogenous transcripts. (B) Humanization studies replace the expression of the fly ortholog with a human ortholog. This can either be accomplished by (1) combining a fly ortholog LOF strategy with cDNA expression, or (2) using CRISPR to knock the human cDNA with the variant into the endogenous fly locus. (C) Overexpression assays express the human/fly cDNA ubiquitously or in a tissue of interest using a GAL4 line, without manipulating the endogenous fly ortholog of the human gene of interest. (D) When the variant of interest affects a conserved amino acid, the fly analogous variant approach can be utilized. This can be accomplished by (1) combining a fly ortholog LOF strategy with fly cDNA expression, (2) generating a knock-in allele by directly targeting the endogenous fly ortholog gene based on genome editing, or (3) overexpressing the fly cDNA ubiquitously or tissue-specifically using various GAL4 lines.
