Strategy to “humanize” a Drosophila gene to assess functional consequences of a novel variant. (A) For most genes, the Drosophila Core of the MOSC performs functional studies of a patient variant by humanizing the orthologous gene in the fly. First, a fly gene that is most likely to be the ortholog of the human gene is identified using the MARRVEL tool. MARRVEL also provides a link to the FlyBase page that displays known biological function, transcriptomics and proteomics data, mutant phenotypes and available resources for the Drosophila gene of interest. If a coding intronic MiMIC is available, the Drosophila Core uses this as an entry point to study the gene. Through recombinase-mediated cassette exchange (RMCE), an artificial exon is integrated that functions as a gene trap, creating a strong LOF allele. This artificial exon contains a T2A ribosomal skipping sequence and a coding sequence for the GAL4 transcriptional activator (T2A-GAL4). (B) By crossing the T2A-GAL4 strain to a transgenic fly that carries a UAS-human cDNA construct (together with a deficiency of the locus or an independent mutant allele of the fly gene, data not shown), the fly gene can be humanized. When the gene of interest is transcribed, the splice acceptor (SA) in the artificial exon splices into the upstream exon. Since a transcription termination sequence (polyA) is present at the 3′ end of this artificial exon, the transcript is terminated, and the remaining portion of the fly gene is not transcribed. When this transcript is translated, a truncated protein that is usually nonfunctional is made together with a GAL4 protein. GAL4 is expressed in the same spatial and temporal pattern as the fly gene, allowing expression of the corresponding human cDNA under the control of the UAS element (GAL4 target sequence). By comparing the ability of the reference (wild type) and variant (mutant) to rescue the fly mutant phenotype, one can assess whether the variant of interest impacts protein function.
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