Abstract
Genomic sequencing efforts can implicate large numbers of genes and de novo mutations as potential disease risk factors. A high throughput in vivo model system to validate candidate gene association with pathology is therefore useful. We present such a system employing Drosophila to validate candidate congenital heart disease (CHD) genes. The protocols exploit comprehensive libraries of UAS-GeneX-RNAi fly strains that when crossed into a 4×Hand-Gal4 genetic background afford highly efficient cardiac-specific knockdown of endogenous fly orthologs of human genes. A panel of quantitative assays evaluates phenotypic severity across multiple cardiac parameters. These include developmental lethality, larva and adult heart morphology, and adult longevity. These protocols were recently used to evaluate more than 100 candidate CHD genes implicated by patient whole-exome sequencing (Zhu et al., 2017).
Highlights
The use of the Drosophila model to elucidate molecular mechanisms underlying human diseases is well documented (Bier and Bodmer, 2004; Cagan, 2011; Zhang et al, 2013; Owusu-Ansah and Perrimon, 2014; Diop and Bodmer, 2015; Na et al, 2015), and 75% of human disease associated genes are represented by functional homologs in the fly genome (Reiter et al, 2001)
Drosophila has been used to study genes related to congenital heart disease (CHD) for over 20 years, based on evolutionarily conserved genetic mechanisms of heart development
We developed a highly efficient cardiac-targeted gene silencing approach in flies to examine effects on heart structure and function for fly homologs of candidate CHD genes (Zhu et al, 2017)
Summary
The use of the Drosophila model to elucidate molecular mechanisms underlying human diseases is well documented (Bier and Bodmer, 2004; Cagan, 2011; Zhang et al, 2013; Owusu-Ansah and Perrimon, 2014; Diop and Bodmer, 2015; Na et al, 2015), and 75% of human disease associated genes are represented by functional homologs in the fly genome (Reiter et al, 2001). Drosophila has been used to study genes related to CHD for over 20 years, based on evolutionarily conserved genetic mechanisms of heart development. 10 μl pipette tips (BioExpress, GeneMate, catalog number: P-1234-10XL) 4. FisherbrandTM plastic Petri dishes (Fisher Scientific, catalog number: S33580A) 6. Microscope slides (VWR, catalog number: 16004-430) 7. Schneider’s Drosophila medium (Thermo Fisher Scientific, GibcoTM, catalog number: 21720001) 14. Triton X-100 (Fisher Scientific, catalog number: BP151-100) 18. Alexa Fluor 555 Phalloidin (Thermo Fisher Scientific, InvitrogenTM, catalog number: A34055) 19. Biotin-conjugated goat anti-mouse antibody (Vector Laboratories, catalog number: SP-1100) 21. Streptavidin (Cy5) (Thermo Fisher Scientific, InvitrogenTM, catalog number: SA1011). VECTASHIELD antifade mounting medium with DAPI (Vector Laboratories, catalog number: H-1200). Gilson P200 pipette classic large plunger (Gilson, model: P200) Vannas spring scissors–3 mm cutting Edge (Fine Science Tools, catalog number: 15000-10) Dumont #5 forceps (Roboz Surgical Instrument, catalog number: RS-4955) Ultimate Flypad (Genesee Scientific, catalog number: 59-172) Stereo microscope (ZEISS, model: Stemi 305) Zeiss ApoTome. microscope using a 20× Plan-Apochromat 0.8 N.A/air objective (ZEISS, model: Apotome.2) Drosophila incubator set to 25 °C and 29 °C (Panasonic Healthcare, model: MIR-154-PA)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
