Aug 29, 2024
Genetic interaction analysis using ERGs in D. melanogaster
- Natalie Kaempf1,
- Ayse Kilic1,
- Patrik Verstreken1
- 1VIB-KU Leuven Center for Brain & Disease Research, 3000 Leuven, Belgium.
Protocol Citation: Natalie Kaempf, Ayse Kilic, Patrik Verstreken 2024. Genetic interaction analysis using ERGs in D. melanogaster. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9256pl3e/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 27, 2024
Last Modified: August 29, 2024
Protocol Integer ID: 106494
Keywords: ASAPCRN, genetic interaction, Drosophila melanogaster, ERG
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: ASAP-000430
EMBO long-term postdoctoral fellowship
Grant ID: ALTF_299-2019
Research project, FWO Vlaanderen
Grant ID: G0A5219N
Research project, FWO Vlaanderen
Grant ID: G0B8119N
Methusalem project
Grant ID: METH/21/05 (3M210778)
Research project, KU Leuven Parkinson Fonds
Grant ID: EQZ-PARFON-O2010
Opening the Future grant, Leuvens Universiteitsfonds (LUF)
Grant ID: EQZ-OPTFUP-O2010
Research project, FWO Vlaanderen
Grant ID: G031324N
Abstract
This protocols describes the recording of electroretinograms (ERGs) and calculation of genetic interactions of fly mutants for a transheterozygous screen.
Electroretinograms (ERGs)
Electroretinograms (ERGs)
For every measurement male flies are collected (including the control) 4±1 d after eclosion from the transheterozugous crosses reared in a temperature and light-controlled incubator
at 25 °C and 12-hour light-dark cycle
Flies are sedated with CO2 and immobilized on glass microscope slides using double-sided tape
Filamented glass micropipettes are used to generate electrodes using the Laser-pipette-puller P2000.
Glass electrodes (borosilicate, 1.5 mm outer diameter) filled with 3 M NaCl are placed in the thorax as a reference and on the fly eye for recordings
The flies are exposed to darkness for 3 seconds, followed by 1 second of LED light illumination inside a Faraday Cage.
This is repeated 4 times for each fly (5 times in total).
Light-evoked signals are amplified by a DC amplifier and the amplified signal is processed
by a data acquisition device (Clampex) and Axosope 10.7, connected to a PC running Clampfit 10.7 software (Molecular Devices).
First control flies are recorded to to assess whether the recording is stable and conditions are comparable to previous recordings
ERG traces are analyzed for depolarisation amplitude and On/Off peaks in IGOR Pro 6.37 (WaveMetrics) using a custom-made macro.
Analysis for genetic interaction using non-interacting model
Analysis for genetic interaction using non-interacting model
For all ERGs the depolarization amplitude is determined and normalized to the mean depolarization amplitude of the control of the individual experiment.
The expected depolarization amplitude for every gene pair is calculated by multiplying the normalized mean depolarization amplitude of the single heterozygous mutants.
The genetic interaction strength is calculated by subtracting the modelled expected depolarization (step 9) from the individual observed depolarization amplitude of double heterozygous gene pairs