Rapid Automated DNA Extraction for Live Zebrafish Embryos
The Zebrafish Embryonic Genotyper (ZEG)
Simple. Rapid. Gentle.
Despite the widespread use of zebrafish, automated research tools for working with zebrafish embryos have not developed at the same pace as the research methodologies. Currently for genotyping, embryos are grown to adult age (two to three months) before manual fin clipping which requires a trained technician four to six hours to prepare cells and genotype 96 fish; as well as the effort and expenses of raising more adult fish than may be ultimately needed.
Alternatively, zebrafish embryos or larvae can be sacrificed and genotyped. If individual animals need to be distinctly genotyped this is even more laborious, and obviously additional testing or use of the animals is not possible.
The Zebrafish Embryonic Genotyper (ZEG) is an automated microfluidic system that extracts genetic material from live zebrafish embryos. The genetic material can then be used for downstream DNA amplification, identification, and analysis. The ZEG has rapid extraction, with 96 embryos being sampled in an hour. Furthermore, the sampling process is non-destructive meaning the embryos can be sampled, and then be raised to adulthood.
- LIVE genotyping of embryos that can be raised to adulthood
- RAPID cell extraction (96 embryos in an 1 hour)
- EASY-TO-USE Minimal training required to get up and running
- UNIQUE capabilities make out-of-reach experiments possible
- SAVE time and facility fees
- Components: 1 Base Unit and disposable chips
- Chip extraction throughput: 24 embryos in 7.5 min
- Size: ~6 x ~6 in
- Extraction Speed: from 96 embryos (~ 72 hpf) within an hour
- Genotyping sensitivity: > 80 %
- Embryo survivability: > 90 %
- Applications: Extracted genetic material can be identified using gel electrophoresis, HRMA (high resolution melting analysis), and Sequencing
- Viability: No observed effects on embryos’ morphology, or behavior at 7 dpf (after processed by ZEG at 72 hpf)
- Lifespan: The ZEG is non-destructive & safe. Embryos can easily be raised to adulthood
Data & Workflow
Presentations Using the ZEG
- Cellular and Molecular Characterization of the Effects of the Zebrafish Embryo Genotyper Protocol
- A Microfluidic Device for DNA Extraction from Live Zebrafish Embryos for Rapid Genotyping
- A Genetic Model Therapy Proposes a Critical Role for Liver Dysfunction in Mitochondrial Biology and Disease 2020
- Vanishing White Matter Disease Expression of Truncated EIF2B5 Activates Induced Stress Response 2020
- Presentation at Zebrafish Interest Group (ZIG), University of Utah 2019
- An automated system for rapid cellular extraction from live zebrafish embryos and larvae: Development and application to genotyping
- Zebrafish Facility Workshop 2018
The story behind the ZEG
Dr. Raheel Samuel, the co-founder of wFluidx which built the ZEG joined us on 17 Minutes of Science to talk more about the ZEG and his work developing tools for zebrafish research.
Frequently Asked Questions
The converter included with the unit will allow you to use an AC power input ranging between 100 – 200 V, 50/60 Hz. The converter output is DC and has an output of 12 V and 2 A. That amount is regulated to 1.4 V and 0.03 A by the controller housed in the base unit.
It depends. Users have reported that both increases and decreases in extraction time have yielded improved genotyping efficiency. We encourage you to figure out what works best for your group– and let us know if any changes make a big impact!
We recommend using fresh primers solutions and a hot start polymerase or a direct from tissue PCR kit. Make sure you are getting the most volume you can off of each well after the extraction is completed, which should be 10 µL or more. Extending the number of PCR cycles may also aid in increasing sensitivity.
Since ZEG is used for live zebrafish embryos, the amount of extracted DNA is going to be smaller than a tissue sample. Getting desired or acceptable results might require tweaking of PCR protocols, extended cycling, fresh primers, etc.
We recommend aiming for 250 bp or smaller gene fragments. Anything over that and you may experience decreased sensitivity or will have to modify your PCR protocols or chemistry. We are currently working to improve ZEG’s sensitivity for larger amplicons.
No. ZEG requires de-chorionated embryos. Sorry :(
ZEG was developed to be used with 72 hpf embryos. It has been reported to us that 24 hpf and 48 hpf embryos can be used with ZEG with >90% sensitivity and survivability. The embryos must be de-chorionated first, though.