ハイスループット DNA の抽出およびフィン クリッピングが 3dpf ゼブラフィッシュ仔のジェノタイピング

This method can help answer key questions in the biomedical field. Such as the use of zebrafish to generate genetic models of disease. The main advantage of this technique is that it allows early identification of genotypes using a simple high-throughput method.

Though this method can enable extensive research using zebrafish models of genetic epilepsies, it can also be applied to other genetic disease models and any type of mutation study. Generally, individuals new to this method may have difficulties as the transection of the larval fin must be precisely performed within the pigment gap of the tail. Prior to starting the fin clipping of the zebrafish larvae, prepare the dissection surface.

Tape a piece of autoclave tape across the interior surface of a nine centimeter Petri dish lid and position the lid under a stereo microscope. Prepare a P1000 pipette tip for accommodating a three dpf larva with minimal stress, by cutting off the end of the pipette tip to a diameter of two millimeters. Place three to five days post fertilization, or dpf, zebrafish larvae in a Petri dish and anesthetize in 1.5 millimolar tricaine in 1x E3 embryo medium.

Using the modified P1000 micro pipette, pick up and anesthetize zebrafish larva and place it onto the positioned Autoclave tape on the Petri dish lid. Remove excess tricaine solution surrounding the larva. The area should be as dry as possible to successfully section and pick up the fin, but still wet to ensure survival.

To avoid bleeding and ensure survival of the larva during fin clipping, the transection must occur within the pigment gap. Distal to the limit of the blood circulation, as indicated in this image. Working under a stereo microscope, use a micro scalpel to section the caudal fin.

During the incision, apply a steady but gentle downward pressure to avoid damaging the notochord. Visualize the sectioned fin under the microscope, and position the piece on top of the tip of the micro scalpel blade. Place the micro scalpel containing the fin onto the surface of a small piece of filter paper.

Due to the presence of melanocytes in the transected tissue, the fin appears as a small black spot. To ensure successful DNA extraction, it is important that a black dot marking the placement of the fin is observed on the filter paper before continuing with the protocol. Using scissors and tweezers, transfer the filter paper containing the fin to a well of a 96-well PCR plate containing 25 microliters of a 50 millimolar sodium hydroxide solution.

Next, prepare a flat bottom 96-well tissue culture plate, numbering the wells according to the label of the PCR plate. Using the modified P1000 pipette, filled with 200 microliters of fresh e3 embryo medium, carefully collect the zebrafish larva using gentle pressure. Dispense the larva in the 96-well plate in the same well number as the PCR plate.

Make sure the filter paper containing the fin is well submerged in the solution. To prevent cross contamination between each cut, clean the blade of the micro scalpel and the tweezers by dipping into a 70%ethanol solution. Wipe the blade with a clean paper tissue or wipe.

When all larvae have been clipped, store the larvae in the 96-well plate at 28 degrees Celsius until their genotypes have been identified. Begin this procedure by sealing the 96-well PCR plate containing the fin pieces and centrifuging the samples at 1000 times G for one minute. To make sure all the filter papers are submerged in the sodium hydroxide solution.

For tissue lysis, heat the samples in a thermal cycler at 95 degrees Celsius for five minutes. Followed by cooling to four degrees Celsius for 10 minutes. Next, add six microliters of 500 millimolar tris-HCL pH8.0 to each sample.

Vortex the samples. Briefly centrifuge the plate at 1500 times G for five minutes at room temperature. To extract genomic DNA using this method, clip the fin as demonstrated earlier, but add the filter paper with the clipped fin to a well of a 96-well PCR plate containing 30 microliters of 5%chelating resin.

After all fin pieces have been clipped, seal the 96-well PCR plate and briefly centrifuge the samples to make sure all filter papers are submerged with the chelating resin. Heat the samples in a thermal cycler at 95 degrees Celsius for 15 minutes, followed by cooling to four degrees Celsius for 10 minutes. Briefly centrifuge the samples to pellet the resin beads and obtain the DNA in the suspension.

A multiplex PCR assay will enable the discrimination between homozygous mutants, heterozygotes, and wild type larvae. Set up the PCR reaction as detailed in the text protocol. Perform the PCR reactions in 96-well thermal cycler.

Prepare a 1%agarose gel in sodium borate buffer, stained with 1x gel red. To facilitate the process, if possible, use a gel system that is compatible with multi channel pipettes to enable higher throughput capabilities by reducing the time to load samples. Run the gel in 1x sodium borate buffer at 250 volts for 15 minutes.

Image the gel under ultraviolet light to allow discrimination of the different genotypes. Start this assay by preparing two 12%polyacrylamide gels using the 1.5 millimeter spacer plate and the 15 sample combs as described in the text protocol. Use a vertical electrophoresis system and 1x TBE buffer as the running buffer.

After performing PCR as described in the text protocol, heat the PCR products at 94 degrees Celsius for five minutes, followed by cooling at four degrees Celsius for 10 minutes. Load 10 microliters per PCR sample, and eight microliters of molecular weight marker. Run the gel at 150 volts for one hour, or until the molecular weight marker almost reaches the foot line of the glass plate.

Image the gel under ultraviolet light to allow discrimination of the different genotypes. This protocol was used to genotype of the offspring of a heterozygous cross, in which the mutant ileal has a 5 base pair insertion in the first coating exon of the aldh7a1 gene. The expected result from PCR amplification of both wild type and mutant ileals is a 434 BP amplicon.

A 293 BP band arises from amplification of the mutant ileal, and a 195 BP band is obtained for the wild type ileal. Analysis of PCR reactions on a 1%agarose sodium borate gel identified wild type genotypes in lanes two and six, heterozygous genotypes in lanes three, four, and five, and a homozygous mutant genotype in lane one. Various mutations where genotyped by a heteroduplex melting assay.

The plpbpot101 and plpbpot102 mutant ileals of the plpbp gene each let to a frame shift and early stop codon. After denaturation and annealing, the PCR fragments for heterozygous animals would contain heteroduplex and homoduplex DNA that are easily separated and visualized using polyacrylamide gel electrophoreses. Compound heterozygous mutant genotypes were identified in lanes two, four, and eight, heterozygous genotypes in lanes three, six, seven, nine, and ten, and a wild type genotype in lane five.

Once mastered, fin clipping 96 larvae using this technique can be done in less than an hour, if performed properly. Following this procedure, other methods, like pooling of zebrafish larvae of the same genotype for tissue extraction, can be performed and ordered to answer additional questions, such as, biomarker investigation and disease models. After it's development, this technique paved the way for research in the field of models of metabolic epilepsy to explore disease path physiology in zebrafish.

After watching this video, you should have a good understanding of how to properly genotype three day old zebrafish larvae by microdissection of a caudal fin.