A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

The overall goal of the following experiment is to generate a possible zebrafish model of polycystic kidney disease. This is achieved by injecting morph against Wind five A in transgenic WT one BGFP zebrafish embryos at the one cell stage. Next, a rescue experiment is performed by microinjection of mouse Wint five A Mr.Nna, with the Wint five a morpho.

Then the injected embryos are anesthetized and embedded at 48 and 72 hours post fertilization in order to monitor kidney structure and cyst formation. After wind five A knockdown, the results show that wind five A knockdown causes prone kidney cyst formation in transgenic WT one BG FP zebrafish based on fluorescence microscopy of prone kidney structures. The implications of this technical extend toward building a model ofcy kidney disease because the transparency of the fish embol covered with the use of kidney specific Fluor protein transgenic allow a direct view of kidney structures in a AL organism.

This method can provide insight into win five a knockdown causing zebra fish kidney cyst formation. It can also be applied to test many other genes that are suspected to cause renal cyst formation. This protocol is simple and less time consuming.

After designing and synthesizing morphos according to the manufacturer's instructions, add high grade sterile water to the glass bottles to thoroughly resuspend to a final concentration of 25 micrograms per microliter. Use a spectrophotometer to measure the concentration and store the stock solution at room temperature on the day of the injection, use high grade sterile water to dilute the morphos to the desired concentration, and at 0.5%phenol red to a final concentration of 0.05%Next turn on the air compressor and adjust the pressure setting to 50 si. Then turn on the dissecting microscope light source and the pico micro injection pump and adjust the settings as follows.

Use five microliters of injection solution to load a pre pulled glass needle and place the needle in a vertical position with a tip pointing down when there are no visible air bubbles, insert the needle into the holder. Adjust the injection angle to 45 degrees. Bring the needle tip into view under the microscope, high off the stage, and focus on the thinnest region of the tip.

Then use fine point tweezers to break off the needle. Tip, place a capillary tube with an inner diameter of 0.15 millimeters under the microscope. Inject the solution into one end.

Adjusting the ejecting time on the pico microinjection pump until 17 drops form a liquid column, one millimeter long. This makes the volume of every drop equal to one nanoliter. Set up transgenic WT one BG FP zebrafish breeding pairs according to published protocols for standard zebrafish husbandry and maintenance.

Following a natural spawn, collect embryos and transfer them into a Petri dish with E three water. Using the embryos between the one and four cell stage, transfer them to a 10 centimeter Petri dish with wedge-shaped auger troughs. After aspirating the medium gently press the embryos into the troughs Under the dissecting microscope, orient the embryos to visualize the cell.

Then with the injection needle penetrate the Corian and then the yolk to inject one or two drops of morpho into the yolk. Transfer the injected embryos to a 10 centimeter Petri dish with E three water and incubate at 28.5 degrees Celsius. Record the number of viable injected embryos to perform a phenotype rescue experiment.

Select an ortho log from a gene of another species that has a different primary base pair structure that is therefore resistant to the morpho. In this example, mouse went five A was chosen. Design a primer set with the forward primer starting at the translational start site and the reverse primer close to the end of the MR NA code region.

Add a T seven promoter sequence at the five prime end of the forward primer sequence with the primers and one microgram of template DNA containing the mouse wind. Five A-C-D-N-A sequence. Set up a 50 microliter PCR reaction and run using the program shown here.

After verifying the size of the PCR product, purifying it and checking the concentration, use a capped RNA synthesis kit to synthesize in vitro capped mRNA. According to the manufacturer's instructions, use 20 microliters of RNAs free water to dilute the MR NA and determine the concentration aliquot and store the RNA at negative 80 degrees Celsius on the day of the injection. Prepare a working solution of the MR NA that will deliver a 40 picogram or lower dose to avoid non-specific or toxic effects to the embryo.

Keeping the MNA solution on ice inject morphoses and then mRNA into the embryos or co inject the solutions. The next day, remove dead embryos and check to make sure that embryos have undergone gas. Then add 0.003%end phenyl THREA or PTU to the E three water to prevent melon at 48 hours Post fertilization.

Use fine tweezers to manually coate the embryos before using a dissecting microscope to take images immediately and at 72 hours post fertilization approximately 10 minutes prior to imaging under a fluorescence microscope, anesthetize the embryos by placing them in a 10 centimeter Petri dish containing 160 micrograms per microliter of buffered trica. Use 3%methylcellulose to mount the embryos and under a dissecting microscope, orient them in a prone position. Then using 10 times and 20 times magnification image.

The embryos under a fluorescence microscope wind five A knockdown was achieved by introducing translation blocking morpho, also called a UG morpho or exon intro border splice morpho, also called splice morpho to zebrafish embryos. At the one cell stage, the A UG morpho targets the start codon and therefore inhibits both maternal and zygotic win five a expression. The splice morph targets the third splice donor site and inhibits only the zygotic transcript of win five A As demonstrated here, the A UG and splice morphines pheno copied each other with multiple defects including curly tail, down body axis and pericardial edema.

This figure shows that mouse went five a mRNA partially rescues the morph phenotype demonstrating that it is not due to off target effects shown here. The transgenic WT one BGFP fish expresses GFP driven by the WT one B promoter and outlines the profic structure. The fish showed glomerular cyst formation after wind five a knockdown at 48 hours post fertilization.

The control fish injected only with phenol red displayed normal prone fric development forming fused glomerular in a U-shape with tubules that extend laterally at 72 hours post fertilization. The glomerular structures further developed with vascular loops and both the A UG and splice morph injected embryos developed cysts in the glomeruli and proximal tubes as seen in this figure HNE staining of the transverse histological sections of 72 hours post fertilization, morant embryos revealed cyst formation, disrupted glomerular structures, and dilated renal tubes. After watching this video, you should have a good understanding of how to development a possible gery phase model of cystic kidney disease by knockdown of the wind aging and observe system formation with the flu report face line.

Once it is demonstrated that gene mutation results in kidney cyst formation in zebrafish, further experiments can be conducted to investigate the detailed mechanisms of kidney cyst formation.