This procedure begins by preparing morpho and in vitro transcribed cap mRNA. A micro pipette is filled by siphoning the morpho containing solution and the injection volume is then calibrated using a micrometer one cell stage. Zebrafish embryos are then collected and arranged in a microinjection chamber for yolk micro injections.
Working solution is injected into the yolk just below the cell and cytoplasmic movements are used to carry the solution for cell micro injections. Inject the solution into the cell cytoplasm following injections. Incubate the embryos at 28.5 degrees Celsius in E three medium and score for phenotypes of interest at subsequent stages.
Hi, I'm Shiloh Yuen from the laboratory, Dr.Sasha's son and the Department of Genetics at Yale University School of Medicine. Today we'll demonstrate a robust microinjection technique for performing mRNA overexpression and morpho oligo nucleotide gene knockdown studies in zebrafish. We utilize this procedure in our laboratory to study the role of the S cilium in cystic kidney diseases.
So let's get started. One powerful approach for gene function studies is to micro inject in vitro transcribed capped RNA in zebra fish embryos. In this demonstration, we will micro inject an EGFP tagged transcript and use live whole embryo GFP expression as a visible readout for a successful injection.
First, we'll perform an in vitro cap RNA transcription reaction on your transcript of interest. In our lab, we routinely insert A-C-D-N-A of interest into a PCs two plus vector and perform an in vitro synthesis reaction of large amounts of capped RNA using the M message M machine SP six kit. Next, we'll purify the RNA sample by running it through the RN easy mini kit or by phenol chloroform extraction and isopropanol precipitation.
Carefully determine the concentration of the RNA preparation and store it minus 80 degrees Celsius until ready for use on the day of the injection. Thaw the RNA sample vortex lightly and spin down briefly. Now prepare a working solution with sterile water and a final concentration of 0.025 to 0.05%phenol red, phenol red serves as a visible marker for the injection of the solution into the embryo.
Here we will prepare a working solution of 100 nanograms per microliter EGFP mRNA with 0.05%phenol red. Lastly, keep the working sample on ice and return the RNA stock to minus 80 degrees Celsius. Proceed to preparation of the morpho when ready to inject this sample.
Morino antisense oligonucleotides are widely used to modify gene expression by blocking translation of a targeted protein or by modifying prem NA.Splicing morphos in the zebra fish serve as a powerful reverse genetics tool by knocking down gene function. Here we will micro inject a morph oligo targeted to the translational initiation site of pkd two. We routinely use 300 M of a morph oligo, specifically designed for a gene of interest obtained from gene tools LLC.
We then add 100 microliters sterile water to make a three millimolar stock solution and then aliquot the solution and store at minus 20 degrees Celsius until ready for use on the day of the injection. We heat the morino solution at 65 degrees Celsius for five minutes. Then we snap cool the solution on ice immediately and spin briefly this step denatures any secondary structures in the oligo and ensures that the solution is completely solubilized.
Next, we'll prepare a working solution by diluting the Morpho solution in sterile water and a final concentration of 0.025 to 0.05%pinal red. In this demonstration, we will be preparing a working solution of 0.5 millimolar PKD two morino with 0.05%phenol red. Keep the working solution at room temperature and proceed to micro pipette filling when ready to inject this sample.
To begin, we'll need to fabricate micro pipettes to be used for the injections. This is done by heating and pulling BOS silicate glass capillary tubes in a micro pipette polar device. Once the needles are pulled, they're kept in a Petri dish on top of a small amount of clay or adhesive tape tape.
Next, we'll prepare microinjection chamber plates by pouring 1.5%aros in one XE three medium plates molded with wedge-shaped troughs. That will serve as an easy method for holding the embryos during the injection. The agro chamber plates are then allowed to solidify and stored at four degrees Celsius until they're ready to be used.
To begin filling the micro pipette. Cut the distal tip with forceps or a surgical razor blade to create an opening that is just visible under a dissecting microscope at 50 x magnification. Next place two microliters of your working solution.
As a drop onto a cover slip, attach the back of a micro pipette to a five milliliter syringe fitted with tubing on a hypodermic needle and slightly submerged the distal tip of the micro pipette into your drop of working solution. Take care not to break the tip of the micro pipette. Siphon the working solution through the distal end of the micro pipette by pulling the plunger of the syringe.
Next, we'll calibrate the micro pipette injection volume. Place a drop of mineral oil on a micrometer slide and turn on the power and air supply to the pressure pulse micro injector apparatus. Next, attach the micro pipette to the micro pipette holder of the micro injector apparatus.
The micro injector is pressure regulated and discharges are activated by pressing the foot pedal under the dissection microscope. Test the injection volume by using the micro injector to place a drop of your working solution onto the micrometer oil. Measure the diameter of the drop as it floats as a sphere on top of the oil.
Adjust the duration and pressure of injection to carefully calibrate your volume of injection. This step aids in the reproducibility of the micro injection experiment. In this demonstration, all micro injections will be done with a drop diameter of 0.15 millimeters.
Finally, once your injection volume has been calibrated, use the hold knob on the micro injector to prevent back filling and leaking of the micro pipet. Now it's time to prepare the zebrafish embryos. Zebrafish will randomly mate, in the first few hours of each morning to begin the preparation, collect embryos at the one cell stage and place them in one XE three medium.
Using a three milliliter transfer pipette, arrange the embryos along the wedge-shaped troughs in the microinjection chamber plates. Remove the medium so that the embryos are shallowly, submerged and not flooded. This step aids in settling the embryos to the bottom of the troughs as well as in the penetration of the corion by the micro pipette.
Now we will begin the injections. First, we'll manipulate the embryos with the micro pipette so that the cytoplasm of the one cell stage embryo is visible under the dissecting microscope. Take care not to break the tip of the micro pipette, penetrate the corion and then the yolk with the micro pipette in order to inject into the embryo.
In this demonstration, we will be first micro injecting into the yolk directly below the cell and allowing cytoplasmic and diffusion to bring the working solution into the cell. This flow is visible due to the phenol red added to the working solution. Since this is a single cell embryo, it can be injected freehand or with a manipulator.
We will also demonstrate direct injection into the cell cytoplasm. Direct microinjection into the cell is more robust, but time consuming as proper embryo orientation and microinjection technique are required. As the cell membrane is more rigorous than the yolk membrane, it is often quicker to enter the micro pipette through the yolk to reach the cell.
Cytoplasm orienting the animal pole towards the bottom of the trough and working with steady hands may aid in this method of injection. After microinjection, place the embryos into a Petri dish with E three medium and incubate them at 28.5 degrees Celsius for normal development. Over the next several hours and days of embryo development, observe the embryos for your phenotypes of interest.
To verify the success of the microinjection, we will monitor the expression of GFP in the developing embryos beginning at shield stage by in vivo whole embryo fluorescence microscopy expression of the construct is most strong during the early events of embryonic development and will often diminish during development. As the injected capped RNA is gradually degraded and depends on the stability of the expressed protein. Based on previously published results, we expect the PKD two morphine to mimic the dorsal body axis curvature as found in PKD two mutant zebrafish and present kidney cysts at approximately two to three days post fertilization.
We've just shown you a robust microinjection technique for performing mRNA overexpression and morpho oligonucleotide gene knockdown studies in zebrafish when doing this procedure, remember that's important. Inject with a steady hand. So that's it.
Thanks for watching and good luck with your experiments.
