The overall goal of this procedure is to determine gene protein function by disrupting normal expression patterns of your gene or protein of interest. This is accomplished by first making the aros mold and injection needles. The second step of the procedure is to collect mid embryos at the one cell stage.
The third step of the procedure is orienting one cell stage embryos in the correct position for injection. The final step of the procedure is injecting cell tracers, mRNAs, or antisense oligonucleotides into the one cell stage midar embryos. Ultimately, results can be obtained through microscopic observation that show morphological, physiological or molecular changes associated with the protein affected by the substance injected.
Hi, I'm Sean Brazinski from the lab of Dr.Makoto Fki in the Department of Biology and Biochemistry at the University of Bath. I'm wan also from the full Seki lab. Today we'll show you a procedure for the microinjection of Meca embryos with cell tracers, mRNAs and antisense oligonucleotides.
We use this procedure in our lab to study the function of gene of interest in vivo at a single cell level. So let's get started. Unlike zebrafish, male and female, maka can easily be distinguished by their dimorphic fins.
Female fins have a round shape with a smooth margin. Both the anal and dorsal fins of the male are larger than those of the female, and the dorsal fin has a deep notch between the last two rays. Keep a mating pair of meca in a mating tank with or without water flow.
This P can be kept in the tank with running water for up to two months For daily injection, meka lay up to 40 eggs daily, which cluster at the belly by attachment filaments. Use a net to catch and remove a female out from the tank and hold the fish with one hand to secure it inside the net. Gently tease the eggs away with an index finger.
Return the female to the tank. Use forceps or an index finger to transfer the eggs to a six centimeter Petri dish filled with embryo medium. Under a dissecting microscope, use two pairs of forceps to cut the attachment filaments and uncluster the eggs.
Pick up embryos with a pasta pipette to separate from feces and algae and transfer a maximum of 40 eggs to a dish of fresh embryo.Medium. Place the eggs on ice if micro injecting immediately. Alternatively, manipulate the speed of development into later stages with variations in incubation temperature.
A plexiglass mold is required to make agarose plates for holding embryos during injection. Refer to the written portion of this protocol for details. Pour 0.5 centimeters of 1.5%aros in water into a nine centimeter Petri dish.
When the aase has solidified, cut out an area a little bit smaller than the mold so that the mold does not sink to the bottom. Build the cut area with more 1.5%aase and place the plexiglass mold over it. Avoid introducing air bubbles when the Aris has solidified.
Generally release the mold to create troughs. Cover the Aris with embryo medium. Cover the dish and store at four degrees Celsius until ready for use.
Prepare a strong wide injection needle from a filament containing glass capillary. Use a micro loader to backfill the injection needle with injection solution, including phenol red As a tracer, connect the needle to an air pump injector stand. Set up next to a dissecting microscope.
Transfer eggs of the appropriate stage to the arose injection plate. Embryos can be kept on ice for up to 30 minutes to halt development whilst carrying out the injection under the stereo microscope. Use forceps to align seven to 10 eggs in each trough.
Rotate the eggs so that the injection needle hits the eggs perpendicularly. The one cell of the embryo is opposite the dent soil droplets. Therefore, the oil droplet should be orientated towards the left, such that the one cell is on the right for injection.
In meca nucleic acids and tracer dyes are micro injected through the Corian into the cytoplasm of the embryo rather than into the yolk. As for zebra fish, move the needle close to the eggs. Touch the tip of the needle to the Corian or use micro forceps to break open the needle tip so that a small amount of the injecting solution flows outta the end.
Re-break the needle in case of blockage, puncture the Corian and move the tip of the injection needle just inside the inner layer to reduce the likelihood of reflux into the needle. Adjust the holding pressure of the injector to approximately 80 to 100 hectare Pascals sharply. Poke the membrane and insert the needle into the cytoplasm.
Inject the embryo. View the embryo from the side to confirm the even distribution of injected material within the cytoplasm. Liquids that have been correctly injected into the cytoplasm have a blurry boundary material injected too deeply into the yolk, has a distinct boundary and will not be transferred to the cytoplasm.
Transfer the injected embryos to a clean dish of embryo medium and allow them to develop as required In an incubator of suitable temperature panel A shows a representative image of correct injection of rumine dextran into the cytoplasm of a one cell stage Meca embryo. Panel B shows a representative image of incorrect injection of rumine dex str into the yolk sac of a one cell stage. Meca embryo dotted lines indicate the outline of the cytoplasm of one cell.
Panel C and D show the embryos from panels A and B respectively at approximately 30 hours post-injection. Note the body of the embryo in panel D is not red as the dye was incorrectly injected into the yolk sack. We've just shown you how to micro inject nucleic acids or tracers into the one cell stage of the darker embryos.
When doing this procedure, it's important to have correctly staged embryo for successful injection. So that's it. Thanks for watching and good luck with your experiments.
