胚胎显微注射和电的脊索动物

The overall goal of transient transfection techniques in Ciona embryos is to study gene regulation and function required to form tadpole like larvae, taking advantage of their invariant cellular lineage, and compact invertebrate genome, in order to gain insight into chordate invertebrate origins. This method can help answer key questions in the field of molecular genetics, such as the conserved mechanisms of the chordate developmental program that are relevant to stem cell research, and for drug discovery. The main advantage of this technique is that with embryo electroporation you can handle hundreds or thousands of synchronized embryos in one day.

Generally individual new to this method will struggle, because of the fragile embryo's dechorionation, and because it is tricky to develop an efficient micro-injection technique. Begin by dissecting Ciona adults in an 18 degree Celsius cooled embryo room. Use small scissors to make an incision at the opposite end of the siphons and on the side of the shorter siphon, under which runs the oviduct and sperm duct.

Elongate the incision to expose the oviduct. Then, use the tip of the scissors to make a precise incision in the oviduct. Gently press the oviduct with the closed scissors and strip off the eggs.

Allow the eggs to drop directly into a six well plate containing artificial sea water with HEPES. Use a pasteur pipette, pre-rinsed with ASWH, to collect and transfer the remaining eggs. To collect sperm, cut the sperm duct with the scissors, then use a separate pasteur pipette to collect concentrated sperm, and transfer into a 1.5 milliliter tube.

Activate the sperm by combining one milliliter of ASWH and 50 microliters of Tris in a 1.5 milliliter micro centrifuge tube. Then, add 20 microliters of concentrated sperm, close the cap, and gently mix by inverting the tube, or using a pasteur pipette. Next, add 100 to 200 microliters of the activated sperm solution to each well of eggs, and mix well by pipetting up and down, so that the eggs are floating in the medium.

Begin dechorionation by collecting the zygotes and dispensing them into glass tubes. With the hand centrifuge, spin the zygotes at twelve hundred times G for about 20 seconds, then slowly stop the centrifuge. Remove the ASWH from the pellets with a pasteur pipette.

Add four milliliters of activated dechorionation solution to the pellet in each tube. Suspend the zygotes by gently pipetting up and down using a tap water treated pasteur pipette, topped with a small rubber pear. The solution should turn yellowish after 1 to 3 minutes.

During dechorionation, remove a small aliquot of the dechorionating suspension, using the pasteur pipette. Deposit a drop on a slide, and observe it under the dissecting microscope. Pipette the zygote suspension up and down, and check the slide every 20 to 30 seconds.

First the follicle cells detach, then the chorion turns yellow and opaque, and finally, it detaches from the zygotes. Pink dechorionated zygotes will sink to the bottom of the glass tube. Once more than 50%of the zygotes are dechorionated, fill the tube with ASWH.

Very gently centrifuge for about 10 to 15 seconds, just enough to sediment the dechorionated zygotes. Stop the centrifuge slowly. Remove nearly all the liquid from the glass tube, including any floating material, and replace with ASWH.

Slowly pipette up and down to wash, and then centrifuge gently as before. Wash again until no chorion debris is left. Gravity sediment the wash to dechorionated zygotes in siliconized 1.5 milliliter tubes.

After sedimentation, remove the ASWH down to the 100 microliter mark on the tube. Now use a pasteur pipette to add 250 microliters of previously prepared DNA in mannitol solution to one tube of zygotes. Gently mix, and immediately transfer the mixture to a four millimeter electroporation cuvette.

Place the cuvette into the electroporation holder, and give a single pulse of 16 milliseconds at 50 volts. Then remove the zygotes from the cuvette using the same pasteur pipette, and expel them into a culture dish containing fresh, filtered ASWH. Agitate the dish to spread out the zygotes.

Rinse the pipette in a beaker of ASWH, and then move on to the next sample. After elecroporating all zygotes, culture the zygotes at 15 to 20 degrees Celsius. Set up the micro-manipulator in an 18 degree Celsius cooled room.

Connect the plastic tubing and the needle holder to a 10 milliliter glass syringe filled with mineral oil. Backfill the tubing and the needle holder with oil, and expel any air bubbles. Insert the injection needle contain 0.5 microliters of injection solution with green vital dye in the needle holder.

And position the holder on the micro-manipulator. Adjust the needle holder so that it moves along a straight line at an angle of 45 degrees relative to the surface. Orient dechorionated eggs in a small agarose coated culture dish along an indentation, so that the eggs can be injected one by one under the dissection microscope.

If necessary, break the needle tip by gently pushing the needle against a piece of glass cover slip, placed on the agarose. Apply slight pressure to verify that the needle tip is open. Inject the unfertilized eggs one by one, by first introducing the needle into the egg, and slightly aspirating to break the egg membrane.

Then, inject the green injection solution into the middle of the egg, to a maximum of one third of the cell diameter. After injecting all the eggs, remove the needle, transfer the injected, unfertilized eggs to a fresh culture dish, and incubate at 15 to 18 degrees Celsius until fertilization. Micro-injection was utilized to study regulation of Ciona intestinalis myelin transcription factor or, MyT, at the gastrula stage of Ciona embryos.

Monitored by insitu hybridization, endogenous expression is observed in the sixth row neural plate precursors in wild type. Micro-injection of morpholinos to knock down early embryonic factors, such as Forkhead box A, a transcription factor, eliminates overall MyT expression. Conversely, MyT expression is ectopically activated upon down regulation of nodal, suggesting that nodal normally represses MyT expression in these lateral nerve cord precursors.

The transcription factor GATAa was tagged with a Venus tag, and electroporated into ectodermal blastomeres with a pfog driver. As seen here, GATAa is mostly localized to nuclei, as expected for a transcription factor. Whereas Venus expression is ubiquitous.

After watching this video, you should have a good understanding of how to perform transient transfection by electroporation into synchronized Ciona zygotes, how to handle the fragile dechorionated embryos, and how to find the correct angle of micro-injection. After their development, these techniques have paved the way towards functional genomics. To explore gene function, gene regulatory networks, and conserve developmental modules, important for tissue formation also in humans.