Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

The aim of the following experiment is to knock down maternal proteins stored in oocytes or overexpress proteins of interest at the point of fertilization of frog eggs. This is achieved by injecting antisense oligos or mRNA into enzymatically deregulated immature oocytes to degrade or overexpress a specific protein. As a second step, oocytes are transferred to progesterone containing medium, which induces maturation of the oocytes to eggs.

Next sperm is injected into the in vitro matured eggs in order to observe the effect of knockdown or overexpression on embryonic development. Ultimately, the development of oligo injected oocytes to swimming tadpoles is the functional test of gene knockdown or overexpression. The main advantage of this technique over existing vessels like host to transfer, is that we can skip the frog surgery process Using standard protocols.

Collect a opus over SAC containing healthy cytes in a nine centimeter petro dish containing MBS plus ps. Then tease apart the ovary into square pieces that are one to two centimeters wide. Collect about five milliliters of the torn ovary with cytes to a new 50 milliliter tube.

Then rinse the tissues with MBS plus PS a few times, and collect the eggs in 15 milliliters of fresh MBS plus ps. Now, add seven units of defoliation enzyme to the suspension and incubate the suspension with gentle vegetation until they are at least partially defoliated. This will take at least an hour after the incubation.

Fill the tube with MBS plus Ps to stop the reaction. Pull the solution onto the tube wall and not directly onto the cytes. Let the tissues settle and discard the soup and agent.

Small, immature cytes will float and they should also be discarded. Repeat this wash Step a total of 10 times after the washes. Transfer the cytes to a nine centimeter dish with MBS plus ps.

Transfer the dish to a dissecting microscope and from here forward, maintain the cytes at 16 to 18 degrees Celsius as much as possible. Select good quality Stage six cytes using Dumont classification using a glass pipette, collect them to a new dish with MBS plus ps. Good quality cyte should have even pigmentation in the animal hemisphere and show clear separation between the animal and vegetal hemisphere.

They should also all be of about the same size, which would be between 1.2 and 1.4 millimeters in diameter. Collect about two to 300 cytes for each injection, making up about 1000 cytes per experiment. The quality of the O site is a key to success.

If the Oside show some abnormality during the preparation, I recommend that you correct an ovary from a different flock and begin again. In preparation for injection of the antisense oligo, set the metal plunger of a micro injector to its lowest position, and insert a glass capillary filled with oil onto the metal plunger. Under a dissection microscope, cut the tip of the needle using small surgical scissors.

Make a smaller tip as possible. Next laser. A strip of paraform on the microscope stage and on it Dispense three microliters of nucleic acid solution at one microgram per microliter.

With this, fill the needle's tip. If air is being trapped, the opening should be made larger. Now make a visible mark on the injection needle about half a millimeter from the tip.

Then proceed with injecting the cytes to inject one first, find an area free of follicle cells where the needle can penetrate in this area. Insert the tip along the equatorial boundary by aiming at the center point under the germinal vesicle. At the same time, steady the cyte with forceps on the opposite side.

Now using foot switch control, eject the solution, eject between 4.6 and 9.2 nanograms of antisense oligos or between 250 picograms to 13.8 nanograms of messenger RNA. After injecting all the cytes, transfer them to incubation medium and let them incubate a few days so that changes can take place. In the proteasome.

Later, transfer the cytes with a minimal amount of media to six centimeter agros coated dishes containing five to eight milliliters of maturation medium. Then let the cytes develop in this media for 16 hours before injecting them with sperm. For this protocol, frozen sperm stock must be prepared.

Consult the text protocol after 16 hours of incubation for maturation. Very carefully transfer the cytes to a six centimeter egg gross coated dish with MBS plus PS to wash them off. If the cytes are mishandled, they may spontaneously activate before the ixy.

Now, count the matured cytes and look for white spots which indicate that the germinal vesicle of the cyte has broken down. If less than 80%are good, they are collectively not healthy enough to survive the ixy procedure. To proceed.

Fill a new dish with injection medium and delicately transfer all the cytes to the dish after half an hour. Proceed with injecting the mature cytes with sperm solution using the same injector preparation described for oligos. Ideally, there should be one or two sperm per 4.6 nanoliters in the injection solution.

To test this injection solution, make drops of DPI solution at 0.3 micrograms DPI per milliliter and inject 4.6 nanoliters into those drops. Then count the number of sperm in each drop by fluorescence microscopy. After confirming the sperm concentration, inject the cytes with 4.6 nanoliters of the sperm solution.

Ensure that the time required to inject the solutions stays consistent and inject the oocytes steadily with minimal pausing between injections. After every 100 injections exchange, the sperm solution. After all the matured oocytes have been injected, the dish should be incubated for four to five hours, then inspected.

For embryos that have undergone cleavage, the cleavage furrows may not be as clear as those of normal fertilized embryos transfer all the cleaved embryos to incubation media and let them incubate overnight. The next day, the surviving embryos should be transferred to 0.1 XMMR embryonic development of ixy embryos. Using in vitro matured oocytes was examined in good experiments, almost 100%of the germinal vesicle oocytes responded to progesterone and showed signs of maturation.

About 25%underwent cleavage. 60 to 80%of cleaved embryos reached the blast gast stage, and about one third of those reached the swimming tadpole stage. The ixy embryos were a mixture of both normal and abnormal embryos showing that injection of the antisense sogo into germinal vecal cytes, followed by IVM and Dixie allowed early embryonic development.

After watching this video, you should have a good understanding of how to manipulate maternal proteins before factorization.