開発にターゲットマイクロインジェクションのテクニック

The overall goal of this procedure is to target microinjections to the developing xenopus kidney. This method can help address key questions in the field of developmental biology such as how the kidney forms and what genes are important for this process. The main advantage of this technique is that it maximizes the delivery of microinjected reagents to a targeted tissue.

Visualization of this method is important because identification of the correct blastomere for targeted microinjection is critical. To begin this procedure both testes were isolated from a single male frog and placed in a 60 millimeter petri dish filled with 10 millileters of testes storage solution. Then store the testes at four degress Celsius.

Next, eggs were collected from a female frog in a 100 millimeter petri dish. Pour off excess water from the petri dish. After that cut off a quarter of a testis in Testes Storage Solution.

Transfer the piece of testis to the petri dish containing the eggs. Subsequently, cut the testis portion into small pieces. Add MMR plus Gentamycin to the petri dish to cover the eggs.

Mix them by swirling the dish and then wait approximately 30 minutes for fertilization to take place at room temperature. After fertilization the pigmented side of the embryos should be facing up. Remove MMR from the petri dish using a transfer pipette and add enough de-jelly solution to cover the embryos.

Afterward, swirl the dish over the next few minutes to disolve the jelly coat on the embryos. Once the embryos are closely touching each other in the center of the dish, the jelly coat has been removed. Remove the de-jelly solution with a transfer pipette.

Next, wash the de-jellied embryos 3 to 5 times in MMR plus Gentamycin by carefully pouring off the MMR and filling the dish with new MMR. Do not remove all of the MMR from the dish, which may damage the embryos. After that, remove any unfertilized eggs or pieces of testis from the petri dish.

To stagger their development, place half of the embryos from a single fertilization in a petri dish kept at 14 degrees Celsius, and the other half of the embryos in a petri dish kept at 18 degrees Celsius. Embryos will develop more slowly at 14 degress Celsius than at 18 degress Celsius. Allowing for multiple sets of injections from a single fertilized clutch.

Prepare the injection solution containing point zero one nanogram per nanoliter Membrane-bound Red Fluorescent Protein mRNA. While the embryos are developing to the 4-cell or 8-cell stage. Keep the injection solution on ice until use.

Then, load a seven inch replacement glass capillary tube into a needle puller with the top of the replacement tube aligned with the top of the needle puller case. Set the heat number two value to 800 and the pull value to 650 and press the pull button to pull the needle. After that, snip off the tip of the pulled needle with a pair of Dumont forceps.

Slip the micropipette collet onto the back of the needle. Next, slip the large hole O-ring onto the back of the needle behind the collet. Now, fill the needle with mineral oil using a 27 gauge hyperdermic needle, being careful not to get air bubbles into the needle.

Slip the needle onto the plunger of the microinjector, seating the needle into the large hole of the white plastic spacer installed on the plunger. Secure the needle by tightening the collet. Next, press and hold the Empty"button on the microinjector control box until there are two beeps.

Afterward, pipette three microliters of injection solution onto a piece of parafilm. Then, insert the tip of the needle into the bead of injection solution on the parafilm. Press and hold the Fill"button on the microinjector control box to draw the injection solution into the needle.

Prior to microinjection, it is important to understand the early cell divisions of the xenopus embryo. The single cell embryo has a darkly pigmented, animal pole and a vegetal pole which is white and yoky. In the two cell embryo the first cleavage typically occurs between the left and right sides of the embryo.

These cells contribute equally to the pronephric lineage. The second cleavage divides the dorsal and ventral halves of the embryo leading to a 4-cell embryo. The dorsal cells are smaller and have less pigment than the ventral cells.

If injecting into a 4-cell embryo, inject the left ventral blastomere to target the left kidney. The third cleavage bi-sects the animal and vegetal sides. Resulting in an 8-cell embryo.

At this point there are four animal blastomeres and four vegetal blastomeres. To target the left kidney of an 8-cell embryo inject into the left ventral vegetal blastomere. Subsequently, fill a 60 millimeter petri dish lined with 500 micron polyester mesh with five percent fycol in MMR plus Gentamycin.

Carefully pipette 20 to 30 8-cell embryos into the dish. Using a hair loop manipulate the embryos so that the blastomere to be injected is facing the needle. To target the left kidney, line up the embryos so that the left ventral vegetal blastomeres of 8-cell embryos face the needle.

Inject 10 nanoliters of injection solution into the selected blastomere of each embryo in the dish. Then, transfer the injected embryos into the wells of a culture plate with five percent fycol in MMR plus Gentamycin. Incubate the injected embryos at 16 degrees Celsius for approximately one to two hours to allow the injected blastomeres to heal.

After that, transfer the microinjected embryos into new wells in the culture plate filled with MMR plus Gentamycin. Incubate the embryos at 14 to 22 degress Celsius until they reach stage 38 to 40. In this procedure transfer 10 to 20 stage 38 to 40 embryos into a glass vial.

Add 10 microliters of five percent benzocaine in 100 percent ethanol to the vial. Mix by inverting the vial and wait 10 minutes to anesthetize the embryos. Next, remove MMR from the vial and fill it with MEMFA.

Then, place it on a three dimensional, rocking platform for one hour at room temperature. After one hour remove MEMFA from the vial and fill it with 100 percent methanol. Then, place the vial on a three dimensional, rocking platform for ten minutes at room temperature.

Repeat this wash step one more time and store the embryos in 100 percent methanol overnight at minus 20 degrees Celsius. Embryos are now fixed and ready for immunostaining. This schematic shows which blastomere to inject for targeting of the left pronephros of xenopus embryos at the 8-cell stage using Membrane-bound Red Fluorescent Protein mRNA as a tracer.

Tracer locatlization after injection in the left ventral vegetal blastomere is shown in red while the kidney is labeled in green. This image shows that the kidney was stained green with antibodies 3G8 and 4A6. Here is a close up view of the kidney region showing tracer loacalization and here is the merged image showing the co-localization of the tracer with the kidney.

Shown here is the confocal image of the kidney of a second embryo stained with 3G8 and 4A6. This is the localization of the red tracer in the kidney and surrounding tissue and this is the merged image showing the co-localization of the tracer 3G8 and 4A6 in the kidney. Prior to performing this procedure it's important to become familiar with the early xenopus embryo.

This will allow you to properly target microinjections through the established xenopus fade map. Once mastered, this technique can be adapted to target other tissues such as the neural tube or heart.