زرع الخلايا مباشرة في الكلى من اسماك الزرد الكبار

The overall goal of this procedure is to perform transplantation of cells directly into the kidney of adult zebrafish to study their progenitor activities. This is accomplished by first conditioning the recipient fish with the radiation. The second step is to prepare the transplanted cells from the transgenic donor fish.

The kidney of recipient fish is then surgically accessed and the donor cells are injected into the kidney. Ultimately, results can be obtained that show progenitor cell engraftment through detection of live EGFP fluorescence. The main advantage of this technique over existing methods like the injection of cells into the blood via the heart, is that the transplant of cells are delivered directly to the kidney.

Select the largest male fish and anesthetize it with 0.02%Trica. Remove the fish when it stops swimming, but the heart is still beating. Place the anesthetized fish on a damp paper towel on top of a stage and use an insulin syringe to inject jettison into the intraperitoneal cavity of the fish.

This induces a favorable environment for enhanced engraftment. Return the fish back into system water for recovery during which it may be docile and sensitive to stimuli like vibrations. After three hours of recovery, treat the fish with 25 gray of gamma irradiation and then return the fish to the system for three days without feeding just prior to the surgical procedure, prepare the donor cells.

Many labeled cell lines can be used, but for renal studies, whole kidney marrow containing renal and hematopoietic progenitor cells are appropriate. Begin by sacrificing three adult transgenic fish by placing them into 0.2%Trica for five minutes. Use a sterilized razor blade to remove the head and tail and use dissecting scissors to make a midline incision along the ventral side.

Under a stereoscope, remove all of the internal organs in the body cavity, including the swim bladder with tweezers. Be careful to not remove the kidney, which is a flat and pigmented organ attached to the dorsal wall of the cavity. Next, dissect out the kidney and place it into a 1.5 milliliter tube containing 300 microliters of supplemented PBS.

Use a micro tube pestle to shear the kidney until it becomes homogenized. Then strain the cells through a 40 micrometer cell strainer inside a 50 milliliter tube. Use the pestle to gently smear the big pieces of tissue that are left on the strainer to further break up the cells.

Now to collect residual cells from the strainer. Wash the strainer with 1.2 milliliters of supplemented PBS. Transfer the cell solution into a new 1.5 milliliter tube centrifuge.

Wash the cell pellet with 1.5 milliliters of supplemented PBS and pass it through a new strainer to filter out clumps of sticky cells. Centrifuge the cells once again in the same manner and resuspend the pellet in 200 microliters of supplemented PBS now transferred the suspension to a PCR tube and centrifuge. It discard the bulk of the supernatant and resuspend the cells in the residuals supernatant.

To get a volume of two to five microliters, store these cells on ice. There should be approximately 2 million cells per kidney to be certain. Count the number of cells with a hemo cytometer and adjust the concentration to 500, 000 cells per microliter.

Now prepare a Hamilton syringe by rinsing it three times with sterile water. Three times with 75%ethanol, and finally three times with PBS with 2%FCS just prior to injection. Load the syringe with one microliter of cell solution.

Anesthetize the conditioned recipient fish in 0.02%trica until the fish has stopped moving, but not too long that the heart stopped speeding. Under a stereoscope, dry the fish on a paper towel ventral side up with the head to the left. Then roll the fish over to expose the dried side.

Using sterilized tweezers, remove the scales in the region immediately posterior to the gills. Make a one centimeter lateral incision in this area at the midline with a scalpel while stabilizing the tissue with tweezers. Then using tweezers.

Open the cut while continuing to make the incision deep enough to expose the head kidney. If bleeding obscures the kidney. Use a new recipient fish now inject one microliter of the cell solution directly into the head kidney, and as the syringe needle is withdrawn, allow the tissue to fall back into place.

This helps prevent the injected cells from leaking out of the kidney. Next, close the opening with a single suture. Hold a suture needle with the needle driver pierce through the muscle on each side of the incision.

Tie two knots and cut off the excess suture. Return the fish to the system for recovery after seven to 18 days, dissect the fish to analyze engraftment of renal progenitor cells by live EGFP fluorescence for renal studies, the kidney is dissected and analyzed by live EGFP fluorescence. Engraftment and differentiation of renal progenitor cells is evident by the presence of EGFP positive renal epithelial cells.

By 18 days post transplant, an average of 24 EGFP positive nephrons are detected, indicating that the renal progenitors have differentiated into new kidney tissue. Generally, individuals new to this method will struggle because it may be difficult to identify the kidney after the incision has been made. However, once mastered, the surgery can be done in three minutes per fish.

While attempting this procedure, it's important to remember that the aorta is closed to the incision site and can be easily cut following this procedure. Other methods like intraperitoneal, injection of fluorescent dex strands can be performed to answer additional questions like whether the new kidney tissue is functional infiltration.