Constructing a Humanized Mouse Model with Engineered Immunity against HIV
Transcription
Pour the thymus pieces and medium from the flask into a 60-millimeter dish. Chill some positive displacement pipette tips by placing them in open 1.5-milliliter sterile screw-cap tubes on ice. Also, place the cell pellets and a volume of gelatinous protein mixture, such as Matrigel, on ice.
Anesthetize the recipient mice with an injectable anesthetic according to an approved animal protocol. Check the anesthesia level of the mouse by squeezing a paw. The absence of a reaction to the pinch indicates a surgical plane of anesthesia.
Shave the left side of each mouse from hip to shoulder, between the center of the back and the stomach. Subcutaneously, inject 6 milligrams per kilogram of diluted carprofen into the animal's shoulder or inguinal triangle. Flush the cannula of the trocar with PBS in a 60-millimeter dish. Using a pair of blunt, curved forceps, place a piece of thymus from the 60-millimeter dish just inside the opening of the cannula, then pull back on the trocar to aspirate the tissue into the cannula.
Next, use a positive displacement pipette and a chilled tip to add 5 microliters of cold gelatinous protein mixture into the tube with the cell pellet, and gently stir to generate the cell suspension. Pipette the cell suspension into the opening of the cannula and slowly pull back on the trocar to load the needle.
Swab the shaved area of the mouse with povidone-iodine followed by isopropanol three times. Determine the darkest spot under the skin, which indicates the location of the spleen. The kidney is approximately 5 millimeters dorsal to the spleen.
After ensuring a suitable level of anesthesia by the absence of a reaction to the paw pinch, use curved forceps to lift the skin, and use surgical scissors to make a 15-millimeter incision in the skin parallel to the spleen. Then, make a similar cut in the peritoneum layer below.
In males, the kidney is easily visible, thus extruded simply by pressing on the abdomen. Support the kidney with a hemostat or a pair of curved blunt forceps. In females, the ovaries tend to block the kidney from easy extraction. Using a hemostat, pick up the ovary and carefully expose the kidney.
Use needle-tipped forceps to pluck a tiny hole at the posterior end of the kidney capsule. Slide the trocar into this hole and along the kidney until the opening of the cannula is completely covered by the kidney capsule. Gently extrude the tissue under the kidney capsule and pull the trocar back out. Use a curved forceps to make sure the thymus piece does not come out with the needle.
Next, lift up the peritoneum with the forceps and gently use the hemostat to push the kidney back into place. Tie one double-knotted stitch with surgical suture in the peritoneum. Also, place two auto clips wound clips to close the skin.
Now, mix the transduced CD34-positive cells and aspirate 0.5 x 106 cells in a 100-microliter volume into an insulin syringe. Inject these cells into the mouse through retro-orbital vein injection.
After injection, swab a small drop of eye lubricant onto each eye and lay the mouse on its side back in a cage. After implanting all the mice, confirm that the animals have regained consciousness and are ambulatory before returning them to the housing room.
