The overall aim of the following experiment is to generate mice carrying an engineered human immune system. This is achieved by first preparing the mouse for surgery and then loading the trocar with a piece of thymus in the second step. Matrigel is mixed with CD 34 negative and CD 34 positive transduced cells, and the mixture is then added to the trocar.
Next, the matrigel constructs are transplanted under the kidney capsule. Ultimately, rejection of the target tumors by the genetically engineered CD eight T cells can be monitored by live pet imaging and physical tumor measurements. The main advantage of this technique over existing meth methods like fully neuron models, is that this method allows for the study of genetically modified human stem cell development within the human thymus and subsequent in vivo efficacy testing of derived lineages demonstrating the procedure will be three technicians from our laboratories, Gregory Bristol, Bernard Levin, and Sean Kim.
In this schematic diagram, the modified BLT model used in these studies for the generation of chimeric mice carrying mart, one specific T cells is outlined. The th implant is reconstructed from transduced and non transduced CD 34 cells isolated from an autologous fetal liver. A fraction of the transduced cells is then frozen and injected into the irradiated mice four to six weeks later.
In this video, implantation of the th live construct is demonstrated once the animals have become sluggish from the anesthesia. Begin by shaving the left side of each mouse from hip to shoulder between the center of the back and the belly. After recording the weight of each animal, punch their ears for numbering and subcutaneously, inject 0.3 milliliters of carprofen into each of their shoulders.
Then lay the mice on their right sides facing left. Now flush the trocar of a 16 gauge cancer implant needle with a round file tip with PBS. Using a pair of needle nose forceps, add a piece of thymus to the dish of PBS and then holding the trocar horizontal with the rod.
Just inside the tip aspirate the tissue. Sliding the trocar under the kidney capsule and injecting the tissue is one of the most challenging aspects of this procedure. Next, have a helper.
Use an eend orph positive displacement pipette to mix five microliters of cold matrigel into one tube of cells with gentle stirring. Keeping the trocar horizontal. Pull slowly back on the rod while the helper pipettes the matri gel.
Mix into the trocar for each mouse. First, swab the bare skin of the animal with Betadine and then wipe it off with an isopropanol wipe. Two times determine the darkest spot under the skin, indicating the location of the spleen.
The kidney is found about five millimeter dorsal to the spleen using blunt forceps to pick up the skin over the kidney. Make a cut about 15 millimeters long in the skin parallel to the spleen. Make a similar cut in the peritoneum muscle layer below.
In males, the kidney should be directly visible. Simply squeeze the abdomen to pop it out. Keeping pressure on the abdomen with the left hand to keep the kidney exposed in females first use a hemostat to pick up the ovary and then drag out the kidney to help retain the organ outside of the body.
Pluck a little hole in the posterior end of the kidney capsule and then slide the trocar into this hole and along the kidney until the orifice of the trocar is completely covered by the kidney capsule. Then using the little finger of the right hand, inject the tissue. Now use the closed hemostat to gently push the kidney back into place.
Tie one stitch in the peritoneum with a double bow, squeezing the skin up like a purse, and then put it in two wound clips. Finally, put a drop of PBS onto each eye and lay the mouse on its side in a cage on top of some bedding. After implanting all of the mice in the same way, confirm that the animals have returned to lying on their bellies before leaving them.
This first figure shows a representative image of the THI live implant in humanized mice. In this figure, the normal development of the thymic tissue and the physiological tissue distribution of the human CD four and CD eight positive T is demonstrated. Following reconstitution, the animals carry a human immune system with a normal distribution of CD four and CD eight positive T cells.
Here a representative image of a mouse carrying a melanoma tumor is shown. The gray areas indicate the CT scan image. The colored areas indicate the metabolic activity of the tumor as detected by the PET scan.
The CT scan alone in this experiment indicated a large tumor mass in the area indicated by the white circle. However, as the in vivo PET imaging illustrates this area mostly consists of necrotic and scar tissue, underscoring the utility of PET imaging as a more sensitive and accurate way to assess tumor regression and clearance. Following this procedure, other assays such as T-cell, phenotyping and ex vivo activation can be performed to answer questions relevant to T-cell function.
