Generating a Double Humanized BLT (Bone-Marrow, Liver, Thymus) Mouse Model

To implant the liver and thymus tissues into the left kidney capsule, first, place, the mouse in a hood. On the day of surgery, treat the mice with whole body sublethal irradiation at a 12 centi-gray per gram of body weight dose. Shave each mouse around the left lateral and medial side after confirming an appropriate level of sedation. Apply ophthalmic ointment to the animal's eyes, and apply an ear tag as needed, and disinfect the skin with three sequential iodine and 70% isopropanol scrubs.

When the skin has been prepped, use forceps to load 1 to 1.6-millimeter cubed human fetal liver tissue fragment into a trocar, followed by one 1 to 1.6-millimeter cubed human fetal thymus tissue fragment. Use the forceps to lift the skin, and use a scalpel to make a small longitudinal cut into the skin. Extend the cut to 1.5 to 2 centimeters on the left side of the mouse, and use forceps to lift the muscle layer. Use scissors to open the muscle layer longitudinally, extending the cut as necessary, to expose the kidney, and gently grasp the fatty tissue surrounding the kidney.

Use a scalpel to make a 1 to 2-millimeter incision at the posterior end of the kidney capsule and slowly insert the preloaded trocar into the incision, parallel to the long axis of the kidney. Release the tissues between the kidney capsule and kidney, and return the kidney and bowel to their normal positions. Then, use sutures to close the muscle layer and surgical staples to close the skin. Place the mouse into an autoclaved micro-isolator cage with monitoring until full recumbency. Six hours after the surgery, place the mice under a heat lamp and disinfect the tales with 70% isopropanol.

Inject 1.5 to 5 x 10⁵ CD34-positive human fetal liver tissue isolated hematopoietic stem cells in 200 microliters of PBS into one dilated tail vein per animal. Then, stop any bleeding with gentle pressure and return the mice to their home cage. 9 to 12 weeks after the surgery, use an appropriately sized plastic cone restraint to isolate one leg of one humanized mouse and spray the medial side of the isolated leg with 70% isopropanol. Spread antibiotic and pain-relieving ointment onto the collection site.

Using a 25-gauge needle held at a 90-degree angle, puncture the saphenous vein to collect 50 to 100 microliters of blood into an EDTA-coated blood collection tube. When a suitable volume of blood has been obtained, apply pressure to the site with the sterile piece of gauze to stop the bleeding before returning the mouse to its cage. Then, use the collected peripheral blood sample to assess the level of human immune cell reconstitution by flow cytometry, using antibodies against human CD45, CD3, CD4, CD8, CD19, and mouse CD45.

For fresh fecal sample collection, place individual autoclaved paper bags into a fume hood, and place one mouse into each bag until each animal defecates. Then, return the mice to their cage, and use sterile forceps to transfer each fecal sample into one 1.5-milliliter tube per mouse for minus 80 degrees Celsius storage.

At the end of the 14-day antibiotic treatment, change the drinking water to autoclaved sterilized water and transfer the mice into a new autoclaved cage. 24 and 48 hours after the cessation of antibiotics, fill properly prepared sources of fecal microbiota transplant material and aliquot the samples in an anaerobic chamber under anaerobic conditions.

To perform a fecal transplant, deliver 200 microliters of fecal microbiota transplant material via oral gavage to each experimental animal, and spread any remaining thawed material on the fur of the humanized mice or onto the cage bedding.