A Double Humanized BLT-mice Model Featuring a Stable Human-Like Gut Microbiome and Human Immune System

Our protocol is significant, because it can be use to reproducibly create mice with both a human gut microbiome and a human immune system. We call these mice, double humanized mice. The double humanized mouse model can be used for both basic and translational biomedical research, including the study of in vivo relationships between the human gut microbiome and human immune system.

Demonstrating the procedure will be Jianshui Zhang, a postdoc, and Yilun Chang, a PhD grad student from our laboratory. 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 centigrays 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. Disinfect the skin with three sequential iodine and 70%isopropanol scrubs.

When the skin has been prepped, use forceps to load one to one-to-six millimeter cubed human fetal liver tissue fragment into a trocar, followed by one one-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 two 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 one-to-two 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 microisolator cage with monitoring until full recumbancy.

Six hours after the surgery, place the mice under a heat lamp and disinfect the tails with 70%isopropanol. Inject 1.5 to five times 10-to-the-five CD34 positive human fetal liver tissue isolated hemopoietic 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.

Nine to 12 weeks after the surgery, use an appropriately sized plastic comb 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 a 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 thaw 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 humanized mice or onto the cage bedding. Here, an example of flow cytometry analysis of peripheral blood from a humanized BLT mouse 10 weeks post surgery is shown. T and B cell populations can both be identified, as well as CD4 and CD8 positive T cells subsets.

In this graph the relative abundance of the human fecal donor samples used to transfer a gut microbiome to create double humanized mice can be observed. The phenotypic changes to the spleen and cecum induced by antibiotic treatment is similar to that observed in germ-free animals. This principal component analysis plot of representative 16S ribosomal RNA sequencing data reveals that double humanized mice have human-like gut microbiomes that are unique to the human donor sample.

The most critical aspect of this protocol is to limit the introduction of unwanted bacteria to ensure the mice remain healthy and maintain the human gut microbiome. This model has the potential to advance personalized medicine and to allow us to look at how human disease impacts the gut microbiome while controlling for other mitigating factors.