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

Begin with an anesthetized, immunodeficient mouse that lacks immune cells.

Treat the mouse with a sub-lethal dose of X-rays to eliminate existing stem cells from the bone marrow.

Dissect the mouse and deliver fragments of human fetal liver and thymus tissue between the kidney and its capsule.

Close the incision.

The implanted liver and thymus tissue grow, forming organoids, which are cell aggregates resembling an organ.

After recovery, inject human hematopoietic stem cells or HSCs into the tail vein.

Injected HSCs migrate to the thymus organoid and bone marrow, differentiating into various human immune cells.

Provide water supplemented with antibiotics to the mouse to kill existing gut microbes.

Using an oral tube, introduce human fecal microbes directly into the mouse's gut to promote microbial colonization.

This results in the development of a double humanized BLT-mouse model featuring a human immune system and gut microbes.