TFR1 humanization (B-hTFR1 mice):

• The extracellular domain of the mouse Tfr1 gene (exons 4–19) is replaced with the corresponding human sequence.

FcRn humanization (B-hFcRn mice):

• The human full-length FCGRT cDNA was inserted into the Fcgrt exon 2 of wild-type mice.

Protein expression analysis of TFR1 in homozygous B-hTFR1/hFcRn mice. Various tissue lysates were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTFR1/hFcRn mice (H/H), and then analyzed by western blot with anti-transferrin receptor antibody (abcam, ab214039). 40 μg total protein was loaded for western blotting analysis. TFR1 was detected in heart, liver, spleen, lung, kidney, brain, skeletal muscle, stomach, colon, testis and eyeballs from both wild-type C57BL/6JNifdc mice and homozygous B-hTFR1/hFcRn mice, as the antibody was cross-reactive between human and mouse.

Protein expression analysis of FcRn in homozygous B-hTFR1/hFcRn mice. Various tissue lysates were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTFR1/hFcRn mice (H/H), and then analyzed by western blot with species-specific anti-FcRn antibody (mFcRn: R&D, AF6775; hFcRn: Novus Biologicals, NBP1-89128). 40 μg total protein was loaded for western blotting analysis. mFcRn was only detected in heart, liver, spleen, lung, kidney, skeletal muscle, stomach, colon, testis and eyeballs from wild-type C57BL/6JNifdc mice. hFcRn was exclusively detected in heart, liver, spleen, lung, kidney, skeletal muscle, stomach, colon, testis and eyeballs from homozygous B-hTFR1/hFcRn mice.

Mouse total IgG and mouse albumin expression in wild-type C57BL/6JNifdc and homozygous B-hTFR1/hFcRn mice. Serum were collected from wild-type C57BL/6JNifdc (+/+) (3 male and 3 female, 7-week-old), homozygous B-hTFR1 mice (H/H), homozygous B-hFcRn mice (H/H) (3 male and 3 female, 7-week-old) and homozygous B-hTFR1/hFcRn mice (H/H, H/H) (3 male and 3 female, 6-week-old),  and analyzed by ELISA kit (abcam, ab151276; abcam, ab207620). (A) Mouse total IgG in FcRn humanized mice was lower than that in B-hTFR1 mice and wild-type mice. (B) Mouse albumin levels in TFR1 humanized mice and  FcRn humanized mice remained comparable with those in wild-type mice. Values are expressed as mean ± SEM.