Gene targeting strategy for B-hCD4 mice. The full coding sequence of mouse Cd4 gene was replaced by human CD4 counterpart gene sequences in B-hCD4 mice.

Strain specific CD4 expression analysis in heterozygous B-hCD4 mice by flow cytometry. Thymocytes were collected from wild type C57BL/6 (+/+) and heterozygous B-hCD4 mice (H/+) and analyzed by flow cytometry with species-specific anti-CD4 antibody. Mouse CD4 was detectable in WT mice and heterozygous B-hCD4 mice. Human CD4 was exclusively detectable in heterozygous B-hCD4 mice but not in WT mice.

Strain specific CD4 expression analysis in B-hCD4 mice by flow cytometry. Blood cells were collected from wild-type C57BL/6 (+/+) and homozygous B-hCD4 mice (H/H) and analyzed by flow cytometry with species-specific anti-CD4 antibody. Mouse CD4 was detectable in wild-type mice. Human CD4 was exclusively detectable in homozygous B-hCD4 mice but not in wild-type mice.

Strain specific CD4 expression analysis in B-hCD4 mice by flow cytometry. Lymph nodes were collected from wild-type C57BL/6 (+/+) and homozygous B-hCD4 mice (H/H) and analyzed by flow cytometry with species-specific anti-CD4 antibody. Mouse CD4 was detectable in wild-type mice. Human CD4 was exclusively detectable in homozygous B-hCD4 mice but not in wild-type mice.

Strain specific CD4 expression analysis in B-hCD4 mice by flow cytometry. Spleen was collected from wild-type C57BL/6 (+/+) and homozygous B-hCD4 mice (H/H) and analyzed by flow cytometry with species-specific anti-CD4 antibody. Mouse CD4 was detectable in wild-type mice. Human CD4 was exclusively detectable in homozygous B-hCD4 mice but not in wild-type mice.

Analysis of leukocyte subpopulations in B-hCD4 mice by FACS. Blood, lymph node and spleen cells were isolated from C57BL/6 and B-hCD4 mice (3 mice/group, 6-week-old). Flow cytometry analysis were performed to assess leukocyte subpopulations. Percent of T cells, B cells, NK cells, macrophages, granulocytes and monocytes in homozygous B-hCD4 mice were similar to those in the C57BL/6 mice, demonstrating that CD4 humanized does not change the overall development, differentiation or distribution of these cell types in blood, lymph node and spleen. Data was shown as Mean ± SEM, and analyzed using T-Test.