Species specific analysis of GPC3 gene expression in wild-type C57BL/6 mice and homozygous humanized B-hCD3E/hGPC3 mice by RT-PCR. Kidney was collected from wild-type C57BL/6 mice (+/+) and homozygous B-hCD3E/hGPC3 mice (H/H; H/H). Mouse Gpc3 mRNA was detectable only in wild-type C57BL/6 mice. Human GPC3 mRNA was detectable only in homozygous B-hCD3E/hGPC3 mice, but not in wild-type C57BL/6 mice. 

Strain specific GPC3 expression analysis in homozygous B-hCD3E/hGPC3 mice by western blot. Liver and kidney were collected from wild type (WT) mice (+/+) and homozygous B-hCD3E/hGPC3 mice (H/H; H/H), and analyzed by western blot with anti-GPC3 antibody. GPC3 was detectable in WT mice (+/+) and homozygous B-hCD3E/hGPC3 mice (H/H; H/H) due to the cross-reactivity of antibodies.

Strain specific CD3E expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hCD3E/hGPC3 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hCD3E/hGPC3 mice (H/H; H/H), and analyzed by flow cytometry. Mouse CD3E was only detectable in wild-type C57BL/6 mice. Human CD3E was exclusively detectable in homozygous B-hCD3E/hGPC3 mice, but not in wild-type C57BL/6 mice.

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 mice and homozygous B-hCD3E/hGPC3 mice (n=4, 8-week-old). A. Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Percentages of T cells, B cells, NK cells, dendritic cells, monocytes, macrophages, granulocytes, CD4+ T cells, CD8+ T cells and Tregs in B-hCD3E/hGPC3 mice were similar to those in C57BL/6 mice, demonstrating that humanization of CD3E and GPC3 do not change the frequency or distribution of these cell types in spleen. The frequency of leukocyte subpopulations in thymus and blood of B-hCD3E/hGPC3 mice were also comparable to wild-type C57BL/6 mice (Data not shown). Values are expressed as mean ± SEM.