Background:

• TFR1, also known as transferrin receptor or CD71, is a 95 kD type II heterodimeric transmembrane glycoprotein, expressed on proliferation cells, reticulocytes, and erythroid precursors. Its expression is very low on resting leukocytes. TFR1 plays a role in the control of cellular proliferation by facilitating the uptake of iron via ferro-transferrin binding and the recycling of apo-transferrin to the cell surface. It is an attractive target for anti-cancer agents, particularly those that aim to induce lethal iron deprivation in malignant hematopoietic cells.
• CD3E (Cluster of Differentiation 3 epsilon) is an important component of the T cell receptor (TCR) complex, playing a crucial role in T cell development, activation, and signal transduction. CD3E gene mutations can lead to severe combined immunodeficiency (SCID) or CD3 ε deficiency, characterized by decreased T cells and impaired immune function. Abnormal CD3E signals may be associated with autoimmune diseases such as rheumatoid arthritis and type 1 diabetes.

Targeting strategy:

• The genome of mouse Tfr1 gene that encode the extracellular domain was replaced by human TFR1 counterparts in B-hCD3EDG/hTFR1 mice.
• The exons 2-8 of mouse Cd3e gene that encode the full-length protein were replaced by human CD3E exons 2-9 in B-hCD3EDG/hTFR1 mice. The exons 1-5 of mouse Cd3d and the exons 1-6 of Cd3g gene that encode the full-length protein were replaced by human CD3D exons 1-5 and CD3G exons 1-7 in B-hCD3EDG/hTFR1 mice, respectively.

Verification: 

• Human TFR1 protein and CD3E protein were exclusively detectable in B-hCD3EDG/hTFR1 mice but not in wild-type mice. Humanization of TFR1 and CD3E did not change the overall frequency or distribution of immune cell types in spleen, blood and lymph nodes.

Application:

This product is used for the pharmacological and safety evaluation of therapeutic drugs, as well as for the evaluation of pharmacodynamics and safety of treatments for tumors or neurodegenerative disease and the potential of drugs to penetrate the blood-brain barrier.

Gene targeting strategy for B-hCD3EDG/hTFR1 mice.

The exons 2-8 of mouse Cd3e gene that encode the full-length protein were replaced by human CD3E exons 2-9 in B-hCD3EDG/hTFR1 mice. The exons 1-5 of mouse Cd3d and the exons 1-6 of Cd3g gene that encode the full-length protein were replaced by human CD3D exons 1-5 and CD3G exons 1-7 in B-hCD3EDG/hTFR1 mice, respectively.

The exons 4-19 of mouse Tfr1 gene that encode extracellular domain are replaced by human counterparts in B-hCD3EDG/hTFR1 mice. The genomic region of mouse Tfr1 gene that encodes cytoplasmic portion is retained. The promoter, 5’UTR and 3’UTR region of the mouse gene are also retained. The chimeric TFR1 expression is driven by endogenous mouse Tfr1 promoter, while mouse Tfr1 gene transcription and translation will be disrupted.

Strain specific TFR1 expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hCD3EDG/hTFR1 mice by flow cytometry. Bone marrow erythrocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hCD3EDG/hTFR1 mice (H/H, H/H). Protein expression was analyzed with anti-mouse TFR1 antibody (Biolegend, 113808) and anti-human TFR1 antibody (Biolegend, 334107) by flow cytometry. Mouse TFR1 was only detectable in wild-type C57BL/6JNifdc mice. Human TFR1 was exclusively detectable in homozygous B-hCD3EDG/hTFR1 mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific CD3E expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hCD3EDG/hTFR1 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hCD3EDG/hTFR1 mice (H/H, H/H). Protein expression was analyzed with anti-mouse CD3E antibody (Biolegend, 100341) and anti-human CD3E antibody (BD Horizon™, 562426) by flow cytometry. Mouse CD3E was only detectable in wild-type C57BL/6JNifdc mice. Human CD3E was exclusively detectable in homozygous B-hCD3EDG/hTFR1 mice.

Strain specific CD3E expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hCD3EDG/hTFR1 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hCD3EDG/hTFR1 mice (H/H, H/H). Protein expression was analyzed with anti-mouse CD3E antibody (Biolegend, 100341) and anti-human CD3E antibody (BD Horizon™, 562426) by flow cytometry. CD3E cannot be detected in B cells of wild-type C57BL/6JNifdc mice and homozygous B-hCD3EDG/hTFR1 mice.

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6JNifdc mice (male, n=3, 9-week-old) and homozygous B-hCD3EDG/hTFR1 mice (male, n=3, 9-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, Neutrophils, monocytes, macrophages, CD4+ T cells, CD8+ T cells and Tregs in B-hCD3EDG/hTFR1 mice were similar to those in C57BL/6JNifdc mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.001.

Frequency of leukocyte subpopulations in blood by flow cytometry. Blood cells were isolated from wild-type C57BL/6JNifdc mice (male, n=3, 9-week-old) and homozygous B-hCD3EDG/hTFR1 mice (male, n=3, 9-week-old). A. Flow cytometry analysis of the blood cells 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, Neutrophils, monocytes, macrophages, CD4+ T cells, CD8+ T cells and Tregs in B-hCD3EDG/hTFR1 mice were similar to those in C57BL/6JNifdc mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.001.

Frequency of leukocyte subpopulations in Lymph nodes by flow cytometry. Lymph nodes cells were isolated from wild-type C57BL/6JNifdc mice (male, n=3, 9-week-old) and homozygous B-hCD3EDG/hTFR1 mice (male, n=3, 9-week-old). A. Flow cytometry analysis of the lymph nodes cells was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Percentages of T cells, B cells, NK cells, CD4+T cells, CD8+T cells and Tregs in B-hCD3EDG/hTFR1 mice were similar to those in C57BL/6JNifdc mice. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.001.