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Animal Models and Cell Lines

>Single Humanized Immune-Checkpoint Mice

B-hCD3e mice


General  information

Strain description


Strain name

C57BL/6-CD3Etm2(hCD3E)/Bcgen

Common name

B-hCD3E   mice

Background strain

C57BL/6

Product Number

B-EM-026

Application

Animal model for anti-hCD3 or hCD3E antibody/Bispecific antibody efficacy 

evaluation


Project background


The CD3e molecule, epsilon encoded by CD3E gene is a polypeptide, which together with CD3-gamma, -delta and -zeta, and the T-cell receptor alpha/beta and gamma/delta heterodimers, forms the T cell receptor-CD3 complex. The CD3 complex, a common surface marker on T cells, has important functions not only as an essential component in forming the T cell receptor (TCR)-CD3  complex, but also as an external signal transducer; therefore, the CD3 complex is one of the target molecules to modulate T cell functions. The epsilon polypeptide plays an essential role in T-cell development.


Targeting strategy



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mRNA expression analysis


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Figure 1.RT-PCR analysis of B-hCD3E gene

The hCD3E mRNA was detected in splenocytes isolated from the homozygous B-hCD3E mice.

Protein expression analysis


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Figure 2. Splenocytes from both wild type (WT) C57BL/6 and homozygous B-hCD3E mice were analyzed by flow cytometry.

Results: Mouse CD3+ cells were detectable in the WT C57BL/6 mice, while human CD3+ cells were detectable in the homozygous B-hCD3E mice.


Weight of thymuses and spleens


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Figure 3. Weight of thymuses and spleens

Thymus and spleen were weighed after isolated from C57BL/6 and B-hCD3E mice(n=6). The result shows there is no significant difference regarding the spleen weight between B-hCD3E mice and C57BL/6 mice. However, the thymus weight in B-hCD3E is lower than that in the wild type C57BL/6 mice.


Analysis of lymphocyte subpopulation in B-hCD3E mice


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Figure 4. Analysis of lymphocyte subpopulation in thymus

Thymocytes were isolated from C57BL/6 and B-hCD3E mice(n=4). The proportion of lymphocyte subpopulation was tested by flow cytometry. As a result, the expression profile of lymphocyte subpopulation in homozygous B-hCD3E mice is similar to that in the C57BL/6 mice, indicating that T cell differentiation is not affected by the humanization of CD3E.


Analysis of lymphocyte subpopulation in B-hCD3E mice


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Figure 5. Analysis of lymphocyte subpopulation in spleen, peripheral blood and lymph node

Lymphocytes were isolated from spleen, peripheral blood and lymph node in C57BL/6 and B-hCD3E mice(n=4). The proportion of lymphocyte subpopulation was tested by flow cytometry. As a result, the expression profile of lymphocyte subpopulation in homozygous B-hCD3E mice is similar to that in the C57BL/6 mice, indicating that T cell differentiation is not affected by the humanization of CD3E.


Analysis of T cell activation stimulated with anti-CD3 antibody in vitro


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Figure 6. T cells activity detection

T cells (2.5×106) were isolated from the splenocytes of C57BL/6 and B-hCD3E mice (n=4), and incubated in the presence of anti-CD3 antibody (C57BL/6,anti-mCD3; B-hCD3E, anti-hCD3) with anti-mouse CD28 for 24h, 48h and 72h. T cell proliferation were tested using flow cytometry. As a result, the T cell activation in B-hCD3E mice is specifically up-regulated by anti-hCD3 antibody, similar to the activation level as shown in the C57BL/6 treated with anti-mCD3 antibody.


Analysis of T cell activation stimulated with anti-CD3 antibody in vitro



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Figure 7. Detection of cytokine levels

T cells (2.5×106) were isolated from the splenocytes of C57BL/6 and B-hCD3E mice (n=4), incubated in the presence of anti-CD3 antibody (C57BL/6, anti-mCD3; B-hCD3E, anti-hCD3) and anti-mCD28 for 24h, 48h and 72h. IFN-γ and IL-2 productions were then tested using ELISA method. As a result, there is no significant difference regarding the production of IFN-γ and IL-2 between B-hCD3E mice and C57BL/6 mice.






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