Basic Information
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Gene targeting strategy
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Gene targeting strategy for B-hIL12A/hIL12B/hIL12RB1/hIL12RB2 mice.
The exons 1-7 of mouse Il12a gene that encode the full length coding sequence were replaced by human IL12A exons 1-7 in B-hIL12A/hIL12B/hIL12RB1/hIL12RB2 mice.
The exons 2-8 of mouse Il12b gene that encode the full length coding sequence and 3’UTR were replaced by human IL12B exons 2-8 in B-hIL12A/hIL12B/hIL12RB1/hIL12RB2 mice.
The extracellular and transmembrane region coding sequences of human IL12RB1 gene plus the mouse Il12rb1 cytoplasmic coding sequences were inserted into the exon1 of B-hIL12A/hIL12B/hIL12RB1/hIL12RB2 mice.
The extracellular region coding sequences of human IL12RB2 gene plus the mouse Il12rb2 transmembrane and cytoplasmic coding sequences were inserted into the exon2 of B-hIL12A/hIL12B/hIL12RB1/hIL12RB2 mice.
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mRNA expression analysis
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Strain specific analysis of IL12A, IL12B, IL12RB1 and IL12RB2 gene expression in B-hIL12A/hIL12B/hIL12RB1/hIL12RB2 mice by RT-PCR. Mouse Il12a was detectable in splenocytes of wild-type mice (+/+). Human IL12A was detectable only in homozygous B-hIL12A/hIL12B/hIL12RB1/hIL12RB2 mice but not in wild-type mice. Mouse Il12b, Il12rb1 and Il12rb2 were detectable in thymocytes of wild-type mice (+/+). Human IL12B, IL12RB1 and IL12RB2 were only detectable in homozygous B-hIL12A/hIL12B/hIL12RB1/hIL12RB2 mice but not in wild-type mice.
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Protein expression analysis
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Strain specific IL12p70 (IL-12A (p35) and IL-12B (p40) active heterodimer referred to as ‘p70’) expression analysis in homozygous B-hIL12A/hIL12B/hIL12RB1/hIL12RB2 mice by ELISA.
Serum was collected in wild-type (+/+) and homozygous B-hIL12A/hIL12B/hIL12RB1/hIL12RB2 mice (H/H) stimulated with anti-mCD3ε and anti-mCD28 in vivo (n=3), and analyzed by ELISA with species-specific IL12 ELISA kit. Mouse IL12p70 was detectable in wild-type mice. Human IL12p70 was exclusively detectable in homozygous B-hIL12A/hIL12B/hIL12RB1/hIL12RB2 mice but not in wild-type mice. ND: Not detectable.
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Functional analysis
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IL12 induced the IFN-γ production in CD4+ T cells sorted from splenocytes.
CD4+ T cells were sorted from the splenocytes in the wild-type (+/+) and homozygous B-hIL12A/hIL12B/hIL12RB1/hIL12RB2 mice (H/H) (n=3), the production of IFN-γ in supernatants were assessed after 48 h of incubation with 0.02 or 0.2 μg/mL rhIL-12 in combination with bead-associated CD3 and CD28 mAbs, under the condition in the panel. For comparison, with 0.01 or 0.05 μg/mL of rmIL-12 as control. Mouse IFN-γ were both increased after responsiveness to mIL-12 in wild-type mice and hIL-12 in homozygous B-hIL12A/hIL12B/hIL12RB1/hIL12RB2 mice (H/H). The humanized mice were successfully constructed.
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Analysis of leukocytes cell subpopulation in spleen
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Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hIL12A/hIL12B/hIL12RB1/hIL12RB2 mice (n=3, 6-week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for the CD45+ population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hIL12A/hIL12B/hIL12RB1/hIL12RB2 mice were similar to those in the C57BL/6 mice, demonstrating that the humanization does not change the overall development, differentiation or distribution of these cell types in spleen. Values are expressed as mean ± SEM.
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Analysis of T cell subpopulation in spleen
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Analysis of spleen T cell subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hIL12A/hIL12B/hIL12RB1/hIL12RB2 mice (n=3, 6-week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live CD45+ cells were gated for TCRβ+ T cell population and used for further analysis as indicated here. B. Results of FACS analysis. The percent of CD4+ T cells, CD8+ T cells and Tregs in homozygous B-hIL12A/hIL12B/hIL12RB1/hIL12RB2 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hIL12A/hIL12B/hIL12RB1/hIL12RB2 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in spleen. Values are expressed as mean ± SEM.
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Blood tests
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Complete blood count (CBC). Blood from female C57BL/6 and B-hIL12A/hIL12B/hIL12RB1/hIL12RB2 mice (n=6, 7 week-old) was collected and analyzed for CBC. The measurements of B-hIL12A/hIL12B/hIL12RB1/hIL12RB2 mice were similar to that in C57BL/6 mice, indicating that humanization does not change blood cell composition and morphology. Values are expressed as mean ± SEM.
Blood chemistry tests in wild-type and humanized B-hIL12A/hIL12B/hIL12RB1/hIL12RB2 mice. Serum from the C57BL/6 and B-hIL12A/hIL12B/hIL12RB1/hIL12RB2 mice (n=6, 7 week-old) was collected and analyzed for levels of indicators. The measurements of B-hIL12A/hIL12B/hIL12RB1/hIL12RB2 mice were similar to that in C57BL/6 mice, indicating that humanization does not change the health of related tissues, such as liver. Values are expressed as mean ± SEM.
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Poster
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AACR 2022: B-hIL12A/hIL12B/hIL12RB1/hIL12RB2: A Novel Animal Model for Generation of IL12 Therapies
