Basic Information
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Targeting strategy
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Gene targeting strategy for 112518. IL12RB1: the extracellular region sequences of mouse Il12rb1 were replaced by human IL12RB1 counterpart gene sequences. IL12RB2: the extracellular and transmembrane region sequences of mouse Il12rb2 were replaced by human IL12RB2 counterpart gene sequences.
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mRNA expression analysis
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Strain specific analysis of IL12RB1 mRNA expression by RT-PCR. Spleen RNA were isolated from wild-type C57BL/6 mice (WT), homozygous B-hIL12RB1/hIL12RB2 mice plus and homozygous B-hIL12RB1/hIL12RB2 mice ad, then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse/human strain specific IL12RB1 primers. Human IL12RB1 mRNA exclusively detectable in homozygous B-hIL12RB1/hIL12RB2 mice plus and B-hIL12RB1/hIL12RB2 mice ad but not in wild-type mice. Mouse Il12rb1 was only detectable in wild-type C57BL/6 mice but not in humanized mice.
IL12RB1 and IL12RB2 gene expression in wild-type C57BL/6 mice and homozygous B-hIL12RB1/hIL12RB2 mice by RT-qPCR. Splenocytes were collected from wild-type C57BL/6 mice (2 female, 1 male, n=3, 14-week-old) and homozygous B-hIL12RB1/hIL12RB2 mice plus & ad (male, n=3, 14-week-old). Then cDNA libraries were synthesized by reverse transcription, followed by PCR with human-specific IL12RB1 and IL12RB2 primers. Values are expressed as mean ± SEM. Significance was determined by unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001.
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Function 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 of wild-type C57BL/6 mice, homozygous B-hIL12RB1/hIL12RB2 mice plus, homozygous B-hIL12RB1/hIL12RB2 mice ad and homozygous B-hIL12A/hIL12B/hIL12RB1/hIL12RB2 mice. The production of IFN-γ in supernatants were assessed after incubation with rm/hIL-12 in combination with bead-associated 0.4 μg/mL anti-mCD3e and 0.8 μg/mL anti-mCD28 antibodies for 48 hours. Mouse IFN-γ were increased after responsiveness to mIL-12 in humanized mice and wild-type mice. While, only hIL-12 induced mouse IFN-γ increase in humanized mice. Mice information: 1) humanized mice, male, 3 mice/group, 14-week-old. 2) wild-type C57BL/6 mice, 2 female and 1 male mice, 14-week-old.