Basic Information
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Targeting strategy
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Gene targeting strategy for B-hPD-1 plus/hPD-L1/hPD-L2 mice.
Human PD-1 gene encoding the extracellular region and mouse PD-1 gene encoding the transmembrane and cytoplasmic region were inserted after the initiation codon ATG of mouse PD-1 gene in B-hPD-1 mice plus. The exon 3 of mouse Pdl1 gene that encodes the IgV domain was replaced by human PD-L1 exon 3 in B-hPD-L1 mice. B-hPD-1 plus/hPD-L1 mice was developed by cross-mating the B-hPD-1 mice plus and the B-hPD-L1 mice together. The exons 3-4 of human PD-L2 encoding the extracellular region were knocked-in to replace the exon 3-4 sequences of mouse PD-L2 in B-hPD-1 plus/hPD-L1 mice. Thus, the B-hPD-1 plus/hPD-L1/hPD-L2 mice were obtained.
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mRNA expression analysis in humanized B-hPD-1 plus/hPD-L1/hPD-L2 mice
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Species specific analysis of PD-L2 gene expression in wild-type C57BL/6 mice and homozygous humanized B-hPD-1 plus/hPD-L1/hPD-L2 mice by RT-PCR. Thymus was collected from wild-type C57BL/6 mice (+/+) and homozygous B-hPD-1 plus/hPD-L1/hPD-L2 mice (H/H). Mouse PD-L2 mRNA was detectable only in wild-type C57BL/6 mice. Human PD-L2 mRNA was only detectable in homozygous B-hPD-1 plus/hPD-L1/hPD-L2 mice, but not in wild-type C57BL/6 mice.
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Protein expression analysis in spleen
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Strain specific PD-L2 expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hPD-1 plus/hPD-L1/hPD-L2 mice by flow cytometry. Bone marrow was collected from wild-type C57BL/6 mice (+/+) and homozygous B-hPD-1 plus/hPD-L1/hPD-L2 mice (H/H) and cultured in vitro to induce the bone marrow-derived dendritic cells (BMDCs). The successfully induced BMDCs were stimulated with or without LPS in vitro. Expression of PD-L2 on BMDCs was analyzed by flow cytometry. Mouse PD-L2 was only detectable in wild-type C57BL/6 mice. Human PD-L2 was exclusively detectable in homozygous B-hPD-1 plus/hPD-L1/hPD-L2 mice, but not in wild-type C57BL/6 mice.
