Basic Information
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Description
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- CRBN interacts with the DNA damage-binding protein-1 (DDB1), Cullin 4 (Cul4A or Cul4B), and regulator of Cullins1 (RoC1) to form the functional E3 ubiquitin ligase complex. In this complex, CRBN functions as a substrate receptor of E3 ubiquitin ligase complex and targets proteins for proteolysis through a ubiquitin-proteasome pathway.
- The CDS of human CRBN gene that encodes the full-length protein was inserted into the mouse Crbn exons 2-3. The B-hCRBN mice will express the human CRBN protein, while mouse Crbn will no longer be expressed.
- The cortex, liver, lung, kidney, spleen were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hCRBN mice (H/H), and then analyzed by western blot with anti-CRBN antibody. Mouse CRBN was detectable in wild-type mice. Human CRBN was exclusively detectable in homozygous B-hCRBN mice. The antibody is cross-recognize both human and mouse CRBN.
- Tumor cell lines inoculated in B-hCRBN mice can be used to study the in vivo efficacy and safety evaluation of CRBN small molecule drugs, molecular glue drugs based on CRBN, or PROTAC drugs based on CRBN.
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Targeting strategy
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Gene targeting strategy for B-hCRBN mice. The CDS of human CRBN gene that encodes the full-length protein was inserted into the mouse Crbn exons 2-3. The B-hCRBN mice will express the human CRBN protein, while mouse Crbn will no longer be expressed.
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mRNA expression analysis
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Species specific analysis of CRBN gene expression in wild-type C57BL/6 mice and homozygous B-hCRBN mice by RT-PCR. Cortex was collected from wild-type C57BL/6 mice (+/+) and homozygous B-hCRBN mice (H/H). Mouse Crbn mRNA was detectable in wild-type C57BL/6 mice. Human CRBN mRNA was only detectable in homozygous B-hCRBN mice, but not in wild-type mice.
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Protein expression analysis
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Strain specific CRBN expression analysis in homozygous B-hCRBN mice by Western blot. The cortex, liver, spleen, lung, kidney were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hCRBN mice (H/H), and then analyzed by western blot with anti-CRBN antibody. Mouse CRBN was detectable in wild-type mice. Human CRBN was exclusively detectable in homozygous B-hCRBN mice. The anti-CRBN antibody was cross-reactive between human and mouse.
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Functional analysis
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Naïve CD4+ T cells from B-hCRBN mice have increased production of IL2 when treated with Lenalidomide, but no change in cells of wild-type mice. Naïve CD4+ T cells were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hCRBN mice (H/H), then stimulated with DMSO or Lenalidomide in vitro for 24 hours. The supernatants were collected, and IL2 production was measured by ELISA. The results show that Lenalidomide significantly up-regulates the production of IL2 in B-hCRBN mice, but not in wild-type C57BL/6 mice.
