Description

• 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.
• CRBN were detectable in both wild-type mice and homozygous B-hCRBN mice. The anti-CRBN antibody was cross-reactive between human and mouse.
• Mouse Crbn mRNA was detectable in cortex of wild-type mice (+/+). Human CRBN mRNA was detectable in B-hCRBN mice (H/H) but not in wild-type mice.
• Naïve CD4+ T cells from B-hCRBN mice have increased production of IL-2 when treated with Lenalidomide, but no change in cells of wild-type mice.
• The body weight, complete blood count and blood biochemistry of male and female B-hCRBN mice were analyzed. The main organs were dissected, weighed and analyzed by H&E staining. No obvious abnormalities were found in all the organs detected (brain, heart, lung, liver, spleen, stomach, small intestine, colon, kidney, uterus, ovary and testis).
• 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.

Targeting strategy

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.

mRNA expression analysis in humanized B-hCRBN mice

Strain specific analysis of CRBN mRNA expression in wild-type C57BL/6 mice and homozygous B-hCRBN mice by RT-PCR. Heart, liver, spleen, lung, kidney, small intestine, colon and cortex RNA was isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hCRBN mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human CRBN primers. Mouse Crbn mRNA was detectable in wild-type C57BL/6 mice. Human CRBN mRNA was detectable only in homozygous B-hCRBN mice but not in 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.

Hematology analysis

Complete blood count (CBC) of C57BL/6 and B-hCRBN mice. Values are expressed as mean ± SD.

Biochemistry analysis

Biochemical test of C57BL/6 and B-hCRBN mice. Values are expressed as mean ± SD.

Gross anatomy of female and male C57BL/6 mice

The organs of female and male C57BL/6 mice (10-week-old, n=10).

Gross anatomy of female and male B-hCRBN mice

The organs of female and male B-hCRBN mice (10-week-old, n=10).

Organ weight

Average weight of the main organs of B-hCRBN mice. Values are expressed as mean ± SD.

H&E staining in C57BL/6

Histopathological analysis of organs in C57BL/6 mice. The main organs of C57BL/6 were isolated at 10 weeks of age and analyzed with H&E staining (male, n=10; female, n=10). Results showed that no obvious abnormalities were found in all of the organs (brain, heart, lung, liver, spleen, stomach, small intestine, colon, kidney, ovary, uterus and testis). Scale bar: 100 μm.

H&E staining in B-hCRBN mice

Histopathological analysis of organs in B-hCRBN mice. The main organs of B-hCRBN mice were isolated at 10 weeks of age and analyzed with H&E staining (male, n=10; female, n=10). Results showed that no obvious abnormalities were found in all of the organs (brain, heart, lung, liver, spleen, stomach, small intestine, colon, kidney, ovary, uterus and testis). Scale bar: 100 μm.