Rag2-KO/hCRBN

Fig.1 Detection of CRBN expression in small intestine, brain, muscle, thymus, liver and spleen by RT-PCR. Small intestine, brain, muscle, thymus, liver and spleen RNA was extracted from 10-week-old male wild-type C57BL/6 (WT)(n=1), homozygous Rag2-KO mice(HO)(n=1) and homozygous Rag2-KO/hCRBN mice (HO/HO)(n=2), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mRNA primers. Mouse Crbn mRNA (185 bp) was detectable in homozygous Rag2-KO mice and wild-type C57BL/6 mice. Human CRBN mRNA (374 bp) was detectable only in homozygous Rag2-KO/hCRBN mice but not in homozygous Rag2-KO mice and wild-type mice. 

Fig.2 Detection of Rag2 expression in small intestine, brain, muscle, thymus, liver and spleen by RT-PCR. Small intestine, brain, muscle, thymus, liver and spleen RNA was extracted from 10-week-old male wild-type C57BL/6 (WT)(n=1), homozygous Rag2-KO mice(HO)(n=1) and homozygous Rag2-KO/hCRBN mice (HO/HO)(n=2), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mRNA primers. Mouse Rag2 mRNA (163 bp) was detectable only in wild-type C57BL/6 mice but not in homozygous Rag2-KO mice and homozygous Rag2-KO/hCRBN mice. 

Fig.3 Detection of CRBN expression in Rag2-KO/hCRBN mice by WB. The spleen, liver, brain, skeletal muscle and small intestine tissue lysates were collected from 7-week-old male wild-type C57BL/6 mice and 7-week-old male homozygous Rag2-KO/hCRBN mice, and then analyzed by western blot with anti-CRBN antibody. CRBN was detectable in spleen, liver, brain, skeletal muscle and small intestine from both WT C57BL/6 and HO/HO Rag2-KO/hCRBN mice, as the antibody was cross-reactived between human and mouse CRBN. CRBN was detectable in spleen, liver, brain, skeletal muscle and small intestine from both WT C57BL/6 and HO/HO Rag2-KO/hCRBN mice, as the antibody was cross-reactived between human and mouse CRBN. 

Abbr. HO, homozygous; WT, wild type; PC, positive control, NIH/3T3 cells.

Fig.4 T and B cell frequencies in the spleen and peripheral blood of WT C57BL/6 mice, HO Rag2-KO and HO/HO Rag2-KO/hCRBN mice. The spleen and peripheral blood were collected from 8-week-old male wild-type C57BL/6 mice, 10-week-old male homozygous Rag2-KO/hCRBN mice and Rag2-KO mice. Deficiency of T and B cells in the spleen and peripheral blood of Rag2-KO/hCRBN mice.

Fig.5 The degradation of GSPT1, IKZF3 and CK1a by CC-885 in Rag2-KO/hCRBN and Rag2-KO mice in vivo. The skeletal muscle, heart and spleen were collected from 8-week-old female homozygous Rag2-KO/hCRBN mice and 6-week-old female homozygous Rag2-KO mice 6h post vehicle or CC-885 (5mg/kg) treatment, and then analyzed by western blot with anti-GSPT1 antibody, anti-IKZF3 antibody and anti-CK1a antibody. 

Fig.6 In vivo hepatotoxicity evaluation of CC-885 in Rag2-KO and Rag2-KO/hCRBN mice. 

Serum biochemistry was analyzed 6 h post-treatment with vehicle or CC-885 (5 mg/kg) in 8-week-old female homozygous Rag2-KO/hCRBN mice and 6-week-old female homozygous Rag2-KO mice.