• RAGE is expressed in more than 300 cells in addition to lung tissue, and studies have shown that tumors can increase the expression of this gene in a variety of cells.
• Exons 1-11 including 3’UTR of the mouse RAGE gene that encodes the full-length protein was replaced by human RAGE exons 1-11 including 3’UTR in B-hRAGE mice plus.
• Mouse RAGE protein was only detectable in wild-type C57BL/6 mice. Human RAGE protein was detectable in homozygous B-hRAGE mice plus but not in wild-type mice.
• Mouse RAGE mRNA was only detectable in wild-type C57BL/6 mice. Human RAGE mRNA was detectable in homozygous B-hRAGE mice plus but not in wild-type mice.

Gene targeting strategy for B-hRAGE mice plus. Exons 1-11 including 3’UTR of the mouse RAGE gene that encodes the full-length protein was replaced by human RAGE exons 1-11 including 3’UTR in B-hRAGE mice plus.

Strain specific analysis of RAGE mRNA expression in wild-type C57BL/6 mice and B-hRAGE mice plus by RT-PCR. Lung RNA were isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hRAGE mice plus (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human RAGE primers. Mouse RAGE mRNA was detectable only in wild-type C57BL/6 mice. Human RAGE mRNA was detectable only in homozygous B-hRAGE mice plus but not in wild-type mice.

Strain specific analysis of RAGE expression in wild-type C57BL/6N mice and heterozygous humanized B-hRAGE mice plus by RT-qPCR. Blood, and blood RNA were isolated from wild-type C57BL/6N mice (+/+) (n=3, 9-week-old) and B-hRAGE mice plus (n=5, 9-week-old), then cDNA libraries were synthesized by reverse transcription, followed by real-time quantitative PCR with RAGE primers. The mRNA levels of RAGE was in detectable in B-hRAGE mice plus with Applied Biosystems SYBR, but not wild-type C57BL/6N mice. The mRNA levels of RAGE in lung from B-hRAGE mice plus was higher than that in blood from B-hRAGE mice plus.

Western blot analysis of RAGE protein expression in homozygous B-hRAGE mice plus. Lung tissue lysates were collected from wild-type C57BL/6N mice (+/+), homozygous B-hRAGE mice plus (H/H) and B-Rage KO mice(RM), and then analyzed by western blot with species-specific anti-RAGE antibody. 40 μg total proteins were loaded for western blotting analysis. RAGE was detected in lung.

Strain specific RAGE expression analysis in wild-type C57BL/6N mice and heterozygous humanized B-hRAGE mice plus by ELISA. Serum was collected from wild-type C57BL/6N mice (+/+) (male, n=3, 7-week-old) and heterozygous B-hRAGE mice plus (H/+) (male, n=3, 15-week-old). Expression level of mouse and human RAGE were analyzed by ELISA (Human RAGE Quantikine ELISA Kit: R&D, DRG00). Human RAGE was exclusively detectable in heterozygous B-hRAGE mice plus(n=3). Values are expressed as mean ± SEM.

Strain specific RAGE expression analysis in wild-type C57BL/6N mice and heterozygous humanized B-hRAGE mice plus by ELISA. Lung homogenates were collected from wild-type C57BL/6N mice (+/+) (male, n=3, 7-week-old) and heterozygous B-hRAGE mice plus (H/+) (male, n=3, 15-week-old). Expression level of mouse and human RAGE were analyzed by ELISA (Human RAGE Quantikine ELISA Kit: R&D, DRG00). Human RAGE was exclusively detectable in heterozygous B-hRAGE mice plus(n=3). Values are expressed as mean ± SEM.