RAGE (receptor for advanced glycation end products) is expressed in more than 300 cell types, including lung tissue, and is involved in signaling pathways associated with proliferation, inflammation, and tumor progression. The AGE–RAGE axis activates intracellular signaling cascades that promote ROS generation and NF-κB activation, contributing to chronic degenerative and neoplastic diseases.

In RAGE humanized mice (B-hRAGE mice plus), exons 1–11 including the 3’UTR of the mouse Rage gene, which encode the full-length protein, were replaced by the corresponding human RAGE exons 1–11 including the human 3’UTR. This design enables expression of human RAGE in place of the endogenous mouse protein.

Validation data demonstrate species-specific expression of RAGE in this model. Mouse Rage mRNA and protein are detectable only in wild-type C57BL/6 mice, whereas human RAGE mRNA and protein are exclusively detectable in homozygous RAGE humanized mice (B-hRAGE mice plus). These findings support the utility of this model for in vivo pharmacological evaluation of human RAGE-targeted therapeutics.

Key Advantages

• Species-specific target expression: Human RAGE mRNA and protein are exclusively detectable in RAGE humanized mice (B-hRAGE mice plus), while mouse Rage expression is restricted to wild-type controls.
• Physiologically relevant tissue distribution: Human RAGE expression is detectable in lung tissue and serum of RAGE humanized mice (B-hRAGE mice plus), with higher mRNA levels observed in lung compared to blood.
• Validated for in vivo therapeutic studies: Human RAGE-targeted nucleic acid drugs reduce RAGE mRNA and protein levels in lung tissue of RAGE humanized mice (B-hRAGE mice plus), demonstrating suitability for in vivo efficacy and pharmacodynamic evaluation.

Validation

• Human RAGE mRNA expression: RT-PCR and RT-qPCR analyses confirmed that human RAGE mRNA is detectable in lung and blood of RAGE humanized mice (B-hRAGE mice plus), but absent in wild-type mice. Mouse Rage mRNA is detectable only in wild-type controls.
• Human RAGE protein expression: Western blot and ELISA analyses demonstrated that human RAGE protein is detectable in lung tissue and serum of RAGE humanized mice (B-hRAGE mice plus), while mouse RAGE protein is detected only in wild-type mice.
• Efficacy validation: Administration of human RAGE-targeted nucleic acid drugs in RAGE humanized mice (B-hRAGE mice plus), reduced human RAGE mRNA levels at day 7 and decreased human RAGE protein levels in lung tissue at day 28, supporting the model's application for in vivo drug evaluation.

Applications

This product is used for pharmacodynamics and safety evaluation of drugs for various chronic degenerative diseases and neoplastic diseases

In RAGE humanized mice plus (B-hRAGE Mice Plus), exons 1–11 of the mouse Rage gene, which encode the full-length protein and include the native 3′UTR, are replaced with human RAGE exons 1–11 together with the human 3′UTR. This genomic replacement enables expression of human RAGE in place of mouse Rage.

Species-specific analysis of RAGE mRNA expression was performed in lung tissues from wild-type C57BL/6 mice and homozygous RAGE humanized mice (B-hRAGE mice plus) by RT-PCR. Following reverse transcription, PCR was conducted using primers specific for mouse Rage and human RAGE transcripts. Mouse Rage mRNA was detectable only in wild-type C57BL/6 mice (+/+), whereas human RAGE mRNA was exclusively detectable in homozygous RAGE humanized mice (B-hRAGE mice plus) and absent in wild-type controls. These results confirm species-specific gene replacement and transcriptional specificity in this humanized mouse model.

Species-specific analysis of RAGE mRNA expression was performed in wild-type C57BL/6N mice and heterozygous homozygous RAGE humanized mice (B-hRAGE mice plus) by RT-qPCR. Lung and blood RNA were isolated from wild-type C57BL/6N mice (+/+) (n=3, 9-week-old) and homozygous RAGE humanized mice (B-hRAGE mice plus, n=5, 9-week-old). cDNA libraries were generated by reverse transcription, followed by real-time quantitative PCR using human RAGE-specific primers and Applied Biosystems SYBR. Human RAGE mRNA was detectable in B-hRAGE mice plus but was not detectable in wild-type C57BL/6N mice. In homozygous RAGE humanized mice (B-hRAGE mice plus) , RAGE mRNA levels in lung tissue were higher than those in blood.

Western blot analysis of RAGE protein expression in lung tissues from RAGE humanized mice (B-hRAGE mice plus). Lung tissue lysates were collected from wild-type C57BL/6N mice (+/+), homozygous RAGE humanized mice (B-hRAGE mice plus, H/H), and RAGE KO mice, followed by western blot analysis using species-specific anti-RAGE antibodies. mRAGE protein was detected in wild-type mice, whereas hRAGE protein was detected in RAGE humanized mice (B-hRAGE mice plus). GAPDH was used as a loading control.

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

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

The inhibitory efficiency of RAGE-targeted nucleic acid drugs was evaluated in homozygous RAGE humanized mice (B-hRAGE mice plus). RAGE humanized mice (B-hRAGE mice plus, 5–6 weeks old, female) were randomly divided into six groups and administered either PBS or human RAGE-targeted nucleic acid drugs (provided by a client) at the indicated doses via intratracheal (i.t.) administration (50 μL/animal, single dose). Lung tissues were collected at day 7 (D7) and day 28 (D28) following administration. (A) Schematic representation of the dosing regimen and sample collection timeline. (B) Relative human RAGE mRNA expression levels in lung tissue at day 7 after treatment. (C) Human RAGE protein levels in lung tissue at day 28 after treatment. Values are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA, *P < 0.05.

Q1: What are RAGE humanized mice (B-hRAGE mice plus)?

RAGE humanized mice (B-hRAGE Mice Plus) are genetically engineered mice in which exons 1–11 of the mouse Rage gene, including the native 3′UTR, are replaced with the corresponding human RAGE exons 1–11 and the human 3′UTR, enabling expression of the full-length human RAGE protein.

Q2: How is human RAGE expression validated in RAGE humanized mice (B-hRAGE mice plus)?

RT-PCR, RT-qPCR, Western blot, and ELISA analyses demonstrate that mouse RAGE mRNA and protein are detectable only in wild-type mice, whereas human RAGE is detectable exclusively in RAGE humanized mice (B-hRAGE mice plus) .

Q3: In which tissues is human RAGE expression validated in RAGE humanized mice (B-hRAGE mice plus)?

Human RAGE mRNA and protein are detectable in lung tissue and serum of RAGE humanized mice (B-hRAGE mice plus), with higher mRNA levels observed in lung compared to blood.

Q4: What are the main applications of RAGE humanized mice (B-hRAGE mice plus)?

RAGE humanized mice (B-hRAGE mice plus) are used for pharmacodynamic and safety evaluation of therapeutics targeting chronic degenerative diseases and neoplastic diseases, supporting in vivo assessment of RAGE-targeted interventions.