C57BL/6N-Igf1rtm5(IGF1R)Bcgen/Bcgen • 111974
Key Advantages
Validation
Application
In B-hIGF1R mice plus, a chimeric CDS encoding the mouse Igf1r signal peptide, human IGF1R extracellular domain, human IGF1R transmembrane domain, and mouse Igf1r cytoplasmic domain followed by the mouse Igf1r 3' UTR was inserted after mouse Igf1r exon 2 to replace exon 2 of the mouse Igf1r gene.
This targeting strategy drives chimeric IGF1R expression under the endogenous mouse Igf1r promoter while disrupting endogenous mouse Igf1r transcription and translation. B-hIGF1R mice plus therefore provide a humanized IGF1R platform for evalsuating species-specific anti-IGF1R antibodies and human IGF1R-targeted therapeutics.
Strain-specific analysis of IGF1R mRNA expression was performed in wild-type C57BL/6N mice, B-hIGF1R mice, and B-hIGF1R mice plus by RT-PCR.
Kidney RNA was isolated from wild-type C57BL/6N mice (+/+), homozygous B-hIGF1R mice (H/H), and homozygous B-hIGF1R mice plus (H/H). cDNA libraries were synthesized by reverse transcription followed by PCR with IGF1R primers. Mouse Igf1r mRNA was detectable in wild-type C57BL/6N mice. Human IGF1R mRNA was detectable in homozygous B-hIGF1R mice and homozygous B-hIGF1R mice plus, but not in wild-type mice. No-RT indicates no reverse transcription.
Western blot analysis of IGF1R protein expression was performed in wild-type C57BL/6N mice and homozygous B-hIGF1R mice plus.
Heart, liver, spleen, lung, kidney, brain, and colon tissues were collected from wild-type C57BL/6N mice (+/+) and homozygous B-hIGF1R mice plus (H/H) and analyzed by western blot using anti-IGF1R antibody (CST, 3027S). A total of 40 μg protein was loaded for western blot analysis, and GAPDH was used as an internal control. IGF1R was detectable in both C57BL/6N and B-hIGF1R mice plus because the antibody cross-reacts between human and mouse IGF1R. Relative IGF1R protein levels were normalized to GAPDH.
Immunofluorescence analysis of IGF1R expression in isolated brain micro-vessels was performed in wild-type C57BL/6 mice and homozygous B-hIGF1R mice plus.
Brain micro-vessels were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hIGF1R mice plus (H/H) and analyzed by immunofluorescence using anti-mCD31 antibody and anti-hIGF1R antibody. Human IGF1R was detectable in brain micro-vessels of homozygous B-hIGF1R mice plus, but not in wild-type mice.
Immunofluorescence analysis of IGF1R expression in isolated brain micro-vessels was performed using an anti-IGF1R antibody that cross-reacts between human and mouse IGF1R.
Brain micro-vessels from wild-type C57BL/6 mice (+/+) and homozygous B-hIGF1R mice plus (H/H) were stained with anti-mCD31 antibody and anti-IGF1R antibody (CST, 3027S). IGF1R signal was detectable in both wild-type C57BL/6 mice and homozygous B-hIGF1R mice plus because the antibody recognizes both human and mouse IGF1R.
Strain-specific IGF1R expression in wild-type C57BL/6N mice and homozygous B-hIGF1R mice plus was analyzed by flow cytometry.
Brain cells were collected from wild-type C57BL/6N mice (+/+) and homozygous B-hIGF1R mice plus (H/H) (male, 7-week-old, n=1), and analyzed with anti-human IGF1R antibody (BioLegend, 351805). Human IGF1R was detectable in total brain cells and brain endothelial cells of homozygous B-hIGF1R mice plus, supporting use of B-hIGF1R mice plus for brain endothelial IGF1R and CNS delivery studies.
Frequency of leukocyte subpopulations in spleen was analyzed by flow cytometry in B-hIGF1R mice plus.
Splenocytes were isolated from wild-type C57BL/6N mice (male, n=3, 7-week-old) and homozygous B-hIGF1R mice plus (male, n=3, 7-week-old). Flow cytometry was used to evalsuate T cells, B cells, NK cells, dendritic cells, neutrophils, monocytes, macrophages, CD4+ T cells, CD8+ T cells, and Tregs. Leukocyte subpopulation frequencies in B-hIGF1R mice plus were similar to wild-type C57BL/6N mice. Blood and lymph node immune cell profiles were also comparable (data not shown). Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.
Western blot analysis of phospho-IGF1R/AKT protein expression was performed to evalsuate functional IGF1R signaling in B-hIGF1R mice plus.
Kidney cells from wild-type C57BL/6N mice (+/+), homozygous B-hIGF1R mice (H/H), and homozygous B-hIGF1R mice plus (H/H) were cultured with figitumumab (1 μg/mL) and/or recombinant human IGF1 protein (200 ng/mL). Cells were analyzed by western blot using anti-IGF1R, anti-phospho-IGF1R, and anti-phospho-AKT antibodies. IGF1 induced IGF1R phosphorylation in wild-type and B-hIGF1R mice plus, while figitumumab-analog suppressed IGF1-induced activation of humanized IGF1R in B-hIGF1R mice plus. Phosphorylation of endogenous mouse IGF1R remained unaffected, reflecting the species-related binding profile of figitumumab.
Complete blood count (CBC) was performed to evalsuate hematological characteristics of B-hIGF1R mice plus.
Blood was collected from male and female C57BL/6JNifdc mice and B-hIGF1R mice plus (n=10, 6-week-old) for CBC analysis. No differences were observed in measured hematology parameters between C57BL/6JNifdc mice and B-hIGF1R mice plus, indicating that humanization of IGF1R does not alter blood cell composition or morphology. Values are expressed as mean ± SEM.
Blood chemistry tests were performed to evalsuate serum biochemistry in B-hIGF1R mice plus.
Serum from male and female C57BL/6JNifdc mice and B-hIGF1R mice plus (n=10, 6-week-old) was collected for biochemistry analysis. No differences were observed in measured blood chemistry parameters between male C57BL/6JNifdc mice and male B-hIGF1R mice plus, indicating that humanization of IGF1R does not change blood biochemistry. Values are expressed as mean ± SEM.
Body weight growth curve analysis was performed in B-hIGF1R mice plus.
Ten male and ten female homozygous B-hIGF1R mice plus were randomly selected, and body weight was collected once per week. The minimum and maximum weights in the table are calculated as average ± SD. The body weight growth curve of B-hIGF1R mice plus was consistent with that of wild-type mice, supporting normal growth and development after IGF1R humanization.
Efficacy of anti-IGF1R antibody was evalsuated in high-fat diet (HFD)-induced B-hIGF1R mice plus.
B-hIGF1R mice plus were fed a high-fat diet to induce obesity. Body weight change was monitored after HFD induction and after treatment with anti-IGF1R antibody provided by a client. Anti-IGF1R antibody significantly inhibited HFD-induced body weight gain, as shown by lower body weight and reduced body weight change in the treatment group compared with the HFD-vehicle group. ***p < 0.001. Values are expressed as mean ± SEM.
Note: This experiment is a collaborative validation project with the client.
Metabolic efficacy of anti-IGF1R antibody was evalsuated in HFD-induced B-hIGF1R mice plus.
B-hIGF1R mice plus were fed a high-fat diet to induce obesity and treated with anti-IGF1R antibody provided by a client. Food intake, blood glucose, intraperitoneal glucose tolerance test (IPGTT, 15% D-glucose), and insulin tolerance test (ITT, 0.5 U/kg) were analyzed after treatment. Anti-IGF1R antibody affected food intake and metabolic response in HFD-induced B-hIGF1R mice plus. Values are expressed as mean ± SEM. Significance was determined by ordinary one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.
Note: This experiment is a collaborative validation project with the client.
Q1: What are B-hIGF1R mice plus?
B-hIGF1R mice plus are target-humanized mice expressing a chimeric IGF1R protein with human IGF1R extracellular and transmembrane regions, enabling evalsuation of human IGF1R-targeted therapeutics in vivo.
Q2: Why is IGF1R an important therapeutic target?
IGF1R is a receptor tyrosine kinase involved in IGF signaling, cell growth, survival, metabolism, and transformation, making IGF1R relevant to oncology, metabolic disease, obesity, diabetes, and CNS delivery research.
Q3: How was IGF1R expression validated in B-hIGF1R mice plus?
Human IGF1R mRNA was validated by RT-PCR, IGF1R protein was detected by western blot across multiple tissues, and human IGF1R expression was confirmed in brain endothelial cells and brain micro-vessels.
Q4: Can B-hIGF1R mice plus be used for functional antibody studies?
Yes. Figitumumab-analog suppressed IGF1-induced activation of humanized IGF1R in B-hIGF1R mice plus, supporting functional evalsuation of human IGF1R-targeted antibodies.
Q5: What are the main applications of B-hIGF1R mice plus?
Applications include anti-IGF1R antibody efficacy studies, IGF1R pathway pharmacology, obesity and metabolic disease research, oncology studies, brain endothelial IGF1R research, and preclinical safety evalsuation.
