Basic Information
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Targeting strategy
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Gene targeting strategy for B-hTFR1 mice. The exons 4-19 of mouse Tfr1 gene that encode the extracellular region were replaced by human TFR1 exons 4-19 in B-hTFR1 mice.
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mRNA expression analysis
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Strain specific analysis of TFR1 gene expression in wild type (WT) mice and B-hTFR1 mice by RT-PCR. Mouse Tfrc mRNA was detectable only in splenocytes of WT mice (+/+). Human TFRC mRNA was detectable only in homozygous B-hTFR1 mice (H/H) but not in WT mice (+/+).
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Protein expression analysis in erythroid cells
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Strain specific TFR1 expression analysis in homozygous B-hTFR1 mice by flow cytometry. Bone marrow was collected from wild type (WT) mice (+/+) and homozygous B-hTFR1 mice (H/H), and analyzed by flow cytometry with species-specific anti-TFR1 antibody. Mouse TFR1 was detectable in WT mice (+/+). Human TFR1 was exclusively detectable in homozygous B-hTFR1 mice (H/H) but not in WT mice (+/+).
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Protein expression analysis in brain
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Strain specific TFR1 expression analysis in homozygous B-hTFR1 mice by Immunofluorescence staining. Brain was collected from wild type (WT) mice and homozygous B-hTFR1 mice (female,8 week old) and processed into paraffin sections. The paraffin sections were stained using an species-specific anti-human TFR1 antibody (green). Brain tissues were co-stained with an anti-mouse CD31 antibody to visualize microvascular endothelial cells (red). Representative results of WT mice (A) and homozygous B-hTFR1 mice (B) are shown. The results indicated that human TFR1 was exclusively detectable on brain microvascular endothelium of homozygous B-hTFR1 mice, but not in WT mice.
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Protein expression analysis in bone marrow
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Strain specific TFR1 expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hTFR1 mice by flow cytometry. Bone marrow were collected from wild-type C57BL/6 mice and homozygous B-hTFR1 mice (male and female, 9-week-old, n=3). Protein expression was analyzed with anti-TFR1 antibody by flow cytometry. Mouse TFR1 was only detectable in wild-type C57BL/6 mice. Human TFR1 was exclusively detectable in homozygous B-hTFR1 mice, but not in wild-type C57BL/6 mice. The results showed that the expression level of human TFR1 in B-hTFR1 mice is gender independent, similar to the expression level of mouse TFR1 in wild-type C57BL/6 mice .
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Protein expression analysis in blood
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Strain specific TFR1 expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hTFR1 mice by flow cytometry. Blood cells were collected from wild-type C57BL/6 mice and homozygous B-hTFR1 mice (male and female, 9-week-old, n=3). Protein expression was analyzed with anti-TFR1 antibody by flow cytometry. Mouse TFR1 was only detectable in wild-type C57BL/6 mice. Human TFR1 was exclusively detectable in homozygous B-hTFR1 mice, but not in wild-type C57BL/6 mice. The results showed that the expression level of human TFR1 in B-hTFR1 mice is gender independent, similar to the expression level of mouse TFR1 in wild-type C57BL/6 mice .
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Protein expression profile of TFR1
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Immunohistochemical (IHC) analysis of TFR1 protein expression in wild-type C57BL/6 mice and homozygous humanized B-hTFR1 mice. Mouse tissues were collected from wild-type C57BL/6 mice and homozygous B-hTFR1 mice (female, 8-week-old, n=3). Protein expression was analyzed with anti-TFR1 antibody by IHC. Human TFR1 was detected in the heart, liver, spleen, lung, kidney, brain, colon and stomach of B-hTFR1 mice, which is similar to the expression pattern of mouse TFR1 in wild-type C57BL/6 mice. Red arrow: positive cells expressing TFR1.
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Frequency of leukocyte subpopulations in spleen
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Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 mice and homozygous B-hTFR1 mice (female, 6-week-old, n=3). A. Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequencies of T cell subpopulations. Percentages of T cells, B cells, NK cells, DCs, monocytes, macrophages, neutrophils, CD4+ T cells, CD8+ T cells and Tregs in B-hTFR1 mice were similar to those in C57BL/6 mice, demonstrating that humanization of TFR1 does not change the frequency or distribution of these cell types in spleen. Values are expressed as mean ± SEM.
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Frequency of leukocyte subpopulations in blood
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Frequency of leukocyte subpopulations in blood by flow cytometry. Blood cells were isolated from wild-type C57BL/6 mice and homozygous B-hTFR1 mice (female, 6-week-old, n=3). A. Flow cytometry analysis of the blood cells was performed to assess the frequency of leukocyte subpopulations. B. Frequencies of T cell subpopulations. Percentages of T cells, B cells, NK cells, DCs, monocytes, macrophages, neutrophils, CD4+ T cells, CD8+ T cells and Tregs in B-hTFR1 mice were similar to those in C57BL/6 mice, demonstrating that humanization of TFR1 does not change the frequency or distribution of these cell types in blood. Values are expressed as mean ± SEM.
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Frequency of leukocyte subpopulations in lymph node
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Frequency of leukocyte subpopulations in lymph node by flow cytometry. Leukocytes were isolated from wild-type C57BL/6 mice and homozygous B-hTFR1 mice (female, 6-week-old, n=3). A. Flow cytometry analysis of the leukocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequencies of T cell subpopulations. Percentages of T cells, B cells, NK cells, CD4+ T cells, CD8+ T cells and Tregs in B-hTFR1 mice were similar to those in C57BL/6 mice, demonstrating that humanization of TFR1 does not change the frequency or distribution of these cell types in lymph node. Values are expressed as mean ± SEM.
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Growth curve
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Growth curve of B-hTFR1 mice. Select mice aged 3 to 8 weeks, and randomly sample and weigh 50 males and 50 females from each age group. The minimum and maximum weights of the mice in the table are calculated as the average ± SD. The growth curve follows a normal distribution, with a 68% probability that random errors fall within the ± SD range.
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Hematology analysis
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Humanization of TFR1 does not alter hematological parameters. Values are expressed as mean ± SD.
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Biochemistry analysis
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Humanization of TFR1 does not alter various blood biochemical parameters. Values are expressed as mean ± SD.
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Hematoxylin and eosin staining of femur
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Hematoxylin and eosin (H&E) staining of femur. The Femur from wild-type C57BL/6 mice and B-hTFR1 mice (female, 10 week-old, n=10) were collected and analyzed by H&E staining. Representative results of WT mice (A) and homozygous B-hTFR1 mice (B) are shown, and no abnormalities were found in the bone marrow of both mice. The results indicated that Humanization of TFR1 does not change the normal morphology of mouse bone marrow.
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The uptake of anti-human TFR1 antibody in the brain tissue of B-hTFR1 mice
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B-hTFR1 mice were intravenously injected with AF680-conjugated control hIgG1 or anti human TFR1 antibodies ab1 and ab2 (provided by a client). After 2 days or 3 days post-injection, the mice were perfused and their brains were collected for analysis. (A) Mouse brain images under imaging system. (B) Fluorescence intensity of mouse brain under imaging system. The results indicate that the uptake of anti-human TFR1 antibody ab2 in the brain of B-hTFR1 mice was higher than that of anti-human TFR1 antibody ab1.
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In vivo PK evaluation of anti-human TFR1 BsAbs
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In vivo pharmacokinetic (PK) evaluation of anti-human TFR1 bispecific antibodies (BsAbs).B-hTFR1 mice were injected with control IgG (10 mpk) and anti-human TFR1 BsAbs (10.9 mpk) provided by a client via tail vein. Brain and serum were taken for in vivo PK evaluation. Brain concentrations(A), serum concentrations (B), and brain-to-serum ratio (C) of anti-human TFR1 BsAbs were quantified. As shown in panel, anti-human TFR1 BsAbs exhibited higher serum clearance and enhanced brain exposure after dose. The results confirmed that brain of B-hTFR1 mice enables uptake of an intravenously administered anti-human TFR1 BsAbs and B-hTFR1 mice provide a powerful preclinical model for in vivo evaluation of effective delivery of protein therapeutics to the central nervous system (CNS).Graphs represent mean ± SEM.
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Summary
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- mRNA expression analysis:
Mouse Tfrc mRNA was detectable only in splenocytes of WT mice (+/+). Human TFRC mRNA was detectable only in homozygous B-hTFR1 mice (H/H) but not in WT mice (+/+).
- Protein expression analysis:
Human TFRC was exclusively detectable on erythroid cells of homozygous B-hTFR1 mice (H/H) but not in WT mice (+/+), and mouse TFRC was detectable only in WT mice (+/+).
Human TFR1 was exclusively detectable on brain microvascular endothelium of homozygous B-hTFR1 mice, but not in WT mice.
- Leukocytes cell subpopulation analysis：
TFR1 humanized does not change the overall development, differentiation or distribution of immune cell types in spleen, thymus and blood.
- Blood routine test and Blood biochemical test
TFR1 humanized does not change the blood cell composition and morphology, ALT and AST levels or health of liver.
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Poster
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Publications
