Name: hTFRC Mouse
Brand: サイヤジェン（Cyagen）
SKU: C001584

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hTFRC Mouse

製品名

hTFRC Mouse

製品ID

C001584

系統名

C57BL/6NCya-Tfrc^tm1(hTFRC)/Cya

背景情報

C57BL/6NCya

状況

このマウス系統を論文で使用する場合は、「hTFRC Mouse（カタログ番号C001584）はサイアジェンから購入しました。」と引用してください。

Other Target Humanized Mouse Models

Blood-Brain Barrier

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遺伝子型

性別

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Other Target Humanized Mouse Models

Blood-Brain Barrier

基本情報

検証 Data

系統詳細

The Transferrin receptor (TFRC) gene encodes Transferrin Receptor 1 (TFR1), a protein that is expressed at low levels in most normal cells but shows increased expression in highly proliferative cells, such as basal epidermal cells, intestinal epithelium, and certain activated immune cells. Brain capillary endothelial cells, which constitute the blood-brain barrier (BBB), also express this receptor at high levels^[1]. TFR1 plays a critical role in maintaining iron metabolism and homeostasis by facilitating receptor-mediated endocytosis of iron-bound transferrin (Tf) via Tf cycling, thereby promoting iron uptake^[2]. Cellular iron deficiency can lead to apoptosis, while cellular transformation requires substantial iron to sustain proliferation, with iron overload contributing to tumor progression. The high expression of TFR1 in many tumors makes it a potential tumor marker, offering a target for therapies to inhibit tumor growth and metastasis^[1]. Moreover, TFR1 is implicated in anemia and iron metabolism disorders. Studies have shown that elevated TFR1 expression in cardiomyocytes is associated with exacerbated inflammation in myocarditis patients^[3].

As a target for antibody-mediated cancer therapy, TFR1 can be leveraged through two approaches: one involves the use of antibodies conjugated to anti-cancer drugs, which are indirectly internalized via receptor-mediated endocytosis; the other employs antibodies that directly disrupt receptor function or induce Fc effector functions such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC). Various clinical drugs targeting TFR1 are currently under development, including antisense oligonucleotides (ASOs), antibody-drug conjugates (ADCs), and antibody-oligonucleotide conjugates, applicable to diseases such as cancer, anemia, and neurodegenerative disorders. Research indicates that enhancing antibody transport across the blood-brain barrier via TFR1, by forming specific bispecific antibodies with anti-β-amyloid antibodies, can improve therapeutic outcomes in Alzheimer's patients^[4-5]. As research progresses, TFR1 is expected to become an effective clinical target for multiple diseases and a synergistic target for drug delivery across the blood-brain barrier (BBB).

The hTFRC mouse model was generated by inserting the human TFRC gene sequence into the mouse Tfrc gene locus using gene-editing technology. To minimize interference from mouse gene sequences or proteins, part of the mouse Tfrc gene sequence was knocked out, resulting in a model expressing only the human TFR1 protein. This model is valuable for studying iron metabolism disorders, neurodegenerative diseases, and tumor development, supporting the development of TFR1-targeted therapeutics and preclinical pharmacological evaluations. Compared with the genome humanized huTFRC mice (Cat. No.: C001860), the CDS humanized hTFRC mice in this datasheet (Cat. No.: C001584) exhibited higher TFRC-mediated delivery efficiency in the central nervous system (CNS) and presented an anemic phenotype.

参考文献

Candelaria PV, Leoh LS, Penichet ML, Daniels-Wells TR. Antibodies Targeting the Transferrin Receptor 1 (TfR1) as Direct Anti-cancer Agents. Front Immunol. 2021 Mar 17;12:607692.

Xu W, Barrientos T, Mao L, Rockman HA, Sauve AA, Andrews NC. Lethal Cardiomyopathy in Mice Lacking Transferrin Receptor in the Heart. Cell Rep. 2015 Oct 20;13(3):533-545.

Kobak KA, Franczuk P, Schubert J, Dzięgała M, Kasztura M, Tkaczyszyn M, Drozd M, Kosiorek A, Kiczak L, Bania J, Ponikowski P, Jankowska EA. Primary Human Cardiomyocytes and Cardiofibroblasts Treated with Sera from Myocarditis Patients Exhibit an Increased Iron Demand and Complex Changes in the Gene Expression. Cells. 2021 Apr 6;10(4):818.

Bray, Natasha. "Transferrin'bispecific antibodies across the blood–brain barrier." Nature Reviews Drug Discovery 14.1 (2015): 14-15.

Pardridge, William M. "Blood–brain barrier drug delivery of IgG fusion proteins with a transferrin receptor monoclonal antibody." Expert opinion on drug delivery 12.2 (2015): 207-222.

系統作製戦略

TurboKnockout targeting technology was used to replace part of exon 2 of the mouse Tfrc gene with a human TFRC chimeric cDNA WPRE-BGH pA cassette. Gene-editing techniques were employed to knock out exons 10-13 of the mouse Tfrc gene.

Figure 1a. Diagram of the gene editing strategy for the generation of hTFRC mice. *TFRC chimeric cDNA: Mouse cytoplasmic and helical-Human extracellular.

Figure 1b. Diagram of the gene editing strategy for the generation of hTFRC mice.

適用分野

Studies on iron metabolism disorders, neurodegenerative diseases, and tumor development;

Development, screening, and efficacy evaluation of TFRC-targeted therapies;

Research and evaluation of drug delivery across the blood-brain barrier (BBB).

検証 Data

1. Gene Expression

RT-qPCR results showed that the human TFRC gene was significantly expressed in the liver, hippocampus, cerebral cortex and kidney tissues of hTFRC mice, with no detectable murine Tfrc gene expression. In contrast, only the murine Tfrc gene was expressed in all tissues of WT mice, and the human TFRC gene was not detected.

Figure 2. Gene Expression Analysis of Liver, Hippocampus, Cerebral Cortex and Kidney Tissues in hTFRC Mice and Wild-Type (WT) Mice (6 weeks old, male, homozygous, n=4). ND: Not detected.

2. Protein Expression

（1）TFRC Protein Expression in Brain Tissues (Western Blot)

Using a human-murine cross-reactive antibody, the expression levels of TFRC protein in the cerebral cortex, hippocampus and striatum of brain tissues from 6-week-old male mice were detected, with the results compared against those of wild-type (WT) mice.

Figure 3. TFRC Protein Expression in Brain Tissues of hTFRC Mice and Wild-Type (WT) Mice (6 weeks old, male).

（2）TFRC Protein Expression in Peripheral Tissues (Western Blot)

Using a human-murine cross-reactive antibody, the expression levels of TFRC protein in the kidney, liver, spleen, heart, quadriceps and eye tissues of 6-week-old male mice were detected, with the results compared against those of wild-type (WT) mice.

Figure 4. TFRC Protein Expression in Peripheral Tissues of hTFRC Mice and Wild-Type (WT) Mice (6 weeks old, male).

（3）TFRC Protein Expression in the Cerebral Cortex and Hippocampus (Western Blot)

Human-specific antibodies were used to evaluate human TFRC protein expression in hTFRC and wild-type (WT) mice. Western blot analysis revealed human TFRC protein in the cerebral cortex and hippocampus of homozygous hTFRC mice, whereas no signal was detected in WT mice.

Figure 5. Human TFRC protein expression in the cerebral cortex and hippocampus of hTFRC and WT mice (7 weeks old, males, homozygous, n=2).

3. Flow Cytometry

The expression of TFRC protein was analyzed in TER-119+ cell populations. Flow cytometry results showed that hTFRC mice exhibited significant expression of human TFRC protein in the bone marrow, spleen, and whole blood, with no expression of murine TFRC protein. In contrast, only significant expression of murine TFRC protein was detected in WT mice, with no expression of human TFRC protein (Bars represent mean ± SD).

Figure 6. TFRC protein expression in blood, bone marrow, and spleen of hTFRC mice and wild-type (WT) mice (6-week-old, male, n=5).

4. Co-immunostaining of Human TFRC and Endothelial Cell Marker mCD31

Brain tissue sections from 6-week-old male mice were subjected to co-immunostaining for human TFRC (red) and endothelial cell marker mCD31 (green), with nuclei counterstained with DAPI (blue). Immunofluorescence (IF) results showed that human TFRC protein was specifically expressed in the cerebral microvascular endothelium of homozygous hTFRC mice.

Figure 7. Co-immunostaining Results of Human TFRC and Endothelial Cell Marker mCD31 in hTFRC Mice and Wild-Type (WT) Mice (6 weeks old, male, homozygous). Scale bar: 50 μm

5. 2% Evans Blue (EB) Blood-Brain Barrier Permeability Assay

2% Evans Blue (EB) was administered via tail vein injection at a dose of 40 µL/10 g body weight. Thirty minutes post-injection, physiological saline perfusion was performed to eliminate circulating EB in the blood. Brain tissue observations showed that compared with wild-type (WT) mice, hTFRC mice exhibited no obvious signs of blood-brain barrier damage.

Figure 8. Evaluation of Blood-Brain Barrier Integrity in hTFRC Mice and Wild-Type (WT) Mice (6–12 weeks old, female, homozygous, n=4 per group).

6. Complete Blood Count (CBC)

（1）Erythrocyte Parameters and Platelet Counts

The hemoglobin content of homozygous hTFRC mice was significantly lower than that of wild-type (WT) mice, indicating that hTFRC mice exhibited an anemic phenotype.

Table 1. Erythrocyte parameters and platelet counts in WT and hTFRC mice. Values are expressed as mean±SEM.

（2）Reticulocyte Parameters

Table 2. Reticulocyte parameters in WT and homozygous hTFRC mice. Values are expressed as mean ± SEM.

7. Serum Iron Level

The results showed that the serum iron level of homozygous hTFRC mice was significantly higher than that of wild-type (WT) mice. Data are presented as mean±SEM, and statistical analysis was performed using the Mann-Whitney U test (*p＜0.05).

Figure 9. Detection of Serum Iron Levels in hTFRC Mice and Wild-Type (WT) Mice (8 weeks old, male, homozygous, WT n=4, hTFRC n=5).

8. Pharmacodynamic Validation: TFRC-Mediated Central Nervous System (CNS) Delivery Efficacy

To evaluate the in vivo function of the human TFRC receptor, 8–10 week-old homozygous hTFRC mice and wild-type (WT) mice were administered a single tail vein injection of control antibody (Vehicle IgG) or anti-human TFRC bispecific antibody (TfR BsAb)*. Twenty-four hours after drug injection, drug concentrations in the cerebral cortex and plasma were detected. Results showed that compared with WT mice, hTFRC mice had higher accumulation of TfR BsAb in the cerebral cortex, while the drug concentration in plasma was significantly reduced. This result indicates an effective and specific TFRC-dependent blood-brain barrier (BBB) transport process in hTFRC mice. Values are presented as mean ± standard error of the mean (SEM).

Figure 10. Validation of TFRC-mediated central nervous system delivery efficacy in hTFRC mice (8–10 weeks old, homozygous, n=6, 3 males and 3 females). *This data and the test drugs (Vehicle IgG and TfR BsAb) were provided by a Cyagen collaborator.

9. hTFRC Mice Enable Efficient Brain-Targeted Delivery of AAV9-BI-hTfR1

(A) Four weeks after intravenous injection of AAV9-BI-hTfR1 virus into hTFRC mice, strong mCherry reporter protein (red) signals were detected in the sagittal sections of brain tissues, demonstrating that humanized TFRC mediated efficient virus transcytosis across the blood-brain barrier in this model. In contrast, no obvious signals were observed in brain parenchyma when the control virus AAV9-Control was administered, or when either virus was injected into wild-type (WT) mice.

(B) The distribution levels of viral genomic DNA (gDNA) in brain tissues were quantified by quantitative PCR (qPCR), with data expressed as vg/dg (viral genome copies per diploid genome). The distribution of AAV9-BI-hTfR1 in the brain tissues of hTFRC mice was higher than that in WT mice, and also significantly higher than that of the control virus AAV9-Control in the brain tissues of both mouse strains.

These results confirm that hTFRC mice are suitable for screening AAV capsid variants that cross the blood-brain barrier via human TFRC-dependent transcytosis.

Figure 11. Application of hTFRC Mice for Screening AAV Capsid Variants That Cross the Blood-Brain Barrier via Human TFRC-Dependent Transcytosis (6–8 weeks old, female, n=2, data presented as mean values). *This data was provided by a collaboration partner of Cyagen Biosciences.

hTFRC Mouse

基本情報

検証 Data

関連リソース

遺伝子名

遺伝子別名

NCBI ID

染色体

MGI ID

さらに

データシート

系統詳細

参考文献

系統作製戦略

適用分野

1. Gene Expression

2. Protein Expression

3. Flow Cytometry

4. Co-immunostaining of Human TFRC and Endothelial Cell Marker mCD31

5. 2% Evans Blue (EB) Blood-Brain Barrier Permeability Assay

6. Complete Blood Count (CBC)

7. Serum Iron Level

8. Pharmacodynamic Validation: TFRC-Mediated Central Nervous System (CNS) Delivery Efficacy

9. hTFRC Mice Enable Efficient Brain-Targeted Delivery of AAV9-BI-hTfR1

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