Name: huTFRC Mouse
Brand: サイヤジェン（Cyagen）
SKU: C001860

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huTFRC Mouse

製品名

huTFRC Mouse

製品ID

C001860

系統名

C57BL/6NCya-Tfrc^tm2(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、「huTFRC Mouse（カタログ番号C001860）はサイアジェンから購入しました。」と引用してください。

HUGO-GT Humanized Models

Blood-Brain Barrier

製品タイプ

年齢

遺伝子型

性別

数量

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HUGO-GT Humanized 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 huTFRC mouse model was generated by replacing the mouse Tfrc endogenous extracellular domain with the human TFRC extracellular domain. The murine cytoplasmic and helical will be kept. 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 CDS humanized hTFRC mice (Cat. No.: C001584), huTFRC mice (Cat. No.: C001860) exhibited normal serum iron levels, while the TFRC-mediated delivery efficiency in the central nervous system (CNS) was lower than that of hTFRC mice (Cat. No.: C001584).

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

The mouse Tfrc endogenous extracellular domain was replaced with the human TFRC extracellular domain. The murine cytoplasmic and helical regions were kept.

Figure 1. Gene editing strategy of huTFRC mice.

適用分野

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

Development, screening, and 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 huTFRC mice had significant expression of human TFRC mRNA in the liver, hippocampus, cerebral cortex, and kidney tissues, with no murine Tfrc mRNA detected. In contrast, WT mice only expressed murine Tfrc mRNA in each tissue, with no human TFRC mRNA expression. (Bars represent mean ± SEM)

Figure 2. Gene expression detection in the liver, hippocampus, cerebral cortex, and kidney of huTFRC and wild-type (WT) mice (6-week-old, male, homozygous, n=4). ND: Not detected

2. Protein Expression (Western Blot)

（1）TFRC Protein Expression in Brain Tissues

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

Figure 3. TFRC protein expression levels in the Cerebral Cortex, Hippocampus, and Striatum of brain tissues from huTFRC mice and wild-type (WT) mice (6 weeks old, male).

（2）TFRC Protein Expression in Peripheral Tissues

TFRC protein expression levels in the kidney, liver, spleen, heart, quadriceps, and eye of 6-week-old male huTFRC mice were detected using a human-murine cross-reactive antibody, with wild-type (WT) mice as the control group for comparison.

Figure 4. TFRC protein expression in peripheral tissues of huTFRC mice and wild-type (WT) mice (6 weeks old, male).

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

Human-specific antibodies were used to evaluate human TFRC protein expression in 6-week-old male huTFRC and wild-type (WT) mice. Western blot analysis revealed human TFRC protein in the cerebral cortex and hippocampus of homozygous huTFRC mice, whereas no signal was detected in WT mice.

Figure 5. Human TFRC protein expression in the cerebral cortex and hippocampus of huTFRC and WT mice. (6 weeks old, males, homozygous, n=2).

3. Human TFRC Protein Expression (Flow Cytometry)

The expression of TFRC protein (hCD71) was analyzed in the TER-119+ positive cell population. FACS results showed that significant expression of human TFRC protein was detected in the whole blood, bone marrow, and spleen of huTFRC mice, with no murine TFRC protein expressed. In contrast, WT mice only expressed murine TFRC protein in each tissue, with no human TFRC protein expressed. (Bars represent mean ± SD)

Figure 6. Detection of TFRC protein expression in blood, bone marrow, and spleen of huTFRC and wild-type (WT) mice (6-week-old, male, n=4).

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). The results showed that human TFRC protein was specifically expressed in the cerebral microvascular endothelium of homozygous huTFRC mice.

Figure 7. Co-immunostaining results of human TFRC and endothelial cell marker mCD31 in brain tissues of huTFRC mice and wild-type (WT) mice (6 weeks old, male). (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 huTFRC mice exhibited no obvious signs of blood-brain barrier damage compared with wild-type (WT) mice.

Figure 8. Evaluation of blood-brain barrier integrity in huTFRC mice and wild-type (WT) mice (5-6 weeks old, female, n=4 per group).

6. Complete Blood Count (CBC)

（1）Erythrocyte Parameters and Platelet Counts

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

（2）Reticulocyte Parameters

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

7. Serum Iron Level

Results showed no significant difference in serum iron levels between huTFRC mice and wild-type (WT) mice. Data are presented as mean±SEM, and statistical analysis was performed using the Mann-Whitney U test; ns indicates no significant difference (p>0.05).

Figure 9. Detection of serum iron levels in huTFRC mice and wild-type (WT) mice (8 weeks old, homozygous, male, WT n=4, huTFRC 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 huTFRC 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, huTFRC 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 huTFRC mice. Values are presented as mean ± standard error of the mean (SEM).

Figure 10. Validation of TFRC-mediated central nervous system delivery efficacy in huTFRC 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.

huTFRC 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 (Western Blot)

3. Human TFRC Protein Expression (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

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