B6-hTFRC Mice

Catalog Number: I001185

Strain Name: C57BL/6NCya-Tfrctm1(hTFRC)/Cya

Genetic Background: C57BL/6NCya

Reproduction: Homozygote x Homozygote

 

Strain Description

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 B6-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.

Strain Strategy

STEP 1. Humanization of Mouse Tfrc Gene: 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.



STEP 2. Knockout of Residual Mouse Tfrc Gene Sequences: Gene-editing techniques were employed to knock out exons 10-13 (~3.9 kb) of the mouse Tfrc gene.



Strain Application

  • 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)

 

Validation Data

1. Human TFRC gene and mouse Tfrc gene expression detection


Figure 1. Detection of human TFRC and mouse Tfrc gene expression in various tissues of 7-week-old male homozygous B6-hTFRC mice (hTFRC) and wild-type mice (WT). RT-qPCR analysis showed significant expression of the human TFRC gene in the liver, hippocampus, cerebral cortex, spleen, kidneys, and heart of male B6-hTFRC mice, while wild-type mice showed no expression of the human TFRC gene. The mouse Tfrc gene was expressed in various tissues of wild-type mice but not in B6-hTFRC mice (Bars represent mean±SEM, n=3).

ND: Not detected.

2. Human TFRC protein expression detection (Flow Cytometry)


Figure 2. Flow cytometry analysis of human TFRC and mouse TFRC protein expression in erythrocytes (TER-119+) of 7-week-old male homozygous B6-hTFRC and wild-type (WT) mice. Species-specific TFRC antibodies were used to detect TFRC protein expression in the erythrocytes of wild-type and B6-hTFRC mice. Results showed significant expression of human TFRC protein in bone marrow (BM), spleen, and peripheral blood (PB) erythrocytes of B6-hTFRC mice, while no expression was detected in wild-type mice. Mouse TFRC protein was significantly expressed in the bone marrow, spleen, and peripheral blood erythrocytes of wild-type mice but not in B6-hTFRC mice (Bars represent mean±SEM, n=3).

3. Human TFRC protein expression detection (Western Blot)


Figure 3. Western Blot analysis of human TFRC and mouse TFRC protein expression in the cerebral cortex and hippocampus of 7-week-old male homozygous B6-hTFRC and wild-type (WT) mice. The results showed expression of human TFRC protein in the cerebral cortex and hippocampus of B6-hTFRC mice (Bars represent mean±SEM, n=3).

Note: The human TFRC antibody used in detection is a polyclonal antibody, resulting in multiple bands observed during detection. Additionally, the high homology between the human and mouse regions recognized by this antibody reduced detection specificity, leading to non-specific human TFRC protein bands in wild-type mice.

 


References

[1]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.
[2]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.
[3]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.
[4]Bray, Natasha. "Transferrin'bispecific antibodies across the blood–brain barrier." Nature Reviews Drug Discovery 14.1 (2015): 14-15.
[5]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.