Background:

• TFR1, a membrane protein expressed across various cell and tissue types in the human body, plays a crucial role in facilitating iron transport and metabolism. TFR1 is markedly expressed in numerous types of cancer cells. Studies have demonstrated that targeting TFR1 can effectively suppress tumor growth and metastasis. Moreover, TFR1 is also implicated in other conditions such as anemia and neurodegenerative disorders, etc.
• Given its high expression of TFR1 on brain endothelial cells, TFR1 can be leveraged as a receptor for receptor-mediated transcytosis (RMT), which allows for the efficient transport of large molecules across the BBB. This makes it an ideal target for delivering biotherapeutics to the central nervous system (CNS).

Target strategy:

• The genome of mouse Tfr1 gene that encodes the extracellular domain was replaced by human TfR1 counterparts in B-hTF/hTFR1 mice.
• The exons 1-14 of mouse Trf gene were replaced by human exons 1-17 that encode whole domains of TF protein in B-hTF/hTFR1 mice. The promoter, 5’UTR and 3’UTR regions of the mouse gene are also retained. The human TF expression is driven by endogenous mouse Trf promoter, while mouse Trf gene transcription and translation will be disrupted.

Verification:

• Human TF was exclusively detectable in homozygous B-hTF/hTFR1 mice, but not in wild-type C57BL/6 mice.

Application:

• This product is used to evaluate the pharmacodynamics and safety of treatments for tumors and neurodegenerative diseases, as well as to assess the potential of drugs to penetrate the blood-brain barrier.

Gene targeting strategy for B-hTF/hTFR1 mice. The exons 1-14 of mouse Trf gene were replaced by human exons 1-17 that encode whole domains of TF protein in B-hTF/hTFR1 mice. The promoter, 5’UTR and 3’UTR regions of the mouse gene are also retained. The human TF expression is driven by endogenous mouse Trf promoter, while mouse Trf gene transcription and translation will be disrupted.

Strain specific transferrin (TF) expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hTF/hTFR1 mice by ELISA. Serum was collected from wild-type C57BL/6JNifdc mice (+/+) (female, n=3, 6-week-old) and homozygous B-hTF/hTFR1 mice (H/H) (1 male and 2 female, n=3, 6-week-old). Expression level of human Transferrin was analyzed by ELISA (anti-human transferrin ELISA kit: abcam, ab187391; anti-mouse transferrin ELISA kit: abcam, ab157724). Mouse TF was only detectable in wild-type mice. Human TF was exclusively detectable in homozygous B-hTF/hTFR1 mice, but not in wild-type C57BL/6JNifdc mice. Values are expressed as mean ± SEM.

Western blot analysis of TFR1 protein expression in wild-type C57BL/6JNidc mice and homozygous B-hTF/hTFR1 mice by WB. Various tissues were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTF/hTFR1 mice (H/H), and then analyzed by western blot with anti-TFR1 antibody (abcam, ab214039). 30 μg total protein was loaded for western blotting analysis. GAPDH was detected as internal control. TFR1 was detectable in heart, liver, spleen, lung, kidney, stomach, colon, eyes and brain from C57BL/6JNifdc and homozygous B-hTF/hTFR1 mice, as the antibody was cross-reactive between human and mouse. M, marker.