• Background: EPO encodes erythropoietin, which is produced by the liver during the fetal stage and primarily synthesized by the kidneys in adults before being secreted into the plasma. It plays a crucial role in erythropoiesis. The main function of EPO is to stimulate stem cells in the bone marrow to differentiate into erythroid lineage cells, thereby increasing red blood cell count, elevating hemoglobin levels, and enhancing blood oxygen content to address hypoxemic conditions such as hypoxia. In tumors, EPO promotes the self-renewal and expansion of cancer stem cells in an autocrine/paracrine manner by enhancing the JAK/STAT signaling pathway. Consequently, EPO represents a promising therapeutic target for the development of novel anti-tumor drugs.
• Targeting strategy: The exons 1-5 of mouse Epo gene that encode the whole molecule (ATG to STOP codon), including 3’UTR were replaced by human counterparts in B-NDG hEPO mice. The promoter and 5’UTR region of the mouse gene were retained. The human EPO expression was driven by endogenous mouse Epo promoter, while mouse Epo gene transcription and translation will be disrupted.
• Validation: Human EPO protein were detectable in the B-NDG hEPO mice but not in B-NDG mice.
• Application: B-NDG hEPO mice provide sustained human EPO support to engrafted human HSCs, enabling enhanced human erythroid development and serving as an in vivo platform to study anemia/erythropoiesis and to evaluate EPO–EPOR pathway therapeutics or gene/cell therapies.

Gene targeting strategy for B-NDG hEPO mice. The exons 1-5 of mouse EPO gene that encode the whole molecule (ATG to STOP codon), including 3’UTR are replaced by human counterparts in B-NDG hEPO mice. The promoter and 5’UTR region of the mouse gene are retained. The human EPO expression is driven by endogenous mouse Epo promoter, while mouse Epo gene transcription and translation will be disrupted.

Strain-specific EPO expression analysis in B-NDG mice and homozygous humanized B-NDG hEPO mice by ELISA. Serum was collected from B-NDG mice (+/+) and homozygous B-NDG hEPO mice (H/H) (male, 7-week-old, n=3). Expression level of mouse and human EPO were analyzed with anti-mouse EPO antibody: R&D, MEP00B and anti-human EPO antibody: R&D, DEPRU0. Mouse EPO was only detectable in B-NDG mice. Human EPO was exclusively detectable in homozygous B-NDG hEPO mice. Values are expressed as mean ± SEM.

Note: 1mIU≈8pg, provided by the technical support of R&D.