CRISPR/Cas9-mediated targeted knock-in of human erythropoietin at the β-casein locus results in lactogenic hormone-responsive expression in HC11 mammary epithelial cells

Precise genomic integration strategies are essential for achieving stable and regulated transgene expression. In this study, we established a CRISPR/Cas9-mediated targeted knock-in system to integrate the human erythropoietin (hEPO) gene into the endogenous regulatory region of the mouse β-casein locus in HC11 mammary epithelial cells. A donor vector carrying hEPO was designed for homology-directed repair and successfully introduced into the β-casein locus. A heterozygous single-cell knock-in clone was isolated and validated by genomic analysis. Upon stimulation with lactogenic hormones, the integrated hEPO transgene exhibited hormone-responsive expression, resulting in an approximately 20-fold increase in protein levels compared to non-induced conditions. To enable downstream purification and activity assessment, a GST-tagged hEPO construct was employed. The fusion protein was purified using glutathione affinity chromatography, followed by proteolytic cleavage to obtain recombinant hEPO. The purified protein displayed measurable biological activity with a specific activity of 53.4 mIU/μg. These findings demonstrate that targeted integration at the β-casein locus results in lactogenic hormone-responsive expression driven by endogenous regulatory elements in mammary epithelial cells. This approach provides a controlled gene expression platform that may be applicable to hormone-inducible expression systems for therapeutic protein studies.