SP2/0-mRosa26-Lox71-GFP-High-Lox2272-KI

In the SP2/0 mouse myeloma cell line, we employed CRISPR/Cas9-mediated homologous recombination to achieve site-specific integration of a transgene into the genomic safe harbor locus, Rosa26. This strategy ensures stable and sustained transgene expression while minimizing unpredictable disruptions to the native cellular genome. For the specific construct, a green fluorescent protein (GFP) reporter gene was inserted downstream of a strong promoter. This GFP expression cassette is flanked by precisely engineered, asymmetric LoxP site variants (e.g., Lox71 and Lox2272), thereby establishing a conditional gene-editing platform. This design enables the efficient, site-specific replacement of the initial GFP reporter with other functional genes (e.g., light or heavy chain genes of a therapeutic antibody) in the presence of Cre recombinase. Consequently, this engineered cell line not only facilitates real-time tracking of cell status and enrichment of positive populations but, more importantly, provides a foundation for a versatile and efficient "cell factory." It allows for the subsequent flexible and controlled high-level expression of various proteins of interest, making it applicable for recombinant protein production and antibody engineering optimization studies.