Optimized Southern blotting for enhanced and precise detection of transgenes in CHO cells from transposon-based expression systems

The genetic stability of recombinant CHO cell lines producing therapeutic proteins is critical for ensuring consistent quality in biopharma-ceutical products. Southern blotting remains the gold standard for evaluating transgene integrity and stability in these cell lines. In the biopharmaceutical industry, transposon-based expression systems are widely utilized to generate highly productive and genetically stable CHO cell lines. However, evaluating transgene integration sites and integrity in such cell lines is challenging with standard Southern blotting protocols. This difficulty arises because transposon-mediated transfection often results in multiple independent integration sites in the host genome, each typically harboring a single transgene copy. Upon restriction enzyme digestion, similar-sized DNA fragments are generated, reducing resolution and complicating the separation and detection of the transgenes using standard blotting protocols. Here, we present a modified Southern blotting protocol that significantly improves the resolution of integration banding patterns by refining key steps, including purification of digested DNA prior to electrophoresis and an enhanced DNA transfer method. This protocol was successfully applied to analyze multiple transposon-derived CHO cell lines with high transgene copy numbers, enabling more precise and efficient detection of transgene integration.