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 biopharma­ceutical 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. We present an enhanced Southern blotting protocol for detecting transgene integration sites and assessing transgene integrity in CHO cell lines derived from transposon-based expression system. This protocol provides improved band resolution, facilitating accurate separation and detection of individual transgene insertions and integrity. Optimized key steps of Southern blotting analysis for recombinant Chinese hamster ovary (CHO) cell lines derived from transposon-based expression systemKey steps: post-digest DNA purification, size-matched electrophoresis buffers (Tris-borate-EDTA (TBE) for small, Tris-acetate-EDTA (TAE) for large), and alkaline capillary transfer.Clear performance gains: more resolvable bands (from 19 to 26 bands under TBE) and sharper signals with alkaline transfer.Fit-for-purpose: rapid, lower-cost assay that complements next-generation sequencing (NGS) and targeted locus amplification (TLA) mediated sequencing.Practical utility: improve clone triage, stability monitoring, and regulatory data packages.Actionable workflow provided for routine use. Optimized key steps of Southern blotting analysis for recombinant Chinese hamster ovary (CHO) cell lines derived from transposon-based expression system Key steps: post-digest DNA purification, size-matched electrophoresis buffers (Tris-borate-EDTA (TBE) for small, Tris-acetate-EDTA (TAE) for large), and alkaline capillary transfer. Clear performance gains: more resolvable bands (from 19 to 26 bands under TBE) and sharper signals with alkaline transfer. Fit-for-purpose: rapid, lower-cost assay that complements next-generation sequencing (NGS) and targeted locus amplification (TLA) mediated sequencing. Practical utility: improve clone triage, stability monitoring, and regulatory data packages. Actionable workflow provided for routine use.