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 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.

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.

生产治疗性蛋白的重组中国仓鼠卵巢（CHO）细胞系的遗传稳定性，对于保障生物制药产品的质量一致性至关重要。Southern印迹（Southern blotting）仍是评估此类细胞系中外源转基因整合完整性与稳定性的金标准。在生物制药行业中，基于转座子的表达系统被广泛用于构建高产且遗传稳定的CHO细胞系。然而，采用标准Southern印迹方案评估此类细胞系的转基因整合位点与完整性颇具挑战。这一难点源于转座子介导的转染通常会在宿主基因组中形成多个独立的整合位点，每个位点通常仅携带单个转基因拷贝。经限制性酶切后，会产生大小相近的DNA片段，这会降低分辨率，使得标准印迹方案难以分离和检测转基因。本文提出一种改良的Southern印迹方案，通过优化关键步骤——包括电泳前的酶切后DNA纯化以及升级的DNA转移方法——显著提升了整合条带图谱的分辨率。该方案已成功应用于分析多株携带高转基因拷贝数的转座子来源CHO细胞系，实现了对转基因整合更为精准高效的检测。

本文提出一种改良的Southern印迹方案，用于检测基于转座子表达系统构建的CHO细胞系中的转基因整合位点，并评估转基因完整性。该方案可提升条带分辨率，便于精准分离和检测单个转基因插入片段及其完整性。

针对转座子表达系统来源的重组中国仓鼠卵巢（CHO）细胞系的Southern印迹分析优化关键步骤
关键优化步骤包括：酶切后DNA纯化、适配片段大小的电泳缓冲液（小型片段使用Tris-硼酸-EDTA（TBE）缓冲液，大型片段使用Tris-乙酸-EDTA（TAE）缓冲液）以及碱性毛细管转移法。
性能提升显著：在TBE缓冲液条件下可分辨的条带数从19条增至26条，且采用碱性转移法可获得更清晰的信号。
该方案兼具实用性：操作快速、成本较低，可作为下一代测序（NGS）与靶向位点扩增（TLA）介导测序的补充检测手段。
实际应用价值包括：优化克隆分选、稳定性监测以及监管资料包的筹备。本文提供了可用于常规实验的可落地工作流程。

针对转座子表达系统来源的重组中国仓鼠卵巢（CHO）细胞系的Southern印迹分析优化关键步骤
关键优化步骤包括：酶切后DNA纯化、适配片段大小的电泳缓冲液（小型片段使用Tris-硼酸-EDTA（TBE）缓冲液，大型片段使用Tris-乙酸-EDTA（TAE）缓冲液）以及碱性毛细管转移法。
性能提升显著：在TBE缓冲液条件下可分辨的条带数从19条增至26条，且采用碱性转移法可获得更清晰的信号。
该方案兼具实用性：操作快速、成本较低，可作为下一代测序（NGS）与靶向位点扩增（TLA）介导测序的补充检测手段。
实际应用价值包括：优化克隆分选、稳定性监测以及监管资料包的筹备。本文提供了可用于常规实验的可落地工作流程。