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首页> 外文期刊>Biotechnology Progress >Vector Fragmentation: Characterizing Vector Integrity in Transfected Clones by Southern Blotting
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Vector Fragmentation: Characterizing Vector Integrity in Transfected Clones by Southern Blotting

机译：载体片段化：通过Southern印迹表征转染克隆中载体的完整性

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摘要

The Chinese Hamster Ovary production cell line development process using methotrexate (MTX) amplification is well studied and commonly used for biopharmaceutical processes. However, successful MTX amplification varies from clone to clone and suggested reasons include vector fragmentation during the transfection process and genomic rearrangement of the Chinese Hamster Ovary chromosomes. Here, we elucidated the vector integration patterns of 40 transfected single-cell clones by Southern blotting and showed that vector fragmentation occurs at a significant level in our experiment. This concurs with MTX amplification studies implying that single-cell cloning is necessary to ensure a successful amplification process. Truncations at the ends of the integrated vectors were also observed, whereas gross DNA insertions were not detected in our data. This suggests that end deletions are common, whereas insertion events are rare in animal cells.
机译：使用甲氨蝶呤（MTX）扩增的中国仓鼠卵巢生产细胞系开发过程已得到充分研究，并通常用于生物制药过程。然而，成功的MTX扩增因克隆而异，提示的原因包括转染过程中的载体断裂和中国仓鼠卵巢染色体的基因组重排。在这里，我们通过Southern杂交阐明了40个转染的单细胞克隆的载体整合模式，并表明在我们的实验中载体片段化的发生率很高。这与MTX扩增研究相一致，暗示必须进行单细胞克隆才能确保成功进行扩增。还观察到了整合载体末端的截短，而在我们的数据中未检测到总的DNA插入。这表明末端缺失是常见的，而插入事件在动物细胞中很少见。

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