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Vector fragmentation: Characterizing vector integrity in transfected clones by Southern blotting

Authors

  • Say Kong Ng,

    Corresponding author
    1. Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Centros, Singapore 138668
    2. Singapore-MIT Alliance, National University of Singapore, Singapore 117576
    • Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Centros, Singapore 138668
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  • Wenyu Lin,

    1. Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Centros, Singapore 138668
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  • Rohit Sachdeva,

    1. Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Centros, Singapore 138668
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  • Daniel I.C. Wang,

    1. Singapore-MIT Alliance, National University of Singapore, Singapore 117576
    2. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139
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  • Miranda G.S. Yap

    1. Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Centros, Singapore 138668
    2. Singapore-MIT Alliance, National University of Singapore, Singapore 117576
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Abstract

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. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010

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