Vector fragmentation: characterizing vector integrity in transfected clones by Southern blotting.
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.
Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Centros, Singapore 138668. ng_say_kong@bti.a-star.edu.sg
Say Kong
SK
Ng
Wenyu
W
Lin
Rohit
R
Sachdeva
Daniel I C
DI
Wang
Miranda G S
MG
Yap
D000818:Animals; D015139:Blotting, Southern; D016466:CHO Cells; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media / Q000494:pharmacology; D005434:Flow Cytometry; D005784:Gene Amplification / Q000187:drug effects; D017353:Gene Deletion; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000032:analysis* / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D008727:Methotrexate / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D014162:Transfection
D003470:Culture Media; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D008727:Methotrexate