title: An efficient and targeted gene integration system for high-level antibody expression.
abstract: Random integration linking genomic amplification has been used to generate desired cell lines for stable and high-level expressing recombinant antibodies. But this technique is laborious, and the expression level is unpredictable due to position effects. Here, we have constructed a cell-vector system for high-level antibody expression using an FRT/FLP strategy to overcome position effects. The key is to target the FRT sequence to chromosomal locations where there is a high rate of transcription and gene amplification, and the amplified genes can be maintained. To screen desired loci with high transcriptional activity and amplifiable capacity, dual weakened markers (selectable galactosidase and amplifiable dihydrofolate reductase, DHFR) and the FRT sequence were synchronously cloned into a plasmid. After transfection of a Chinese hamster ovary host cell line with this plasmid, we selected 20 candidate cell lines from 721 individual clones. An antibody gene-targeting vector carrying an FRT-fused hygromycin gene was constructed to target antibody genes into the chromosomal FRT site by FLP recombinase. Three out of 20 cell lines can be used as host cells for site-specific recombination. By using southern blot and fluorescence in situ hybridization (FISH), a candidate engineered cell line, number 37, was chosen. It contains a single FRT-tagged locus in its genome. FISH analysis indicated that the antibody genes were all located at the original FRT-tagged locus in the genome of the gene-targeted and gene-amplified cell lines. Three kinds of recombinant antibodies were successfully expressed in candidate cell line 37. The highest producers produced more than 200 mug/ml of the antibody in 6 days of continuous culture in a spinner flask.
authors.affiliation: Department of Molecular Immunology, Beijing Institute of Basic Medical Sciences, Beijing 100850, PR China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Huang
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.forename: Yu Gang
authors.initials: YG
authors.lastname: Wang
authors.forename: Xin
authors.initials: X
authors.lastname: Gu
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.forename: Bei Fen
authors.initials: BF
authors.lastname: Shen
mesh_terms: D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000032:analysis; D005260:Female; D005784:Gene Amplification; D015870:Gene Expression; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering; D017404:In Situ Hybridization, Fluorescence; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D000906:Antibodies; D011994:Recombinant Proteins; D004247:DNA