An efficient homologous recombination vector pTV(I) contains a hot spot for increased recombinant protein expression in Chinese hamster ovary cells.
We employed reverse genetics to clone a 5.0 kb genomic DNA hot spot HIRPE (hot spot for increased recombinant protein expression) flanking the plasmid integration site from a recombinant Chinese hamster ovary (CHO) cell line. DNA sequence analysis of the 5.0 kb fragment revealed that HIRPE is enriched for repetitive elements, Alu-like sequences and matrix-associated regions that are known to be linked with transcriptionally active regions in a number of mammalian systems. The construction of a homologous recombination vector, pTV1, containing the 5.0 kb HIRPE genomic DNA, a recombinant gene human CTLA4-Ig, and the dhfr gene as a positive selection marker is described. It was observed that the pTV1 vector targeted the CTLA4Ig gene to a preferred locus in the CHO genome contributing to high recombinant gene expression in transfected CHO cells. Preliminary studies suggest that similar to the observation with the parental cell line, pTV1-generated transfectomas that were analyzed appear to harbor an inverted duplication of the genomic DNA at the plasmid integration site.
Koduri
R K
RK
Biotechnology Development, Technical Operations, Bristol-Myers Squibb Company, Syracuse 13221, NY, USA.
Miller
J T
JT
Thammana
P
P
D000069594:Abatacept; D000818:Animals; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D015139:Blotting, Southern; D016466:CHO Cells; D060908:CTLA-4 Antigen; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D018796:Immunoconjugates; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA; D014162:Transfection
D015703:Antigens, CD; D000943:Antigens, Differentiation; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; D018796:Immunoconjugates; D011994:Recombinant Proteins; D000069594:Abatacept; D004247:DNA