[Development of site-specific integration system to high-level expression recombinant proteins in CHO cells].

Stable transformants for mammalian cells from gene transfer often show extreme variability in expression of the introduced transgene. This occurs from the highly variable number of copies integrated into the genome and from position effects on gene expression due to random integration. We constructed engineered CHO strains that can be used for high-level production of foreign proteins by gene-targeting. After transfecting dihydroforate reductase (DHFR)-deficient CHO cells with a newly screening vector plasmid pMCEscan, which carrying a FRT-neo*-IRES-k2tPA fusion gene and a DHFR gene, we screened colonies by k2tPA expression level. We selected 7 clones that expressed high level of k2tPA and carried one copy of the plasmid in their chromosomes. These clones showed in high level k2tPA production without amplification. So we targeted reporter gene (k2tPA) to test the basal expression ability of these cells clones. The clone, 8-1, showed the same effect to high base expression level. In this clone, the FRT-neo*-IRES-tPA gene was integrated at a transcription-active, DHFR-mediated, gene-amplifiable locus in the chromosomes. A gene-targeting vector, carrying a FRT-fused hygromycin-resistance gene, was constructed to target desired genes in chromosomal FRT by FLP recombinase-mediated site-specific recombination. Using this cell-vector system, we could reproducibly obtain high producers of recombinant proteins by gene-targeting and gene amplification. Using the site-specific integration CHO/dhfr- cell line 8-1, the expression level of k2tPA could amount to 17.1 microg/10(6) cell x 24 h.