Abstract 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.