Identification of a Specific Pseudo attP Site for Phage phiC3 Integrase in the Genome of Chinese Hamster in CHO-K1 Cell Line

Abstract

Background: PhiC31 integrase is a DNA site-specific recombinase integrates DNA into the chromosomes between the two sites of attB and attP. Several pseudo attPs have been identified in mammalian genomes with critical features for long-term expression of transgene. In this manuscript, we report a novel intrinsic pseudo attP site named CHOL1 in the Chinese hamster genome implementing an inverse Polymerase Chain Reaction (IPCR). Objectives:Identification of pseudo attP site(s) of Tenecteplase cDNA integration in the genome of stable transformed CHO cell line. Materials and Methods: First, genome was extracted from a stable transformed CHO cell line expressing Tenecteplase. By creating of minicircle DNA in the last step, sequenc‌ing was performed. Results: We obtained one band. BLAST analysis of the respective sequence of inverse PCR band identified a pseudo attP site. Conclusions: Data demonstrated that the phiC31 integrase provides a suitable insertion site in the genome to express the gene of interest.