Construction of eukaryotic integrative vectors by insertion of the integration region attB in pSVM

Different varieties of Chinese Hamster Ovary (CHO) cells are being used frequently for producing most pharmaceutical products for many years. Permanent production in these host cells is a major challenge. The aim of this study was to integrate the vector permanently in the genome. In this study, the phiC31 site-specific recombination system was used for prolonged maintenance of the vector in the CHO cells. In this laboratory experimental study, a restriction enzyme cut site (PvuII) was selected as a cloning site in the pSVM vector using CLC software. Cutting with this enzyme led to create two blunt ends into the vector. Two specific primers were designed for isolation and amplification of attB region from pDrBB2 vector, using Oligo®5 software. Gel electrophoresis and PCR analysis were performed on the amplified fragment in order to confirmation of its structure. The attB region was successfully integrated in to pSVM vector and was shown by gel electrophoresis. Colony PCR technique has revealed the correct structure of the reconstructed vectors. In this study, the phiC31 integrate catalyzes recombination between pseudo-attP sites (in CHO chromosomes) and attB site. A new construct has been made in this project containing attB site. This construct has to be introduced into CHO cells accompanied with pCMV-int vector (co-transfection) containing integrase. This integrase facilitates the incorporation of the pSVM vector into the chromosome, in order to get a permanent existence of the new construct into the CHO cells.