Preparation of Minicircle DNA Vectors for Gene Knock-in to Mammalian Cells.

Minicircle DNA vectors (MCs) are gene carriers with favorable characteristics for gene therapy and the production of recombinant cells using animal cells as hosts. Typically, MCs are prepared by inserting target gene fragments as an expression unit into a standard plasmid vector, followed by the removal of bacterial sequences. In this study, we report a method for MC preparation utilizing the Cre-loxP recombination system. By inserting the target gene fragments between two loxP sites introduced into the plasmid vector, the plasmid backbone can be removed through the action of Cre recombinase. We explored optimal reaction conditions for MC generation using Cre. Furthermore, site-specific knock-ins into the CHO cell genome were performed using the remaining loxP sequence in the generated MCs via the Cre-loxP reaction. When MCs were used as gene donors, significant improvements in gene integration efficiency and enhanced gene expression in CHO cells were observed compared with conventional plasmids. The MC preparation and gene knock-in techniques developed in this study are highly useful for CHO cell engineering.