RESUMO
Bacterial backbone sequences of conventional plasmid vectors have been reported to exhibit negative effects on transgene expression in mammalian cells, such as cytotoxicity and gene silencing. Minicircle DNA vectors can be employed to overcome these issues and to improve the transfection efficiency because of their smaller size. In this study, transgenes are integrated into the hypoxanthine phosphoribosyltransferase (hprt) locus of Chinese hamster ovary (CHO) cells by the Cre-loxP system using minicircle DNA vectors as transgene donors. The targeted transgene integration efficiency is improved 2-3-fold (≈1.4%) using minicircle DNA vectors compared with conventional plasmid vectors. Moreover, clones with expected structures after transgene integration are obtained with a high frequency. When a transgene together with bacterial sequences derived from a plasmid vector is integrated into the hprt locus, the cell growth rate and antibody titer decrease. These results indicate that minicircle DNA vectors are more suitable than conventional plasmid vectors for transgene delivery in recombinant protein production using CHO cells.
Assuntos
Vetores Genéticos/genética , Integrases/genética , Plasmídeos/genética , Transfecção/métodos , Animais , Células CHO , Cricetinae , Cricetulus , Escherichia coli/genética , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transgenes/genéticaRESUMO
We have previously developed an accumulative site-specific gene integration system (AGIS) using Cre-recombinase and mutated loxP sites. AGIS enables repeated transgene integration into a predetermined chromosomal site in mammalian cells. However, the process of establishing cells with multiple integrated copies of the transgene is still time-consuming. In the present study, we describe an improved version of AGIS that facilitates and accelerates the establishment of high-producer Chinese hamster ovary (CHO) cells. Two donor vectors were simultaneously introduced into the cells in a single transfection. Cells with successfully targeted transgene integration were screened based on a change in the color of the reporter fluorescent protein that they express. Repeated rounds of integration allowed the transgene copy number to be increased. As a model, an scFv-Fc antibody gene was integrated into the hprt locus of the CHO cell genome. After three rounds of integration, a high-producer CHO cell clone with six copies of the scFv-Fc gene was successfully established. scFv-Fc productivity was approximately four-fold greater than a control cell line harboring a single copy of the transgene. This newly designed AGIS procedure should facilitate the development of producer cells suitable for biopharmaceutical protein production.
