Retrotransposon-mediated Gene Transfer for Animal Cells

Abstract

Gene delivery methods for animal cells are one of the most important tools in biotechnology fields such as pharmaceutical protein production, generation of transgenic animals and gene therapy. Because retrotransposons can move their own sequences to new genomic locations by a “copy-and-paste” process known as retrotransposition, we attempted to develop a novel gene transfer system based on retrotransposon. A full-length long interspersed element-1 (LINE-1) contains a 5’ untranslated region (5’UTR), two non-overlapping open reading frames (ORFs) separated by a short inter-ORF sequence, and a 3’UTR terminating in an adenosine-rich tract. We constructed a LINE-1 vector plasmid including components necessary for retrotransposition. An intron-disrupted Neo reporter gene and a scFv-Fc expression unit under the control of CMV promoter were added into 3’UTR in order to evaluate retrotransposition and express scFv-Fc. CHO-K1 cells transfected with the plasmids were screened with G418. The established cell clones produced scFv-Fc proteins in the culture medium. To control retrotransposition steadily, we also established retrotransposon systems that supply ORF2 or ORF1–2 separately. Genomic PCR analysis revealed that transgene sequences derived from the LINE-1 vector were positive in all clones. All the clones tested produced scFv-Fc in the culture medium.