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-disruptedNeoreporter 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.
Highlights
Gene delivery procedures for animal cells are one of the most important tools in biotechnology fields such as biopharmaceutical protein production, generation of transgenic animals and gene therapy
We have demonstrated targeted integration of transgenes into Chinese hamster ovary (CHO) cells using the Cre/loxP recombination system (Kameyama et al, 2010; Obayashi et al, 2012) and established high-producer CHO cells mediated by repeatedly introducing transgene expression units in an enzyme-dependent manner (Wang et al, 2017)
By using an efficient knock-in strategy designated precise integration into target chromosome (PITCh) (Nakade et al, 2014), we achieved homologous recombination (HR)independent large gene cassette knock-in for CHO cells using as TALEN- and CRISPR-mediated PITCh system (Sakuma et al, 2015)
Summary
Gene delivery procedures for animal cells are one of the most important tools in biotechnology fields such as biopharmaceutical protein production, generation of transgenic animals and gene therapy. In order to integrate exogenous genes efficiently into cell chromosomes, various methods represented by viral vectors and recombinases have been developed. We have generated transgenic chickens producing recombinant proteins using retroviral vectors for gene transfer (Kamihira et al, 2005; Kawabe et al, 2012). Genome editing tools using target-designable artificial nucleases such as zinc finger nucleases, TALEN and CRISPR/Cas systems have been widely used for genetic engineering of cells. By using an efficient knock-in strategy designated precise integration into target chromosome (PITCh) (Nakade et al, 2014), we achieved homologous recombination (HR)independent large gene cassette knock-in for CHO cells using as TALEN- and CRISPR-mediated PITCh system (Sakuma et al, 2015)
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