A novel system for simultaneous or sequential integration of multiple gene-loading vectors into a defined site of a human artificial chromosome

Teruhiko Suzuki; Yasuhiro Kazuki; Mitsuo Oshimura; Takahiko Hara

doi:10.1371/journal.pone.0110404

A novel system for simultaneous or sequential integration of multiple gene-loading vectors into a defined site of a human artificial chromosome

PLoS One. 2014 Oct 10;9(10):e110404. doi: 10.1371/journal.pone.0110404. eCollection 2014.

Authors

Teruhiko Suzuki¹, Yasuhiro Kazuki², Mitsuo Oshimura³, Takahiko Hara⁴

Affiliations

¹ Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Kamikitazawa, Setagaya-ku, Tokyo, Japan.
² Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Yonago, Tottori, Japan; Chromosome Engineering Research Center, Tottori University, Yonago, Tottori, Japan.
³ Chromosome Engineering Research Center, Tottori University, Yonago, Tottori, Japan.
⁴ Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Kamikitazawa, Setagaya-ku, Tokyo, Japan; Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan.

Abstract

Human artificial chromosomes (HACs) are gene-delivery vectors suitable for introducing large DNA fragments into mammalian cells. Although a HAC theoretically incorporates multiple gene expression cassettes of unlimited DNA size, its application has been limited because the conventional gene-loading system accepts only one gene-loading vector (GLV) into a HAC. We report a novel method for the simultaneous or sequential integration of multiple GLVs into a HAC vector (designated as the SIM system) via combined usage of Cre, FLP, Bxb1, and φC31 recombinase/integrase. As a proof of principle, we first attempted simultaneous integration of three GLVs encoding EGFP, Venus, and TdTomato into a gene-loading site of a HAC in CHO cells. These cells successfully expressed all three fluorescent proteins. Furthermore, microcell-mediated transfer of HACs enabled the expression of those fluorescent proteins in recipient cells. We next demonstrated that GLVs could be introduced into a HAC one-by-one via reciprocal usage of recombinase/integrase. Lastly, we introduced a fourth GLV into a HAC after simultaneous integration of three GLVs by FLP-mediated DNA recombination. The SIM system expands the applicability of HAC vectors and is useful for various biomedical studies, including cell reprogramming.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Base Sequence
Cell Line
Chromosomes, Artificial, Human*
Gene Expression
Gene Order
Gene Transfer Techniques*
Genes, Reporter
Genetic Vectors*
Homologous Recombination*
Humans
Molecular Sequence Data
Sequence Alignment
Transgenes*

Grants and funding

This work was supported in part by the Japan Society for the Promotion of Science KAKENHI Grant numbers 23390256 (T.H.) and 24790696 (T.S.) (http://www.jsps.go.jp/english/index.html). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.