A Method for Producing Transgenic Cells Using a Multi-Integrase System on a Human Artificial Chromosome Vector

et al. (2011) A Method for Producing Transgenic Cells Using a Multi-Integrase System on a Human Artificial Chromosome Vector. PLoS ONE 6(2): e17267. doi:10.1371/journal.pone.0017267 A Method for Producing Transgenic Cells Using a Multi- Integrase System on a Human Artificial Chromosome Vector Shigeyuki Yamaguchi 0 Yasuhiro Kazuki 0 Yuji Nakayama 0 Eiji Nanba 0 Mitsuo Oshimura 0 Tetsuya 0 Joseph Najbauer, City of Hope National Medical Center and Beckman Research Institute, United States of America 0 1 Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Sciences, Tottori University , Yonago , Japan , 2 Division of Laboratory Animal Science, Research Center for Bioscience and Technology, Tottori University , Yonago , Japan , 3 Chromosome Engineering Research Center, Tottori University , Yonago , Japan , 4 Division of Functional Genomics, Research Center for Bioscience and Technology, Tottori University , Yonago , Japan The production of cells capable of expressing gene(s) of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and animal transgenesis. The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and WC31, has major benefits for the efficiency of transgenesis. Recent work on integrases from WC31, R4, TP901-1 and Bxb1 phages demonstrated that these recombinases catalyze sitespecific recombination in mammalian cells. In the present study, we examined the activities of integrases on site-specific recombination and gene expression in mammalian cells. We designed a human artificial chromosome (HAC) vector containing five recombination sites (WC31 attP, R4 attP, TP901-1 attP, Bxb1 attP and FRT; multi-integrase HAC vector) and de novo mammalian codon-optimized integrases. The multi-integrase HAC vector has several functions, including gene integration in a precise locus and avoiding genomic position effects; therefore, it was used as a platform to investigate integrase activities. Integrases carried out site-specific recombination at frequencies ranging from 39.3-96.8%. Additionally, we observed homogenous gene expression in 77.3-87.5% of colonies obtained using the multi-integrase HAC vector. This vector is also transferable to another cell line, and is capable of accepting genes of interest in this environment. These data suggest that integrases have high DNA recombination efficiencies in mammalian cells. The multi-integrase HAC vector enables us to produce transgene-expressing cells efficiently and create platform cell lines for gene expression. - Funding: Funding was provided by the Core Research for Evolutional Science and Technology (CREST) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (http://www.jst.go.jp/kisoken/crest/en/index.html); the New Energy and Industrial Technology Development Organization (NEDO) from the Ministry of Economy, Trade and Industry of Japan (http://www.nedo.go.jp/english/index.html); and Research Fellowships from the Japan Society for the Promotion of Science for Young Scientists (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. Competing Interests: The authors have declared that no competing interests exist. Many methods are available to produce transgenic cells for the functional studies of genes, drug discovery and gene therapy. The most common method used to produce these cells relies on random integration of the gene after transfection of plasmid DNA or transduction with viruses. These methods are followed by antibiotic selection of a stable pool of cells and functional screening to identify individual clones that have the correct function(s). However, random integration into chromosomes is inefficient [1], and the expression levels of genes vary greatly due to positional effects and the number of copies inserted [2,3,4,5]. As a result, the process of generating and selecting gene expression cells can be labor intensive and extremely time consuming. It is a widely held view that new gene expression technology for mammalian cells should optimally include targeting the gene to a transcriptional hot spot in the genome [6]. Although homologous recombination for targeted integration is very specific, it suffers from exceedingly low frequencies [7]. To increase the speed and efficiency of generating transgenic cells, alternative technologies have been considered. The sitespecific gene recombination systems, such as bacteriophage P1derived Cre, yeast-derived FLP, and phage integrases typified by bacteriophage WC31-derived integrase, are example of these. These systems have been used widely for the targeted recombination of transgenes into the genome of mammalian cells [8]. Additionally, these site-specific recombinases can induce the deletion or inversion of DNA sequences leading to conditional gene inactivation or expression [9]. The most powerful tool for site-specific recombination in vitro [10,11] and in vivo [12,13] is Cre recombinase, which catalyzes reciprocal site-specific recombination between two loxP sites. A second site-specific recombinase, FLPe, based on FLP from Saccharomyces cerevisiae, has also been used in mammalian cells and recognizes distinct FRT sites [14]. FLPe is an improved and temperature stable version of the FLP recombinase. However, in assays with chromosomally located FRT sites, the efficiency of FLPe only exhibits 10% Cre recombination activity [15]. A third class of site-specific recombinases, the serine integrases, as typified by WC31 integrase, also displays activity in mammalian cells. Tyrosine integrases such as l phage integrase are also used in mammalian cells [16,17]. However, the recombination efficiency of tyrosine family integrases is lower than that of serine family integrases, and we therefore used serine integrases in this study [18]. The WC31 integrase was originally isolated from a Streptomyces phage [19], and the 605 amino acid WC31 integrase can perform recombination between attP and attB sites, which is different to Cre and FLPe in human cells [20]. Recombination between attP and attB sites generates hybrid attL or attR sites that are no longer substrates for the integrase in the absence of additional cofactors [20,21]. Furthermore, WC31 integrase facilitates integration of attB-bearing plasmids at endogenous sequences with partial identity to attP. These are termed pseudo attP sites [22]. The ability of WC31 integrase to mediate transgene integration into native pseudo attP sites has been used in gene therapy experiments to produce therapeutically useful levels of Factor IX, correct human type VII collagen genes in human keratinocytes that contained mutants of this gene [18], and to produce dystrophin in mouse muscle-derived stem cells, human myob (...truncated)