a method for producing transgenic cells using a multi-integrase system on a human artificial chromosome vector一个方法生产转基因细胞使用multi-integrase系统在人类人工染色体向量.pdfVIP

A Method for Producing Transgenic Cells Using a Multi- Integrase System on a Human Artificial Chromosome Vector 1,2 1,3 4 4 1,3 Shigeyuki Yamaguchi , Yasuhiro Kazuki , Yuji Nakayama , Eiji Nanba , Mitsuo Oshimura , Tetsuya Ohbayashi2* 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 Abstract 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 site- specific 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 h