| Products/Services Used | Details | Operation |
|---|---|---|
| Gene Synthesis | DNA fragments of loxP, FRT, φC31 attP, φC31 attB, Bxb1 attP, Bxb1 attB, and adenovirus genome-derived splicing acceptor sequence were synthesized (GenScript) based on previously reported sequences [16], [28]. Sequences of these elements are listed in Table S1. All the SIM cassettes were cloned in a pUC57-based cloning vector (GenScript). φC31 integrase and Bxb1 integrase expression vectors were a kind gift from Dr. T. Ohbayashi (Tottori University) [16]. For construction of GLVs, an appropriate SIM cassette was inserted into a vector encoding a GOI via conventional restriction digestion and ligation. For the fluorescent gene expression units, PGK, EF1, or CAG promoter-driven EGFP, TdTomato, and Venusexpression units were used. These expression units were encompassed by chicken HS4 insulator sequence [6]. | Get A Quote |
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... More
