Development of novel method for the construction of viral vectors using the site-specific recombinase

近年来，在基因组计划中，许多新的基因被克隆出来，但这些基因的特性还不够。病毒载体可以是这种有效表征的候选者，然而，同时构建许多病毒载体作为表达文库是困难的。在这项研究中，我们证明了一个独特的系统，用于产生病毒载体作为一个库，直接取代一个小的转染质粒（供体质粒）的目的基因与替换基因的复制病毒基因组中的位点特异性重组酶表达细胞使用重组酶介导的盒式交换（RMCE）反应。例如，我们选择复制缺陷型腺病毒载体（第一代rAd）作为病毒载体，Cre/loxP系统用于RMCE。我们已经确定了唯一的突变体loxP（V），它不与野生型loxP（wt loxP）重组，而只与V有效重组。 ...更多信息 在表达Cre的293细胞中，经过4个循环的感染后，目的载体富集到90%以上。另一方面，使用缺乏V的供体质粒检查了RMCE的保真度。在表达Cre的293细胞中感染三个循环后，表达GFP的rAd（其用于本研究中的目的基因并且预期仅在正确的RMCE后产生），被发现了我们通过有限稀释法分离了这些意想不到的rAds，并使用限制性酶切和测序来表征基因组结构。在这些rAds中检测到了RMCE过程中产生的多种非同源重组产物。然而，我们可以通过优化RMCE的效率并最大限度地减少感染循环次数来控制这种意外rAd产物的产生。由于这种利用RMCE反应构建病毒载体的新方法可以有效地产生目的载体，因此该方法不仅适用于其他DNA病毒载体的开发，而且适用于基于病毒的cDNA库。少

Recently, many kind of novel gene have been cloned during genome project, however the characterization of these genes was not sufficient. The virus vector can be a candidate for this efficient characterization, however construction of numerous virus vectors at once, as an expression library, has been difficult. In this research, we demonstrated a unique system for generating viral vectors as a library by directly substituting a gene of interest in a small-transfected plasmid (donor plasmid) with a replaced gene in a replicating viral genome in the site specific recombinase-expressing cells using the recombinase-mediated cassette exchange (RMCE) reaction. For example, we chose the replication-deficient adenovirus vector (1st generation rAd) for the virus vector and Cre/loxP system for RMCE. We have already determined the exclusive mutants loxP (V), which does not recombine with wild type loxP (wt loxP) but efficiently only with V. In combination with a positive selection of the viral ci … More s-acting packaging signal connected with the insert, the purpose vector was enriched to over 90% after four cycles of infection in Cre-expressing 293 cells. And this RMCE reaction was able to substitute DNAs even when wt loxP and V are 30kb apart, though this efficiency decreased.On the other hand, the fidelity of RMCE was examined using a donor plasmid lacking V. After three cycles of infection in Cre-expressing 293 cells, the GFP-expressing rAd, which was used for the gene of interest in this research and was expected to be generated only after correct RMCE, was detected. We isolated these unexpected rAds by limiting dilution and characterized the genome structures using restriction-enzyme digestion and sequencing. We detected many kind of non-homologous recombination products generated during the RMCE in these unexpected rAds. However, we may control generation of this unexpected rAd production by optimizing of the efficiency of RMCE and minimizing the number of cycles infection.Because this novel method to construct the virus vector using RMCE reaction was efficiently generating the purpose vector, this method may also be applicable to not only for the development of the other DNA virus vector but also for viral-based cDNA-library. Less