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

近年来，在基因组计划中克隆出了许多新的基因，但对这些基因的鉴定还不够充分。病毒载体可以作为这种高效表征的候选物，然而，一次构建多个病毒载体作为表达库是困难的。在这项研究中，我们展示了一种独特的系统，通过使用重组酶介导的盒式交换（RMCE）反应，在表达位点特异性重组酶的细胞中，将小转染质粒（供体质粒）中感兴趣的基因直接替换为复制病毒基因组中的替代基因，从而产生病毒载体作为文库。例如，我们选择复制缺陷腺病毒载体（第一代rAd）作为病毒载体，选择Cre/loxP系统作为RMCE。我们已经确定了不与野生型loxP （wt loxP）重组，而只与V重组的排他突变体loxP (V)，结合病毒ci的阳性选择，更多的s-作用包装信号与插入物连接，在表达cre的293细胞中感染4个周期后，目的载体的富集率超过90%。当wt - loxP和V相距30kb时，RMCE反应仍能替代dna，但效率有所下降。另一方面，使用缺乏v的供体质粒检测RMCE的保真度，在表达cre的293细胞中感染三个周期后，检测到表达gfp的rAd，该rAd用于本研究感兴趣的基因，并且只有在正确的RMCE后才能产生。我们通过限制稀释分离出这些意想不到的rad，并使用限制性酶切和测序表征了基因组结构。我们在这些意想不到的rAds中检测到RMCE过程中产生的许多非同源重组产物。然而，我们可以通过优化RMCE的效率和最小化感染周期数来控制这种意外的rAd产生。由于这种利用RMCE反应构建病毒载体的新方法能够高效地生成目的载体，因此该方法不仅适用于其他DNA病毒载体的构建，也适用于基于病毒的cdna文库的构建。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