The production of cells capable of carrying multiple transgenes to Mb-size genomic loci has multiple applications in biomedicine and biotechnology. In order to achieve this goal, three key steps are required: (i) cloning of large genomic segments; (ii) insertion of multiple DNA blocks at a precise location and (iii) the capability to eliminate the assembled region from cells. In this study, we designed the iterative integration system (IIS) that utilizes recombinases Cre, ΦC31 and ΦBT1, and combined it with a human artificial chromosome (HAC) possessing a regulated kinetochore (alphoid^tetO-HAC). We have demonstrated that the IIS-alphoid^tetO-HAC system is a valuable genetic tool by reassembling a functional gene from multiple segments on the HAC. IIS-alphoid^tetO-HAC has several notable advantages over other artificial chromosome-based systems. This includes the potential to assemble an unlimited number of genomic DNA segments; a DNA assembly process that leaves only a small insertion (<60 bp) scar between adjacent DNA, allowing genes reassembled from segments to be spliced correctly; a marker exchange system that also changes cell color, and counter-selection markers at each DNA insertion step, simplifying selection of correct clones; and presence of an error proofing mechanism to remove cells with misincorporated DNA segments, which improves the integrity of assembly. In addition, the IIS-alphoid^tetO-HAC carrying a locus of interest is removable, offering the unique possibility to revert the cell line to its pretransformed state and compare the phenotypes of human cells with and without a functional copy of a gene(s). Thus, IIS-alphoid^tetO-HAC allows investigation of complex biomedical pathways, gene(s) regulation, and has the potential to engineer synthetic chromosomes with a predetermined set of genes.

中文翻译：

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用多整合酶系统在人动染色体上调节动粒体组装基因组DNA片段或基因的方法

能够携带多个转基因至Mb大小的基因组位点的细胞的生产在生物医学和生物技术中具有多种应用。为了实现这一目标，需要三个关键步骤：（i）克隆大的基因组片段；（ii）在精确位置插入多个DNA区块，以及（iii）从细胞中消除组装区域的能力。在这项研究中，我们设计了利用重组酶Cre，ΦC31和ΦBT1的迭代整合系统（IIS），并将其与具有受控动粒（人种^tetO -HAC）的人类人工染色体（HAC）结合在一起。我们已经证明了IIS- aphoid ^tetO -HAC系统是一种有价值的遗传工具，它可以通过重组HAC多个片段上的功能基因来实现。IIS-白斑样^的tetO-HAC与其他基于人工染色体的系统相比，具有许多显着优势。这包括组装无限数量的基因组DNA片段的潜力；DNA组装过程，在相邻的DNA之间仅留下一小段插入（<60 bp）的疤痕，从而使从片段重组而来的基因得以正确剪接；一个标记交换系统，它还能改变细胞的颜色，并且在每个DNA插入步骤都可以进行反选择标记，从而简化了正确克隆的选择；以及存在防错机制以去除带有错误掺入的DNA片段的细胞，从而提高了组装的完整性。此外，IIS的双簧管^tetO带有目标基因座的-HAC是可移动的，从而提供了独特的可能性，可将细胞系还原至其预先转化的状态，并比较具有和不具有基因功能拷贝的人类细胞的表型。因此，IIS-二肽^tetO - HAC允许研究复杂的生物医学途径，基因调控，并具有用预定的基因集改造合成染色体的潜力。