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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使用多整合酶系统在具有调节动粒的人类人工染色体上组装基因组 DNA 片段或基因的方法

能够携带多个转基因到 Mb 大小基因组位点的细胞的产生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) 大基因组片段的克隆；(ii) 在精确位置插入多个 DNA 块和 (iii) 从细胞中消除组装区域的能力。在本研究中，我们设计了利用重组酶 Cre、ΦC31 和 ΦBT1 的迭代集成系统 (IIS)，并将其与具有受调节着丝粒 ( alphoid ^tetO -HAC)的人类人工染色体 (HAC)结合。我们已经证明 IIS- alphoid ^tetO - HAC 系统是一种有价值的遗传工具，通过从 HAC 上的多个片段重新组装功能基因。IIS-alphoid ^tetO-HAC 与其他基于人工染色体的系统相比具有几个显着的优势。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 DNA 之间只留下一个小的插入 (<60 bp) 疤痕，允许从片段重新组装的基因正确拼接；标记交换系统也会改变细胞颜色，并在每个 DNA 插入步骤中进行反选择标记，从而简化正确克隆的选择；并且存在防错机制以去除带有错误掺入的 DNA 片段的细胞，从而提高组装的完整性。此外，IIS-alphoid ^tetO-HAC 携带感兴趣的基因座是可移动的，提供了独特的可能性，将细胞系恢复到其转化前的状态，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- alphoid ^tetO - HAC 允许研究复杂的生物医学途径、基因调控，并有可能用一组预定的基因来设计合成染色体。