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A Versatile System for USER Cloning-Based Assembly of Expression Vectors for Mammalian Cell Engineering

机译：基于用户克隆的哺乳动物细胞工程表达载体组装通用系统。

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摘要

A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique – USER cloning – to rapidly construct mammalian expression vectors of multiple DNA fragments and with maximum flexibility, both for choice of vector backbone and cargo. The vector system includes a set of basic vectors and a toolbox containing a multitude of DNA building blocks including promoters, terminators, selectable marker- and reporter genes, and sequences encoding an internal ribosome entry site, cellular localization signals and epitope- and purification tags. Building blocks in the toolbox can be easily combined as they contain defined and tested Flexible Assembly Sequence Tags, FASTs. USER cloning with FASTs allows rapid swaps of gene, promoter or selection marker in existing plasmids and simple construction of vectors encoding proteins, which are fused to fluorescence-, purification-, localization-, or epitope tags. The mammalian expression vector assembly platform currently allows for the assembly of up to seven fragments in a single cloning step with correct directionality and with a cloning efficiency above 90%. The functionality of basic vectors for FAST assembly was tested and validated by transient expression of fluorescent model proteins in CHO, U-2-OS and HEK293 cell lines. In this test, we included many of the most common vector elements for heterologous gene expression in mammalian cells, in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors.
机译：开发了一种用于蛋白质生产，细胞生物学分析和细胞工厂工程的新型多功能哺乳动物载体系统。该载体系统采用基于无结扎尿嘧啶切除的技术-用户克隆-快速构建具有多个DNA片段且具有最大灵活性的哺乳动物表达载体，从而可以选择载体骨架和货物。载体系统包括一组基本载体和一个工具箱，其中包含多个DNA构件，包括启动子，终止子，选择标记和报告基因以及编码内部核糖体进入位点，细胞定位信号以及表位和纯化标签的序列。工具箱中的构建基块可以轻松组合，因为它们包含已定义并经过测试的柔性装配顺序标签FAST。用户使用FAST进行克隆可以在现有质粒中快速交换基因，启动子或选择标记，并可以简单构建编码蛋白质的载体，将其融合到荧光，纯化，定位或表位标签上。哺乳动物表达载体组装平台目前允许在单个克隆步骤中以正确的方向性和高达90％以上的克隆效率组装多达七个片段。通过在CHO，U-2-OS和HEK293细胞系中瞬时表达荧光模型蛋白来测试和验证用于FAST装配的基本载体的功能。在此测试中，我们包括许多最常见的哺乳动物细胞中异源基因表达载体元件，此外该系统可被其他用户完全扩展。该载体系统旨在通过快速生成可定制基因表达载体的高保真组装能力，促进哺乳动物细胞（如新测序的CHO细胞系）的高通量基因组规模研究。

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