Department of Biological Engineering, Massachusetts Institute of Technology, 40 Ames Street, Cambridge, MA 02142, USA, Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, 40 Ames Street, Cambridge, MA 02142, USA and INRIA Paris-Rocquencourt, Le Chesnay, 78153, France.
Nucleic Acids Res. 2013 Sep;41(16):e156. doi: 10.1093/nar/gkt605. Epub 2013 Jul 11.
We developed a framework for quick and reliable construction of complex gene circuits for genetically engineering mammalian cells. Our hierarchical framework is based on a novel nucleotide addressing system for defining the position of each part in an overall circuit. With this framework, we demonstrate construction of synthetic gene circuits of up to 64 kb in size comprising 11 transcription units and 33 basic parts. We show robust gene expression control of multiple transcription units by small molecule inducers in human cells with transient transfection and stable chromosomal integration of these circuits. This framework enables development of complex gene circuits for engineering mammalian cells with unprecedented speed, reliability and scalability and should have broad applicability in a variety of areas including mammalian cell fermentation, cell fate reprogramming and cell-based assays.
我们开发了一个快速可靠的构建复杂基因回路的框架，用于基因工程改造哺乳动物细胞。我们的分层框架基于一种新的核苷酸寻址系统，用于定义整体回路中每个部分的位置。利用这个框架，我们展示了多达 64kb 大小的合成基因回路的构建，包括 11 个转录单元和 33 个基本元件。我们通过小分子诱导物在瞬时转染和这些回路的稳定染色体整合的人类细胞中展示了对多个转录单元的稳健基因表达控制。这个框架使构建用于工程改造哺乳动物细胞的复杂基因回路具有前所未有的速度、可靠性和可扩展性，并应在包括哺乳动物细胞发酵、细胞命运重编程和基于细胞的测定等多种领域具有广泛的适用性。
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