Ogawa Yoichi, Dansako Tomoko, Yano Kentaro, Sakurai Nozomu, Suzuki Hideyuki, Aoki Koh, Noji Masaaki, Saito Kazuki, Shibata Daisuke
Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan.
Plant Cell Physiol. 2008 Feb;49(2):242-50. doi: 10.1093/pcp/pcm181. Epub 2008 Jan 4.
We established a large-scale, high-throughput protocol to construct Arabidopsis thaliana suspension-cultured cell lines, each of which carries a single transgene, using Agrobacterium-mediated transformation. We took advantage of RIKEN Arabidopsis full-length (RAFL) cDNA clones and the Gateway cloning system for high-throughput preparation of binary vectors carrying individual full-length cDNA sequences. Throughout all cloning steps, multiple-well plates were used to treat 96 samples simultaneously in a high-throughput manner. The optimal conditions for Agrobacterium-mediated transformation of 96 independent binary vector constructs were established to obtain transgenic cell lines efficiently. We evaluated the protocol by generating transgenic Arabidopsis T87 cell lines carrying individual 96 metabolism-related RAFL cDNA fragments, and showed that the protocol was useful for high-throughput and large-scale production of gain-of-function lines for functional genomics.
我们建立了一种大规模、高通量的实验方案，利用农杆菌介导的转化方法构建拟南芥悬浮培养细胞系，每个细胞系携带一个单一的转基因。我们利用日本理化学研究所拟南芥全长（RAFL）cDNA克隆和Gateway克隆系统，高通量制备携带单个全长cDNA序列的二元载体。在所有克隆步骤中，均使用多孔板以高通量方式同时处理96个样品。确定了农杆菌介导转化96个独立二元载体构建体的最佳条件，以高效获得转基因细胞系。我们通过生成携带96个与代谢相关的RAFL cDNA片段的转基因拟南芥T87细胞系来评估该实验方案，结果表明该方案对于功能基因组学中功能获得型细胞系的高通量大规模生产很有用。
