Grozdanov Petar N, MacDonald Clinton C
Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center;
Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center.
J Vis Exp. 2015 Feb 9(96):52235. doi: 10.3791/52235.
Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1α) promoter to drive gene expression. Plasmid vectors containing hEF1α are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1α promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1α promoter part 1, hEF1α promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.
吉布森组装（GA）克隆为传统DNA克隆方法提供了一种快速、可靠且灵活的替代方案。我们使用GA来创建定制质粒，用于在小鼠胚胎干细胞（mESCs）中表达外源基因。在SV40或人巨细胞病毒启动子控制下的外源基因在转染到mESCs后表达迅速减弱。解决这种表达减弱的方法是使用人延伸因子-1α（hEF1α）启动子来驱动基因表达。含有hEF1α的质粒载体不像含有SV40或CMV的质粒那样广泛可得，尤其是那些还含有N端3xFLAG标签的质粒。此处描述的方案是一种快速方法，用于创建在hEF1α启动子表达调控下表达FLAG标签的CstF-64和CstF-64突变体的质粒。GA使用DNA外切酶、DNA聚合酶和DNA连接酶的混合物，使DNA片段重叠末端的克隆成为可能。基于我们可用的模板DNA，我们设计构建体使其组装成单个序列。我们的设计使用了四个DNA片段：pcDNA 3.1载体骨架、hEF1α启动子第1部分、hEF1α启动子第2部分（其包含作为双链合成DNA片段购买的3xFLAG标签），以及CstF-64或特定的CstF-64突变体。将这些片段的序列上传到引物生成工具，以设计用于生成DNA片段的合适PCR引物。PCR后，将DNA片段与含有选择标记的载体混合，并组装GA克隆反应。从单个转化细菌菌落中分离质粒。质粒的初步筛选通过限制性消化进行，随后进行测序。总之，GA使我们能够在5天内创建用于基因表达的定制质粒，包括构建体筛选和验证。
