Udo Hiroshi
Department of Biology, Graduate School of Science, Kyushu University, Fukuoka, Japan.
PLoS One. 2015 Sep 30;10(9):e0139349. doi: 10.1371/journal.pone.0139349. eCollection 2015.
One of the most basic techniques in biomedical research is cDNA cloning for expression studies in mammalian cells. Vaccinia topoisomerase I-mediated cloning (TOPO cloning by Invitrogen) allows fast and efficient recombination of PCR-amplified DNAs. Among TOPO vectors, a pcDNA3.1 directional cloning vector is particularly convenient, since it can be used for expression analysis immediately after cloning. However, I found that the cloning efficiency was reduced when RT-PCR products were used as inserts (about one-quarter). Since TOPO vectors accept any PCR products, contaminating fragments in the insert DNA create negative clones. Therefore, I designed a new mammalian expression vector enabling positive blue white selection in Vaccinia topoisomerase I-mediated cloning. The method utilized a short nontoxic LacZα peptide as a linker for GFP fusion. When cDNAs were properly inserted into the vector, minimal expression of the fusion proteins in E. coli (harboring lacZΔM15) resulted in formation of blue colonies on X-gal plates. This method improved both cloning efficiency (75%) and directional cloning (99%) by distinguishing some of the negative clones having non-cording sequences, since these inserts often disturbed translation of lacZα. Recombinant plasmids were directly applied to expression studies using GFP as a reporter. Utilization of the P2A peptide allowed for separate expression of GFP. In addition, the preparation of Vaccinia topoisomerase I-linked vectors was streamlined, which consisted of successive enzymatic reactions with a single precipitation step, completing in 3 hr. The arrangement of unique restriction sites enabled further modification of vector components for specific applications. This system provides an alternative method for cDNA cloning and expression in mammalian cells.
生物医学研究中最基本的技术之一是用于哺乳动物细胞表达研究的cDNA克隆。痘苗拓扑异构酶I介导的克隆（Invitrogen公司的TOPO克隆）可实现PCR扩增DNA的快速高效重组。在TOPO载体中，pcDNA3.1定向克隆载体特别方便，因为克隆后可立即用于表达分析。然而，我发现当使用RT-PCR产物作为插入片段时，克隆效率会降低（约为四分之一）。由于TOPO载体可接受任何PCR产物，插入DNA中的污染片段会产生阴性克隆。因此，我设计了一种新的哺乳动物表达载体，可在痘苗拓扑异构酶I介导的克隆中实现阳性蓝白斑筛选。该方法利用一种短的无毒LacZα肽作为GFP融合的接头。当cDNA正确插入载体时，融合蛋白在大肠杆菌（携带lacZΔM15）中的最低表达会导致在X-gal平板上形成蓝色菌落。该方法通过区分一些具有非编码序列的阴性克隆提高了克隆效率（75%）和定向克隆效率（99%），因为这些插入片段常常会干扰lacZα的翻译。重组质粒直接用于以GFP为报告基因的表达研究。利用P2A肽可实现GFP的单独表达。此外，简化了痘苗拓扑异构酶I连接载体的制备过程，该过程由连续的酶促反应和单个沉淀步骤组成，3小时内即可完成。独特的限制性酶切位点排列使得载体组件可针对特定应用进行进一步修饰。该系统为哺乳动物细胞中的cDNA克隆和表达提供了一种替代方法。
