LAUSR

© The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Dear Editor, Since the discovery of short hairpin RNA (shRNA) vector-mediated RNA interference (RNAi), this technology has been widely used in cancer research for its specificity, potency, and convenience. However, researchers may find it costly to purchase commercial vectors from biocompanies or timeand labor-consuming to construct their own shRNA vectors using traditional method by inserting annealed duplex into digested vectors. Despite intensive efforts to accelerate shRNA vector cloning in laboratories, the development of a reliable, rapid, convenient, and cost-effective method is still in great demand. To this end, we developed a novel method named SuperSH (Super rapid cloning of shRNA vector) for the effective and rapid construction of shRNA-expressing vectors based on high-performance DNA polymerase and seamless cloning technique [1] (Additional file 1: Figure S1a; the detailed methods can be found in Additional file 1). In our SuperSH method, the shRNA sequences are introduced into the vector via a pair of polymerase chain reaction (PCR) primers rather than via annealed duplex. In detail, the 3′ ends of the primers are designed to bind the template to initiate a PCR to amplify the vector backbone, and the 5′ portions are designed to introduce the sequences of interest as well as to form a short homologous arm for subsequent recombination via seamless cloning [1]. After the seamless recombination reaction, the seamed vector is transformed into competent E. coli using a quick transformation protocol that takes only 5 min [2] (Additional file 1: Figure S1). It’s important to note that the SuperSH method requires a linearized vector template to achieve effective and rapid cloning, skipping the need for purification steps throughout the cloning procedure. For this aim, we created an intermediate vector pSuperSH-MX by introducing restriction enzyme sites for two non-isocaudomers, MluI and XbaI, to allow complete linearization of the template plasmid and to avoid self-ligation in subsequent cloning procedures as well. Our SuperSH method outperforms traditional shRNA cloning method based on annealed complementary oligonucleotide duplex in the following aspects (Additional file 1: Figure S1b). Firstly, the SuperSH method requires only three steps, namely a low-cycle number PCR to amplify the vector backbone, a recombination reaction to seam the vector, and a quick transformation to replicate the vectors in E. coli, which altogether greatly saves researchers’ hands-on work. Secondly, the above procedures also allow users to complete shRNA vector cloning in as short as 30 min. Thirdly, SuperSH uses much shorter oligonucleotides in the application of shRNA cloning, as seamless cloning requires only a short homologous arm (10–15 nucleotides) to cyclize the PCR product, and thus there is no need to synthesize the full length of shRNA sequence (Additional file 1: Figure S2). In addition, we observed this novel method to be much more reliable and Open Access Cancer Communications