An Alternative Method to Facilitate cDNA Cloning for Expression Studies in Mammalian Cells by Introducing Positive Blue White Selection in Vaccinia Topoisomerase I-Mediated Recombination

PLOS ONE, Dec 2019

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.

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An Alternative Method to Facilitate cDNA Cloning for Expression Studies in Mammalian Cells by Introducing Positive Blue White Selection in Vaccinia Topoisomerase I-Mediated Recombination

September An Alternative Method to Facilitate cDNA Cloning for Expression Studies in Mammalian Cells by Introducing Positive Blue White Selection in Vaccinia Topoisomerase I-Mediated Recombination Hiroshi Udo 0 1 0 Department of Biology, Graduate School of Science, Kyushu University , Fukuoka , Japan 1 Editor: Yuntao Wu, George Mason University , UNITED STATES - 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. Vaccinia topoisomerase I-mediated cloning, known as TOPO cloning by Invitrogen, has been widely used in biological and medical research. It was originally developed by Shuman [1], which revolutionized the way to clone PCR-amplified DNAs. It is quick and efficient without relying on restriction enzymes and DNA ligases. Since the cloning procedure is very simple, this method is suitable for large scale cloning as well [2]. The cloning method is based on unique properties of Vaccinia topoisomerase I [3]. The enzyme recognizes a specific pentapyrimidine sequence of 5’-(C/T)CCTT#-3’ and hydrolyzes the phosphodiester bond at the 3’-end of its recognition site [4]. Upon cleavage, a covalent bond is formed between DNA and the enzyme (3’-phosphate of the DNA and tyrosine 274 of the enzyme), and the nicked strand is relaxed in the mean time. The phosphor-tyrosyl bond energy is then utilized to relegate to the 5’-hydroxyl end of the cleaved strand, and the enzyme is released from the DNA after reaction. When the enzyme reacts with a linear double-stranded DNA having its recognition site near the 3’-end, a short terminal fragment is released from the nicked strand, and the enzyme remains covalently bound to the DNA. This enzyme-DNA complex is stable, and is able to recombine with an exogenous DNA having a 5’-hydroxyl end. In the original cloning procedure, insert DNAs were first reacted with Vaccinia topoisomerase I, and the resulting enzyme-insert DNA complex was then added to the dephosphorylated vector DNA [1]. This procedure was later modified to a well-known format, in which the enzyme was linked with the vector DNA (TOPO vector) to accept insert DNAs having 5’hydroxyl ends. This is particularly suitable for cloning of PCR-amplified DNAs, since chemically synthesized primers have a 5’-hydroxyl end. If TOPO vectors are ready, recombination can be done by simply adding PCR products. A variety of TOPO vectors are commercially available today. However, the most popular applications may be cDNA cloning for expression studies in mammalian cells. Since expression vectors require cDNAs to be inserted in the correct orientation, directional TOPO cloning vectors are suitable (such as a pcDNA3.1 directional TOPO cloning vector from Invitrogen). Oriented insertion was facilitated by the inclusion of four bases “CACC” (corresponding to a portion of Kozak sequence [5]) at the 5’-end of the insert DNA and also at the cloning site of the vector. Furthermore, pcDNA3.1 allows expression of cloned cDNAs in mammalian cells under control of a CMV promoter [6]. This is time and cost-efficient since conventional strategies involve two steps, cDNA cloning into a cloning vector and subcloning into an expression vector. I attempted to clone mouse cDNAs using the pcDNA3.1 directional (...truncated)


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Hiroshi Udo. An Alternative Method to Facilitate cDNA Cloning for Expression Studies in Mammalian Cells by Introducing Positive Blue White Selection in Vaccinia Topoisomerase I-Mediated Recombination, PLOS ONE, 2015, Volume 10, Issue 9, DOI: 10.1371/journal.pone.0139349