title: 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.
abstract: 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.
authors.affiliation: Department of Biology, Graduate School of Science, Kyushu University, Fukuoka, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Udo
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D008322:Mammals; D011995:Recombination, Genetic; D012641:Selection, Genetic; D014616:Vaccinia virus / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D049452:Green Fluorescent Proteins; D004264:DNA Topoisomerases, Type I; C528909:TOP1 protein, human