title: High efficiency single step production of expression plasmids from cDNA clones using the Flexi Vector cloning system.
abstract: The success of structural genomics and proteomics initiatives is dependent on the availability of target genes in vectors suitable for protein production. Here, we compare two high-throughput methods for producing expression vectors from plasmid-derived cDNA fragments. Expression vectors were constructed for compatibility with the Gateway recombination cloning system and the Flexi Vector restriction-based cloning system. Cloning protocols for each system were conducted in parallel for 96 different target genes from PCR through the production of sequence-verified expression clones. The short nucleotide sequences required to prepare the target open reading frames for Flexi Vector cloning allowed a single-step PCR protocol, resulting in fewer mutations relative to the Gateway protocol. Furthermore, through initial cloning of the target open reading frames directly into an expression vector, the Flexi Vector system gave time and cost savings compared to the protocol required for the Gateway system. Within the Flexi Vector system, genes were transferred between four different expression vectors. The efficiency of gene transfer between Flexi Vectors depended on including a region of sequence identity adjacent to one of the restriction sites. With the proper construction in the flanking sequence of the vector, gene transfer efficiencies of 95-98% were demonstrated.
authors.affiliation: Department of Biochemistry, Center for Eukaryotic Structural Genomics, University of Wisconsin, Madison, 53706-1549, USA.
authors.forename: Paul G
authors.initials: PG
authors.lastname: Blommel
authors.forename: Peter A
authors.initials: PA
authors.lastname: Martin
authors.forename: Russell L
authors.initials: RL
authors.lastname: Wrobel
authors.forename: Eric
authors.initials: E
authors.lastname: Steffen
authors.forename: Brian G
authors.initials: BG
authors.lastname: Fox
mesh_terms: D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D016366:Open Reading Frames / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D011995:Recombination, Genetic; D014162:Transfection
chemical_list: D018076:DNA, Complementary