A series of Dictyostelium expression vectors for recombination cloning.
We describe a series of Dictyostelium expression vectors for recombination cloning using the Gateway technology. DNA fragments generated by high fidelity polymerase chain reaction are cloned by topoisomerase-mediated ligation, then recombined into any of several Dictyostelium expression vectors using phage lambda LR recombinase. No restriction enzymes are used in this procedure. Coding regions can be expressed from their own promoters, or from a strong actin 15 promoter as a native protein, or with an amino or carboxyl-terminal GFP fusion. Gene promoters of interest can be analyzed by controlled expression of GFP and beta-galactosidase. These vectors allow for rapid and simple characterization of novel DNA, and are ideal for high-throughput studies.
Thomason
Peter A
PA
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. P.Thomason@bham.ac.uk
Brazill
Derrick T
DT
Cox
Edward C
EC
D000818:Animals; D010582:Bacteriophage lambda; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D026942:DNA Topoisomerases; D004023:Dictyostelium / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic / Q000235:genetics; D001616:beta-Galactosidase
D017931:DNA Primers; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase; D026942:DNA Topoisomerases