Creating precise GFP fusions in plasmids using yeast homologous recombination.
Using a combination of primer amplification, homologous recombination, and yeast genetics, we established a method for creating precise promoter and protein fusions in genes originating from organisms other than yeast. One major advantage of this new method is its versatility. Fusions can be produced within a target gene without constraints regarding the site of insertion. Thus, fusions can be generated within a target sequence exactly at the site desired, and all sequences upstream and downstream of the insertion site were preserved. To illustrate the general applicability of this technique, we fused the gene encoding GFP to a Caenorhabditis elegans homologue of the dishevelled gene, dsh-2. This approach is not restricted to GFP fusions but can be utilized to create fusions between almost any two sequences regardless of the source. Therefore, this method provides a flexible alternative to other PCR-mediated techniques.
Hawkins
Nancy C
NC
Simon Fraser University, 8888 University Drive, Burnaby, B.C., Canada V5A 1S6.
Garriga
Gian
G
Beh
Christopher T
CT
D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000096:biosynthesis / Q000235:genetics; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D018390:Gene Targeting / Q000379:methods; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015202:Protein Engineering / Q000379:methods; D011786:Quality Control; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic; D017385:Sequence Homology; D015003:Yeasts / Q000145:classification / Q000235:genetics*
D029742:Caenorhabditis elegans Proteins; D017931:DNA Primers; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins