Homologous recombination-mediated cloning and manipulation of genomic DNA regions using Gateway and recombineering systems.
BACKGROUND
Employing genomic DNA clones to characterise gene attributes has several advantages over the use of cDNA clones, including the presence of native transcription and translation regulatory sequences as well as a representation of the complete repertoire of potential splice variants encoded by the gene. However, working with genomic DNA clones has traditionally been tedious due to their large size relative to cDNA clones and the presence, absence or position of particular restriction enzyme sites that may complicate conventional in vitro cloning procedures.


RESULTS
To enable efficient cloning and manipulation of genomic DNA fragments for the purposes of gene expression and reporter-gene studies we have combined aspects of the Gateway system and a bacteriophage-based homologous recombination (i.e. recombineering) system. To apply the method for characterising plant genes we developed novel Gateway and plant transformation vectors that are of small size and incorporate selectable markers which enable efficient identification of recombinant clones. We demonstrate that the genomic coding region of a gene can be directly cloned into a Gateway Entry vector by recombineering enabling its subsequent transfer to Gateway Expression vectors. We also demonstrate how the coding and regulatory regions of a gene can be directly cloned into a plant transformation vector by recombineering. This construct was then rapidly converted into a novel Gateway Expression vector incorporating cognate 5' and 3' regulatory regions by using recombineering to replace the intervening coding region with the Gateway Destination cassette. Such expression vectors can be applied to characterise gene regulatory regions through development of reporter-gene fusions, using the Gateway Entry clones of GUS and GFP described here, or for ectopic expression of a coding region cloned into a Gateway Entry vector. We exemplify the utility of this approach with the Arabidopsis PAP85 gene and demonstrate that the expression profile of a PAP85::GUS transgene highly corresponds with native PAP85 expression.


CONCLUSIONS
We describe a novel combination of the favourable attributes of the Gateway and recombineering systems to enable efficient cloning and manipulation of genomic DNA clones for more effective characterisation of gene function. Although the system and plasmid vectors described here were developed for applications in plants, the general approach is broadly applicable to gene characterisation studies in many biological systems.
Rozwadowski
Kevin
K
Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, Saskatchewan, Canada, S7N 0X2. RozwadowskiK@agr.gc.ca
Yang
Wen
W
Kagale
Sateesh
S
D017360:Arabidopsis / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000378:metabolism*; D020869:Gene Expression Profiling; D017343:Genes, Plant; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D016678:Genome / Q000235:genetics*; D005966:Glucuronidase / Q000378:metabolism; D016254:Mutagenesis, Insertional; D016366:Open Reading Frames / Q000235:genetics; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D014157:Transcription Factors; D014170:Transformation, Genetic; D019076:Transgenes
C091601:ABI3 protein, Arabidopsis; D029681:Arabidopsis Proteins; D014157:Transcription Factors; D004247:DNA; D005966:Glucuronidase