Chemical regulated production of cDNAs from genomic DNA fragments in plants.
We have developed a new chemical inducible genetic system that allows for the isolation of any cDNA molecule from in vitro generated genomic transgenes in transgenic plants. This system, termed regulated in vivo cDNA generation (RIDE), permits both targeted isolation of individual full-length cDNA molecules and random isolation of any partial or full-length cDNA from in planta genomic libraries. The RIDE system makes use of the 17-beta estradiol-inducible promoter system linked to intron donor and acceptor sites in a new binary vector configuration. In transgenic Arabidopsis and tobacco plants, we show that the RIDE system can isolate low-abundance full-length cDNAs previously unattainable by conventional means at high efficiencies (75-85%). The ability to randomly isolate individual exons and exons spliced together from genomic libraries in planta suggest that this system can be used for the isolation of any cDNA molecules. The RIDE system thus appears to be an efficient and versatile system for the generation of potentially any cDNA molecule. Moreover, the ORF structural data generated will be of value in both verifying and correcting computational ORF predications in the databases available to the scientific community.
Møller
Simon Geir
SG
Laboratory of Plant Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10021-3699, USA.
Chua
Nam-Hai
NH
D017360:Arabidopsis / Q000235:genetics*; D001483:Base Sequence; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000096:biosynthesis* / Q000235:genetics*; D004958:Estradiol / Q000494:pharmacology*; D005091:Exons / Q000235:genetics; D017343:Genes, Plant / Q000235:genetics; D018745:Genome, Plant; D015698:Genomic Library; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics; D014026:Nicotiana / Q000235:genetics*
D018076:DNA, Complementary; D018749:RNA, Plant; D004958:Estradiol