Gene CATCHR--gene cloning and tagging for Caenorhabditis elegans using yeast homologous recombination: a novel approach for the analysis of gene expression.
Expression patterns of gene products provide important insights into gene function. Reporter constructs are frequently used to analyze gene expression in Caenorhabditis elegans, but the sequence context of a given gene is inevitably altered in such constructs. As a result, these transgenes may lack regulatory elements required for proper gene expression. We developed Gene Catchr, a novel method of generating reporter constructs that exploits yeast homologous recombination (YHR) to subclone and tag worm genes while preserving their local sequence context. YHR facilitates the cloning of large genomic regions, allowing the isolation of regulatory sequences in promoters, introns, untranslated regions and flanking DNA. The endogenous regulatory context of a given gene is thus preserved, producing expression patterns that are as accurate as possible. Gene Catchr is flexible: any tag can be inserted at any position without introducing extra sequence. Each step is simple and can be adapted to process multiple genes in parallel. We show that expression patterns derived from Gene Catchr transgenes are consistent with previous reports and also describe novel expression data. Mutant rescue assays demonstrate that Gene Catchr-generated transgenes are functional. Our results validate the use of Gene Catchr as a valuable tool to study spatiotemporal gene expression.
Sassi
Holly E
HE
Department of Medical Genetics and Microbiology, University of Toronto, 1 King's College Circle, Toronto, Canada, M5S 1A8.
Renihan
Stephanie
S
Spence
Andrew M
AM
Cooperstock
Ramona L
RL
D000818:Animals; D030801:Animals, Genetically Modified; D017173:Caenorhabditis elegans / Q000235:genetics* / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis; D015870:Gene Expression; D017238:Genes, Helminth; D017930:Genes, Reporter; D023281:Genomics / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D009154:Mutation; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D016324:Sequence Tagged Sites; D015003:Yeasts / Q000235:genetics
D029742:Caenorhabditis elegans Proteins; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins