Rapid expression screening of Caenorhabditis elegans homeobox open reading frames using a two-step polymerase chain reaction promoter-gfp reporter construction technique

• Published in: Gene Vol. 212; no. 1; pp. 127 - 135
• Main Authors: Cassata, Giuseppe, Kagoshima, Hiroshi, F. Prétôt, René, Aspöck, Gudrun, Niklaus, Gisela, Bürglin, Thomas R
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 28.05.1998
• Subjects: Animals; Animals, Genetically Modified; Base Sequence; Caenorhabditis elegans; Caenorhabditis elegans - embryology; Caenorhabditis elegans - genetics; ceh-38; Cloning, Molecular; DNA Primers - genetics; Expression pattern; Gene Expression; Genes, Helminth; Genes, Homeobox; Genes, Reporter; Green Fluorescent Proteins; Homeobox gene; Long-range PCR; Luminescent Proteins - genetics; Open Reading Frames; Polymerase Chain Reaction - methods; Promoter Regions, Genetic; ceh-38; EtBr, ethidiumbromide; Expression pattern; UV, ultraviolet; DIC, differential interference contrast; GFP, Green fluorescent protein; C., Caenorhabditis; Long-range PCR; Caenorhabditis elegans; WWW, World Wide Web; TAE, Tris(hydroxymethyl)aminomethane-acetate-ethylenediaminetetraacetic acid; NLS, nuclear localization sequence; PCR, polymerase chain reaction; Homeobox gene; ceh, Caenorhabditis elegans homeobox gene
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/S0378-1119(98)00137-1

In this paper a description is given of the expression pattern of the Caenorhabditis elegans homeobox gene ceh-38 using GFP reporter constructs, which were generated using a two-step polymerase chain reaction (PCR) procedure. This method allows fast analysis of genes of interest by looking at their expression in vivo using their putative promoter region to control the expression of a reporter gene. In this case the method was applied to screen C. elegans homeobox-containing genes to identify those that are expressed in the head and nervous system. The C. elegans genome project has made rapid progress, and more than 79 megabases of genomic data with several thousand open reading frames are available. This information can be used to design primers from putative promoter regions, which are amplified using long-range PCR. The long-range PCR product is then directly joined to the vector in a long-range Fill-in PCR. Since many genome projects are advancing rapidly, this approach should also be applicable for other model systems, and the method lends itself to automation, since no gel-purification steps are necessary. ceh-38 is a member of the ONECUT class of homeobox genes. Expression of ceh-38 starts during embryogenesis. In larvae and adults, expression was seen in many different types of tissues, such as the pharynx, gut, hypodermis and many nerve cells.