A dicistronic construct allows easy detection of human CFTR expression from YAC DNA in human cells.
We have made a dicistronic construct where the picornaviral internal ribosome-entry site (IRES) driving the expression of the beta-geo gene has been inserted into the 3'untranslated region of the human CFTR gene present in a YAC. When introduced into the human cell line Caco-2 expressing the CFTR gene, the expression of the dicistronic gene can be detected by lacZ staining and follows the accumulation of the endogenous CFTR mRNA upon differentiation of the cells. These data demonstrate that this IRES-based approach presents an alternative to mRNA in situ hybridisation and allows detection of expression in an autologous system.
Vassaux
G
G
Department of Biochemistry and Molecular Genetics, Imperial College School of Medicine at St Mary's, Norfolk Place, London W2 1PG, UK. g.vassaux@ic.ac.uk
Huxley
C
C
D000230:Adenocarcinoma; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics*; D003110:Colonic Neoplasms; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D004351:Drug Resistance / Q000235:genetics; D015870:Gene Expression; D005796:Genes; D005822:Genetic Vectors; D006801:Humans; D009355:Neomycin; D010849:Picornaviridae / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D012270:Ribosomes; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000235:genetics
C505032:CFTR protein, human; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D001616:beta-Galactosidase; D009355:Neomycin