title: Effect of the rabbit alphas1-casein gene distal enhancer on the expression of a reporter gene in vitro and in vivo.
abstract: Several gene constructs containing the firefly luciferase gene and the herpes simplex virus thymidine kinase gene promoter (TK) were used to evaluate the transcriptional activity of the distal enhancer (-3442, -3285) of the rabbit alphas1-casein gene. Six copies of the enhancer (6i) were added upstream of the TK-luciferase construct in the presence or absence of the chicken beta-globin 5'HS4 insulator. The activity of the constructs was tested by transient transfection in CHO cells and in rabbit primary mammary cell cultured on plastic or on floating collagen. Constructs were also tested in stably transfected mouse mammary HC11 cells. In all cell types the multimerized alphas1-casein enhancer strongly stimulated luciferase gene expression in the presence of lactogenic hormones. It was also sensitive to the extracellular matrix in rabbit primary mammary cells. The constructs were used to generate transgenic mice. The 6i TK transgenic animals expressed the luciferase gene at very low levels irrespectively of the physiological state. No preferential expression in the mammary gland was observed. Addition of 5'HS4 insulator to the 6i TK construct did not prevent silencing in most of the transgenic lines. However, two lines expressed high luciferase levels specifically in the mammary gland. Our data suggest that 6i may confer, when insulated properly, a higher and mammary-specific expression to the TK promoter.
authors.affiliation: Bioprotein Technologies, 63 Boulevard Massena, 75013, Paris, France. pantano@jouy.inra.fr
authors.forename: Thais
authors.initials: T
authors.lastname: Pantano
authors.forename: Geneviève
authors.initials: G
authors.lastname: Jolivet
authors.forename: Sonia
authors.initials: S
authors.lastname: Prince
authors.forename: Celeste
authors.initials: C
authors.lastname: Menck-Le Bourhis
authors.forename: Caroline
authors.initials: C
authors.lastname: Maeder
authors.forename: Céline
authors.initials: C
authors.lastname: Viglietta
authors.forename: Sylvie
authors.initials: S
authors.lastname: Rival
authors.forename: Louis-Marie
authors.initials: LM
authors.lastname: Houdebine
mesh_terms: D000818:Animals; D016466:CHO Cells; D002364:Caseins / Q000096:biosynthesis*; D002460:Cell Line; D002478:Cells, Cultured; D002645:Chickens; D006224:Cricetinae; D004742:Enhancer Elements, Genetic; D005109:Extracellular Matrix / Q000378:metabolism; D005260:Female; D017930:Genes, Reporter; D005914:Globins / Q000235:genetics; D007438:Introns; D007774:Lactation; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008297:Male; D008321:Mammary Glands, Animal / Q000166:cytology; D051379:Mice; D008822:Mice, Transgenic; D008954:Models, Biological; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011817:Rabbits; D018139:Simplexvirus / Q000201:enzymology; D013937:Thymidine Kinase / Q000235:genetics; D014018:Tissue Distribution; D014162:Transfection; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D002364:Caseins; D005914:Globins; D008156:Luciferases; D013937:Thymidine Kinase; D001616:beta-Galactosidase