title: Temperature sensitive simian virus 40 large T antigen immortalization of murine odontoblast cell cultures: establishment of clonal odontoblast cell line.
abstract: During tooth formation instructive epithelial-mesenchymal interactions result in the cytodifferentiation of ectomesenchymal cells into odontoblasts which produce the dentin extracellular matrix (DECM). The purpose of our study was to establish a stable murine odontoblast cell line by immortalization of odontoblasts using retrovirus transfection. In order to accomplish this goal, we utilized a previously characterized odontoblast monolayer cell culture system supportive of odontoblast cytodifferentiation from dental papilla mesenchyme (DPM), expression and secretion of a DECM and dentin biomineralization. First mandibular molars from E-18 Swiss Webster mice were dissected, the DPM isolated, and pulp cells dissociated. Pulp cells (5 x 10(5)/well) were plated as monolayers and grown in alpha-MEM supplemented with 10% FCS, 100 units/ml penicillin and streptomycin, 50 micrograms/ml ascorbic acid. Cultures were maintained for 6 days at 37 degrees C in a humidified atmosphere of 95% air and 5% CO2, with media changes every two days. Immortalization was performed using a recombinant defective retrovirus containing the temperature sensitive SV-40 large T antigen cDNA and the neomycin (G418) resistance gene recovered from CRE packaging cells. Cultures were infected for 24 h with CRE conditioned medium containing 8 micrograms/ml of polybrene, the media was replaced with selective media containing 300 micrograms/ml of G418, and the cultures incubated at 33 degrees C for one month with media changes every 3-5 days. Neomycin resistant cells were cloned by serial dilution to single cells in 96-well culture plates and grown in selection medium at 33 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: University of Texas Health Science Center at San Antonio, Dental School, Department of Pediatric Dentistry 78284-7888, USA.
authors.forename: M
authors.initials: M
authors.lastname: MacDougall
authors.forename: F
authors.initials: F
authors.lastname: Thiemann
authors.forename: H
authors.initials: H
authors.lastname: Ta
authors.forename: P
authors.initials: P
authors.lastname: Hsu
authors.forename: L S
authors.initials: LS
authors.lastname: Chen
authors.forename: M L
authors.initials: ML
authors.lastname: Snead
mesh_terms: D000818:Animals; D000957:Antigens, Viral, Tumor / Q000235:genetics*; D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003771:Dental Papilla / Q000166:cytology / Q000378:metabolism; D003782:Dental Pulp / Q000166:cytology / Q000378:metabolism; D003804:Dentin / Q000166:cytology / Q000378:metabolism; D003810:Dentinogenesis / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D008648:Mesoderm / Q000166:cytology / Q000378:metabolism; D051379:Mice; D009355:Neomycin; D009804:Odontoblasts / Q000378:metabolism*; D009805:Odontogenesis / Q000235:genetics; D013539:Simian virus 40 / Q000235:genetics* / Q000276:immunology; D013696:Temperature; D014074:Tooth Calcification / Q000235:genetics; D014162:Transfection
chemical_list: D000957:Antigens, Viral, Tumor; D009355:Neomycin