title: Transformation and characterization of mutant human fibroblasts defective in peroxisome assembly.
abstract: Human skin fibroblasts deficient in peroxisome biogenesis were transformed by transfecting SV40 ori- DNA with the use of an electroporator, and the biochemical, immunocytochemical, and cytogenetic properties of the transformants were analyzed. Cells (1 x 10(6)) from a patient with Zellweger syndrome and one with neonatal adrenoleukodystrophy were suspended with 2 micrograms of SV40 ori- DNA in PBS; then a high-voltage pulse (2000 V, 30 microseconds) was generated two times. Several colonies expressing large T-antigen were picked up 4 weeks after transfection. Doubling time of the transformants was about half of that and the saturation density was 5 to 10 times greater than that of the parental cells. Biochemical abnormalities including defective lignoceric acid oxidation, dihydroxyacetone phosphate acyltransferase deficiency, and disturbed biosynthesis of peroxisomal beta-oxidation enzymes were preserved in the transformants. Peroxisomes were defective in all colonies, as determined by immunofluorescence staining using anti-catalase IgG. Cell fusion studies confirmed that the transformants belong to the same complementation groups as those of the parental cells. These transformed mutant cell lines are expected to be useful tools for investigating the pathogenesis of inherited diseases related to defects in peroxisome biogenesis.
authors.affiliation: Department of Pediatrics, School of Medicine, Gifu University, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Okamoto
authors.forename: Y
authors.initials: Y
authors.lastname: Suzuki
authors.forename: N
authors.initials: N
authors.lastname: Shimozawa
authors.forename: S
authors.initials: S
authors.lastname: Yajima
authors.forename: M
authors.initials: M
authors.lastname: Masuno
authors.forename: T
authors.initials: T
authors.lastname: Orii
mesh_terms: D000217:Acyltransferases / Q000032:analysis / Q000172:deficiency; D000326:Adrenoleukodystrophy / Q000235:genetics*; D002461:Cell Line, Transformed; D004099:Dihydroxyacetone Phosphate / Q000378:metabolism; D005227:Fatty Acids / Q000032:analysis; D005347:Fibroblasts; D006801:Humans; D008830:Microbodies / Q000378:metabolism*; D009154:Mutation; D014170:Transformation, Genetic; D015211:Zellweger Syndrome / Q000235:genetics*
chemical_list: D005227:Fatty Acids; D004099:Dihydroxyacetone Phosphate; D000217:Acyltransferases; C010210:lignoceric acid