title: Development of cellular models for ribosomal protein S19 (RPS19)-deficient diamond-blackfan anemia using inducible expression of siRNA against RPS19.
abstract: Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia in which 25% of the patients have a mutation in the ribosomal protein S19 (RPS19) gene. No models exist for RPS19-deficient DBA and the molecular pathogenesis is unknown. To establish an in vitro inducible model for DBA, human erythroid leukemic cell lines, TF-1 and UT-7 cells, were cotransduced with a lentiviral vector expressing the green fluorescent protein (GFP) gene and small interfering RNA (siRNA) against RPS19 controlled by a tet operator regulatory element and another transactivator vector containing the red fluorescent protein (RFP) gene and the cDNA encoding a tetracycline-controllable transcriptional repressor. Following transduction, the RFP-positive and GFP-negative cell population was sorted by flow cytometry. Upon incubation with doxycycline (0.5 mug/ml), more than 98% of cells expressed GFP and the siRNA. Significant suppression of erythroid differentiation, cell growth, and colony formation was observed in cells treated with siRNA against RPS19 but not in cells treated with a control vector. These findings show that RPS19 plays an important role in the regulation of hematopoietic cell proliferation and erythroid differentiation. These novel cell lines represent models for RPS19-deficient DBA and can be used to identify the molecular mechanisms in RPS19-deficient DBA.
authors.affiliation: Molecular Medicine and Gene Therapy, Lund University, BMC A12, 221 84 Lund, Sweden.
authors.forename: Koichi
authors.initials: K
authors.lastname: Miyake
authors.forename: Johan
authors.initials: J
authors.lastname: Flygare
authors.forename: Thomas
authors.initials: T
authors.lastname: Kiefer
authors.forename: Taiju
authors.initials: T
authors.lastname: Utsugisawa
authors.forename: Johan
authors.initials: J
authors.lastname: Richter
authors.forename: Zhi
authors.initials: Z
authors.lastname: Ma
authors.forename: Maciej
authors.initials: M
authors.lastname: Wiznerowicz
authors.forename: Didier
authors.initials: D
authors.lastname: Trono
authors.forename: Stefan
authors.initials: S
authors.lastname: Karlsson
mesh_terms: D029503:Anemia, Diamond-Blackfan / Q000209:etiology* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D015536:Down-Regulation; D004318:Doxycycline / Q000494:pharmacology; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism; D041905:Erythroid Cells / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics; D006410:Hematopoiesis / Q000235:genetics / Q000502:physiology; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D022423:Myeloid Cells / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D012269:Ribosomal Proteins / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D013234:Stem Cells
chemical_list: D034741:RNA, Small Interfering; D012269:Ribosomal Proteins; C040343:ribosomal protein S19; D004318:Doxycycline