RESUMO
The task of identifying mutations in nucleic acid sequences is a vital component of research in mammalian genetics. With the advent of the polymerase chain reaction, several useful mutation detection techniques have evolved in recent years. The different methods have complementing strengths and a suitable procedure for virtually any experimental situation is now available.
Assuntos
Técnicas Genéticas , Mutação , Ácidos Nucleicos/genética , Animais , Sequência de Bases , DNA , Humanos , Dados de Sequência Molecular , Polimorfismo Genético , Sensibilidade e EspecificidadeRESUMO
Fanconi anaemia is an autosomal recessive disease for which four known complementation groups exist. Recently, the gene defective in complementation group C (FACC) has been cloned. In order to determine the fraction of Fanconi anaemia caused by FACC mutations, we used reverse transcription PCR and chemical mismatch cleavage (CMC) to examine the FACC cDNA in 17 FA cell lines. 4/17 patients (23.5%) had mutations in this gene. Two Ashkenazi-Jewish individuals were homozygous for an identical splice mutation. Three additional Jewish patients bearing this allele were found upon screening 21 other families. We conclude that a common mutation in FACC accounts for the majority of Fanconi anaemia in Ashkenazi-Jewish families.
Assuntos
Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Anemia de Fanconi/genética , Judeus/genética , Mutação , Proteínas Nucleares , Proteínas/genética , Alelos , Sequência de Bases , Linhagem Celular , Sequência Consenso , DNA/genética , Análise Mutacional de DNA , Éxons , Anemia de Fanconi/etnologia , Proteína do Grupo de Complementação C da Anemia de Fanconi , Proteínas de Grupos de Complementação da Anemia de Fanconi , Frequência do Gene , Genes Recessivos , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Splicing de RNA , Homologia de Sequência do Ácido NucleicoRESUMO
Current strategies for hepatic gene therapy are either quantitatively inefficient or suffer from lack of permanent gene expression. We have utilized an animal model of hereditary tyrosinaemia type I (HT1), a recessive liver disease caused by deficiency of fumarylacetoacetate hydrolase (FAH), to determine whether in vivo selection of corrected hepatocytes could improve the efficiency of liver gene transfer. As few as 1,000 transplanted wild-type hepatocytes were able to repopulate mutant liver, demonstrating their strong competitive growth advantage. Mutant hepatocytes corrected in situ by retroviral gene transfer were also positively selected. In mutant animals treated by multiple retrovirus injections >90% of hepatocytes became FAH positive and liver function was restored to normal. Our results demonstrate that in vivo selection is a useful strategy for hepatic gene therapy and may lead to effective treatment of human HT1 by retroviral gene transfer.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/terapia , Terapia Genética , Hidrolases/genética , Fígado/citologia , Tirosina/sangue , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Animais , Contagem de Células , Transplante de Células , Cicloexanonas/farmacologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Terapia Genética/efeitos adversos , Humanos , Hidrolases/antagonistas & inibidores , Hidrolases/deficiência , Técnicas Imunoenzimáticas , Fígado/efeitos dos fármacos , Fígado/metabolismo , Testes de Função Hepática , Neoplasias Hepáticas/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nitrobenzoatos/farmacologia , Retroviridae/genéticaRESUMO
Hereditary tyrosinaemia type I, a severe autosomal recessive metabolic disease, affects the liver and kidneys and is caused by deficiency of fumarylacetoacetate hydrolase (FAH). Mice homozygous for a FAH gene disruption have a neonatal lethal phenotype caused by liver dysfunction and do not represent an adequate model of the human disease. Here we demonstrate that treatment of affected animals with 2-(2-nitro-4-trifluoro-methylbenzyol)-1,3-cyclohexanedione abolished neonatal lethality, corrected liver function and partially normalized the altered expression pattern of hepatic mRNAs. The prolonged lifespan of affected animals resulted in a phenotype analogous to human tyrosinaemia type I including hepatocellular carcinoma. The adult FAH-/- mouse will serve as useful model for studies of the pathophysiology and treatment of hereditary tyrosinaemia type I as well as hepatic cancer.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/tratamento farmacológico , Cicloexanonas/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Hepatopatias/tratamento farmacológico , Nitrobenzoatos/uso terapêutico , Tirosina/sangue , Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Aminoácidos/sangue , Animais , Cicloexanonas/toxicidade , Modelos Animais de Doenças , Inibidores Enzimáticos/toxicidade , Feminino , Heptanoatos/metabolismo , Humanos , Hidrolases/deficiência , Fígado/efeitos dos fármacos , Fígado/ultraestrutura , Hepatopatias/genética , Hepatopatias/patologia , Neoplasias Hepáticas/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nitrobenzoatos/toxicidade , Pâncreas/patologia , RNA Mensageiro/metabolismo , alfa-Fetoproteínas/metabolismoRESUMO
Fanconi anaemia (FA) is an autosomal recessive disorder characterized by progressive pancytopenia, short stature, radial ray defects, skin hyperpigmentation and a predisposition to cancer. Cells from FA patients are hypersensitive to cell killing and chromosome breakage induced by DNA cross-linking agents such as mitomycin C (MMC) and diepoxybutane (DEB). Consequently, the defect in FA is thought to be in DNA crosslink repair. Additional cellular phenotypes of FA include oxygen sensitivity, poor cell growth and a G2 cell cycle delay. At least 5 complementation groups for Fanconi anaemia exist, termed A through E. One of the five FA genes, FA(C), has been identified by cDNA complementation, but no other FA genes have been mapped or cloned until now. The strategy of cDNA complementation, which was successful for identifying the FA(C) gene has not yet been successful for cloning additional FA genes. The alternative approach of linkage analysis, followed by positional cloning, is hindered in FA by genetic heterogeneity and the lack of a simple assay for determining complementation groups. In contrast to genetic linkage studies, microcell mediated chromosome transfer utilizes functional complementation to identify the disease bearing chromosome. Here we report the successful use of this technique to map the gene for the rare FA complementation group D (FA(D)).
Assuntos
Cromossomos Humanos Par 3 , Anemia de Fanconi/genética , Teste de Complementação Genética , Linhagem Celular , Mapeamento Cromossômico/métodos , Dano ao DNA , Anemia de Fanconi/patologia , HumanosRESUMO
Succinate semialdehyde dehydrogenase (ALDH5A1, encoding SSADH deficiency is a defect of 4-aminobutyric acid (GABA) degradation that manifests in humans as 4-hydroxybutyric (gamma-hydroxybutyric, GHB) aciduria. It is characterized by a non-specific neurological disorder including psychomotor retardation, language delay, seizures, hypotonia and ataxia. The current therapy, vigabatrin (VGB), is not uniformly successful. Here we report the development of Aldh5a1-deficient mice. At postnatal day 16-22 Aldh5a1-/- mice display ataxia and develop generalized seizures leading to rapid death. We observed increased amounts of GHB and total GABA in urine, brain and liver homogenates and detected significant gliosis in the hippocampus of Aldh5a1-/- mice. We found therapeutic intervention with phenobarbital or phenytoin ineffective, whereas intervention with vigabatrin or the GABAB receptor antagonist CGP 35348 (ref. 2) prevented tonic-clonic convulsions and significantly enhanced survival of the mutant mice. Because neurologic deterioration coincided with weaning, we hypothesized the presence of a protective compound in breast milk. Indeed, treatment of mutant mice with the amino acid taurine rescued Aldh5a1-/- mice. These findings provide insight into pathomechanisms and may have therapeutic relevance for the human SSADH deficiency disease and GHB overdose and toxicity.
Assuntos
Aldeído Oxirredutases/genética , Anticonvulsivantes/uso terapêutico , Convulsões/tratamento farmacológico , Convulsões/genética , Animais , Sequência de Bases , Encéfalo/metabolismo , Primers do DNA , Genótipo , Proteína Glial Fibrilar Ácida/metabolismo , Hidroxibutiratos/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Fenobarbital/uso terapêutico , Fenitoína/uso terapêutico , Receptores de GABA-B/metabolismo , Convulsões/enzimologia , Succinato-Semialdeído DesidrogenaseRESUMO
OBJECTIVE: Alkaptonuria (AKU) is a rare genetic disease which results in severe early onset osteoarthropathy. It has recently been shown that the subchondral interface is of key significance in disease pathogenesis. Human surgical tissues are often beyond this initial stage and there is no published murine model of pathogenesis, to study the natural history of the disease. The murine genotype exists but it has been reported not to demonstrate ochronotic osteoarthropathy consistent with the human disease. Recent anecdotal evidence of macroscopic renal ochronosis in a mouse model of tyrosinaemia led us to perform histological analysis of tissues of these mice that are known to be affected in human AKU. DESIGN: The homogentisate 1,2-dioxygenase Hgd(+/)(-)Fah(-)(/)(-) mouse can model either hereditary tyrosinaemia type I (HT1) or AKU depending on selection conditions. Mice having undergone Hgd reversion were sacrificed at various time points, and their tissues taken for histological analysis. Sections were stained with haematoxylin eosin (H&E) and Schmorl's reagent. RESULTS: Early time point observations at 8 months showed no sign of macroscopic ochronosis of tissues. Macroscopic examination at 13 months revealed ochronosis of the kidneys. Microscopic analysis of the kidneys revealed large pigmented nodules displaying distinct ochre colouration. Close microscopic examination of the distal femur and proximal fibula at the subchondral junctions revealed the presence of numerous pigmented chondrocytes. CONCLUSIONS: Here we present the first data showing ochronosis of tissues in a murine model of AKU. These preliminary histological observations provide a stimulus for further studies into the natural history of the disease to provide a greater understanding of this class of arthropathy.
Assuntos
Alcaptonúria/complicações , Condrócitos/patologia , Artropatias/patologia , Nefropatias/patologia , Ocronose/patologia , Animais , Modelos Animais de Doenças , Progressão da Doença , Feminino , Membro Posterior/patologia , Homogentisato 1,2-Dioxigenase/genética , Masculino , Camundongos , Ocronose/complicaçõesRESUMO
AIMS/HYPOTHESIS: We sought to determine the mRNA transcriptome of all major human pancreatic endocrine and exocrine cell subtypes, including human alpha, beta, duct and acinar cells. In addition, we identified the cell type-specific distribution of transcription factors, signalling ligands and their receptors. METHODS: Islet samples from healthy human donors were enzymatically dispersed to single cells and labelled with cell type-specific surface-reactive antibodies. Live endocrine and exocrine cell subpopulations were isolated by FACS and gene expression analyses were performed using microarray analysis and quantitative RT-PCR. Computational tools were used to evaluate receptor-ligand representation in these populations. RESULTS: Analysis of the transcriptomes of alpha, beta, large duct, small duct and acinar cells revealed previously unrecognised gene expression patterns in these cell types, including transcriptional regulators HOPX and HDAC9 in the human beta cell population. The abundance of some regulatory proteins was different from that reported in mouse tissue. For example, v-maf musculoaponeurotic fibrosarcoma oncogene homologue B (avian) (MAFB) was detected at equal levels in adult human alpha and beta cells, but is absent from adult mouse beta cells. Analysis of ligand-receptor interactions suggested that EPH receptor-ephrin communication between exocrine and endocrine cells contributes to pancreatic function. CONCLUSIONS/INTERPRETATION: This is the first comprehensive analysis of the transcriptomes of human exocrine and endocrine pancreatic cell types-including beta cells-and provides a useful resource for diabetes research. In addition, paracrine signalling pathways within the pancreas are shown. These results will help guide efforts to specify human beta cell fate by embryonic stem cell or induced pluripotent stem cell differentiation or genetic reprogramming.
Assuntos
Pâncreas/citologia , Pâncreas/metabolismo , Transcriptoma , Adulto , Células Cultivadas , Biologia Computacional/métodos , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células Secretoras de Glucagon/citologia , Células Secretoras de Glucagon/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Ligantes , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Pâncreas Exócrino/citologia , Pâncreas Exócrino/metabolismo , Ductos Pancreáticos/citologia , Ductos Pancreáticos/metabolismo , Comunicação Parácrina , RNA Mensageiro/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
The characterization of hepatic progenitor cells is of great scientific and clinical interest. Here we report that intravenous injection of adult bone marrow cells in the FAH(-/-) mouse, an animal model of tyrosinemia type I, rescued the mouse and restored the biochemical function of its liver. Moreover, within bone marrow, only rigorously purified hematopoietic stem cells gave rise to donor-derived hematopoietic and hepatic regeneration. This result seems to contradict the conventional assumptions of the germ layer origins of tissues such as the liver, and raises the question of whether the cells of the hematopoietic stem cell phenotype are pluripotent hematopoietic cells that retain the ability to transdifferentiate, or whether they are more primitive multipotent cells.
Assuntos
Diferenciação Celular , Transplante de Células , Células-Tronco Hematopoéticas/citologia , Hepatócitos/citologia , Hidrolases/deficiência , Regeneração Hepática , Fígado/patologia , Tirosinemias/terapia , Animais , Células da Medula Óssea/citologia , Separação Celular/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Tirosinemias/genética , Tirosinemias/patologia , Irradiação Corporal TotalRESUMO
The Bloom protein (BLM) and Topoisomerase IIIalpha are found in association with proteins of the Fanconi anemia (FA) pathway, a disorder manifesting increased cellular sensitivity to DNA crosslinking agents. In order to determine if the association reflects a functional interaction for the maintenance of genome stability, we have analyzed the effects of siRNA-mediated depletion of the proteins in human cells. Depletion of Topoisomerase IIIalpha or BLM leads to increased radial formation, as is seen in FA. BLM and Topoisomerase IIIalpha are epistatic to the FA pathway for suppression of radial formation in response to DNA interstrand crosslinks since depletion of either of them in FA cells does not increase radial formation. Depletion of Topoisomerase IIIalpha or BLM also causes an increase in sister chromatid exchanges, as is seen in Bloom syndrome cells. Human Fanconi anemia cells, however, do not demonstrate increased sister chromatid exchanges, separating this response from radial formation. Primary cell lines from mice defective in both Blm and Fancd2 have the same interstrand crosslink-induced genome instability as cells from mice deficient in the Fancd2 protein alone. These observations demonstrate that the association of BLM and Topoisomerase IIIalpha with Fanconi proteins is a functional one, delineating a BLM-Topoisomerase IIIalpha-Fanconi pathway that is critical for suppression of chromosome radial formation.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , DNA/metabolismo , Anemia de Fanconi/metabolismo , RecQ Helicases/metabolismo , Animais , Linhagem Celular , Reagentes de Ligações Cruzadas/farmacologia , DNA Topoisomerases Tipo I/genética , Anemia de Fanconi/genética , Instabilidade Genômica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitomicina/farmacologia , RNA Interferente Pequeno/genética , RecQ Helicases/genética , Troca de Cromátide IrmãRESUMO
Because ribosome biogenesis plays an essential role in cell proliferation, control mechanisms may have evolved to recognize lesions in this critical anabolic process. To test this possibility, we conditionally deleted the gene encoding 40S ribosomal protein S6 in the liver of adult mice. Unexpectedly, livers from fasted animals deficient in S6 grew in response to nutrients even though biogenesis of 40S ribosomes was abolished. However, liver cells failed to proliferate or induce cyclin E expression after partial hepatectomy, despite formation of active cyclin D-CDK4 complexes. These results imply that abrogation of 40S ribosome biogenesis may induce a checkpoint control that prevents cell cycle progression.
Assuntos
Divisão Celular , Fígado/citologia , Fígado/fisiologia , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas , Proteínas Ribossômicas/fisiologia , Animais , Ciclina D1/biossíntese , Ciclina D1/metabolismo , Ciclina E/genética , Ciclina E/metabolismo , Quinase 4 Dependente de Ciclina , Quinase 6 Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , DNA/biossíntese , Privação de Alimentos , Fase G1 , Deleção de Genes , Marcação de Genes , Hepatectomia , Interferon-alfa/farmacologia , Fígado/metabolismo , Regeneração Hepática , Camundongos , Camundongos Endogâmicos , Fosforilação , Polirribossomos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA Ribossômico/metabolismo , Proteína S6 Ribossômica , Proteínas Ribossômicas/genética , Ribossomos/metabolismo , Fase SRESUMO
The application of new diagnostic techniques has led to improvement in carrier detection and prenatal diagnosis in ornithine transcarbamylase deficiency. Progress has also been made towards somatic gene therapy.
Assuntos
Doença da Deficiência de Ornitina Carbomoiltransferase , Animais , Feminino , Genes , Triagem de Portadores Genéticos , Terapia Genética , Humanos , Masculino , Camundongos , Mutação , Ornitina Carbamoiltransferase/genética , Ornitina Carbamoiltransferase/metabolismo , Diagnóstico Pré-NatalRESUMO
p27(Kip1) is an important regulator of cyclin-dependent kinases. Studies with p27 knockout mice have revealed abnormalities in proliferation and differentiation of multiple cell types. Here we show that primary hepatocytes isolated from livers of adult p27 knockout mice exhibit higher levels of DNA synthesis activity in culture than do wild-type cells. Interestingly, we found that, compared with control hepatocytes, p27 knockout hepatocytes proliferate better after transplantation into diseased livers to reverse liver failure. These results reveal an aspect of p27 that could be used to benefit cell-based therapy.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Hepatócitos/transplante , Hepatopatias/cirurgia , Proteínas Supressoras de Tumor , Animais , Divisão Celular , Células Cultivadas , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/metabolismo , DNA/biossíntese , Hepatócitos/metabolismo , Hepatócitos/patologia , Hidrolases/genética , Hepatopatias/metabolismo , Hepatopatias/patologia , Transplante de Fígado , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Serina-Treonina Quinases/metabolismo , Tirosinemias/genética , Tirosinemias/metabolismo , Tirosinemias/patologia , Tirosinemias/cirurgiaRESUMO
We have analyzed the targeting frequencies and recombination products generated with isogenic vectors at the fah and fgr loci in embryonic stem cells. A single vector which could be linearized at different sites to generate either a replacement or an insertion vector was constructed for each locus. A replacement event predominated when the vectors were linearized at the edge of the homologous sequences, while an insertion event predominated when the vectors were linearized within the homologous sequences. However, the ratio of the targeting frequencies exhibited by the different vector configurations differed for the two loci. When the fgr vector was linearized as an insertion vector, the ratio of targeted to random integrations was four- to eightfold greater than when the vector was linearized as a replacement vector. By contrast, the ratio of targeted to random integrations at the fah locus did not vary with the linearization site of the vector. The different relationships between the targeting frequency and the vector configuration at the fgr and fah loci may indicate a DNA sequence or chromatin structure preference for different targeting pathways.
Assuntos
Marcação de Genes , Vetores Genéticos , Proteínas Proto-Oncogênicas/genética , Recombinação Genética , Animais , Células Cultivadas , Mapeamento Cromossômico , Cromossomos/ultraestrutura , Genes , Hidrolases/genética , Técnicas In Vitro , Camundongos , Proteínas Tirosina Quinases/genética , Mapeamento por Restrição , Quinases da Família srcRESUMO
Following introduction of DNA interstrand cross-links (ICLs), mammalian cells display chromosome breakage or cell cycle delay with a 4N DNA content. To further understand the nature of the delay, previously described as a G(2)/M arrest, we developed a protocol to generate ICLs during specific intervals of the cell cycle. Synchronous populations of G(1), S, and G(2) cells were treated with photoactivated 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and scored for normal passage into mitosis. In contrast to what was found for ionizing radiation, ICLs introduced during G(2) did not result in a G(2)/M arrest, mitotic arrest, or chromosome breakage. Rather, subsequent passage through S phase was required to trigger both chromosome breakage and arrest in the next cell cycle. Similarly, ICLs introduced during G(1) did not cause a G(1)/S arrest. We conclude that DNA replication is required to elicit the cellular responses of cell cycle arrest and genomic instability after psoralen-induced ICLs. In primary human fibroblasts, the 4N DNA content cell cycle arrest triggered by ICLs was long lasting but reversible. Kinetic analysis suggested that these cells could remove up to approximately 2,500 ICLs/genome at an average rate of 11 ICLs/genome/h.
Assuntos
Ciclo Celular , Reagentes de Ligações Cruzadas/farmacologia , Replicação do DNA , DNA/metabolismo , Trioxsaleno/análogos & derivados , Trioxsaleno/farmacologia , Bromodesoxiuridina/metabolismo , Ciclo Celular/efeitos dos fármacos , Separação Celular , Células Cultivadas , Meios de Cultura Livres de Soro/metabolismo , Replicação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fibroblastos/efeitos dos fármacos , Citometria de Fluxo , Humanos , Cinética , Masculino , Mitose/efeitos dos fármacos , Modelos Biológicos , Fatores de Tempo , Raios UltravioletaRESUMO
OBJECTIVE: Fanconi anemia (FA) is a human autosomal-recessive cancer susceptibility disorder characterized by multiple congenital abnormalities, progressive bone marrow failure, and cellular sensitivity to mitomycin C (MMC). FA has at least eight complementation groups (A, B, C, D1, D2, E, F, G), and six of the FA genes have been cloned. Several FA proteins, including FANCA, FANCC, FANCF, and FANCG, interact in a nuclear complex, and this complex is required for the activation (monoubiquitination) of the downstream FANCD2 protein. Activation of FANCD2 results in the assembly of FANCD2/BRCA1 foci. The aim of this study was to analyze the FA pathway in several FA patient-derived cell lines. MATERIALS AND METHODS: We generated an antibody to FANCF and analyzed FANCF expression in human lymphoblasts corresponding to all known FA subtypes. We systematically analyzed the FA pathway (FANCD2 monoubiquitination and assembly of FANCD2 nuclear foci) in patient-derived FA-F and FA-D1 cell lines. RESULTS: FANCF protein expression is normal in cells derived from all FA complementation groups except FA-F and does not vary during cell cycle progression. FANCF, but not FANCD2, is a component of the nuclear FA protein complex and appears to stabilize other subunits of the complex. FANCF is required for the monoubiquitination of the FANCD2 protein following ionizing radiation. FANCD2 is monoubiquitinated in FA-D1 cells, even though these cells are highly sensitive to MMC. CONCLUSIONS: The recently cloned FANCF protein is required for FANCD2 activation, and the yet uncloned FANCD1 protein functions further downstream or independently of the FA pathway.
Assuntos
Anemia de Fanconi/genética , Proteínas Nucleares/genética , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Primers do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Anemia de Fanconi/fisiopatologia , Proteína do Grupo de Complementação A da Anemia de Fanconi , Proteína do Grupo de Complementação D2 da Anemia de Fanconi , Proteína do Grupo de Complementação F da Anemia de Fanconi , Proteína do Grupo de Complementação G da Anemia de Fanconi , Citometria de Fluxo , Regulação da Expressão Gênica/efeitos dos fármacos , Teste de Complementação Genética , Vetores Genéticos/genética , Humanos , Mitomicina/toxicidade , Proteínas Nucleares/fisiologia , Proteínas/genética , Proteínas/fisiologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/fisiologia , Valores de Referência , Transdução de Sinais/fisiologiaRESUMO
OBJECTIVE: Fanconi anemia (FA) is an autosomal-recessive cancer susceptibility syndrome with seven complementation groups. Six of the FA genes have been cloned (corresponding to subtypes A, C, D2, E, F, and G) and the encoded proteins interact in a common pathway. Patient-derived mutations in FA genes have been helpful in delineating functional domains of FA proteins. The purpose of this work was to subtype FA patient-derived cell lines in our repository and to identify FA gene mutations. METHODS: We subtyped 62 FA patients as type A, G, C, or non-ACG by using a combination of retroviral gene transfer and immunoblot analysis. Among these FA patients, we identified six FA-G patients for further analysis. We used a strategy involving amplification of FANCG/XRCC9 exons and direct sequencing to identify novel FANCG mutations in cell lines derived from these FA-G patients. We functionally analyzed FANCG mutant alleles by transducing the corresponding cDNAs into a known FA-G indicator cell line and scoring correction of MMC sensitivity. RESULTS: Our results demonstrate a wide range of mutations in the FANCG gene (splice, nonsense, and missense mutations). Based on this mutational screen, a carboxy terminal functional domain of the FANCG protein appears to be required for complementation of FA-G cells and for normal assembly of the FANCA/FANCG/FANCC protein complex. CONCLUSION: The identification of patient-derived mutant alleles of FA genes can provide important insights to the function of FA proteins. FA subtyping is also a necessary precondition for gene therapy.
Assuntos
Proteínas de Ligação a DNA/genética , Anemia de Fanconi/genética , Adolescente , Alelos , Criança , Pré-Escolar , DNA Complementar/análise , DNA Complementar/genética , Proteína do Grupo de Complementação G da Anemia de Fanconi , Feminino , Heterozigoto , Humanos , Masculino , MutaçãoRESUMO
The sparse fur (spf) and the sparse fur/abnormal skin and hair (spf-ash) mice are two murine models of the human X-linked disorder ornithine transcarbamylase (OTC) deficiency. A defective recombinant retrovirus, delta N2OTC was used to transduce primary hepatocytes derived from these mutant animals. Transduction of the primary cultures was highly efficient, with an average proviral copy number of 0.5-2 per cell in the population of transduced hepatocytes. Northern analysis and slot blots of total RNA isolated from transduced cells showed levels of human OTC mRNA to be equivalent to that present in normal human liver. Enzymatic assays demonstrated that a partial biochemical correction of the defect was achieved. After retroviral transduction, the hepatocytes were trypsinized and replated for long-term culture. Viability after replating exceeded 90%, indicating that the transduced cells might be useful for transplantation. The successful in vitro correction of OTC deficiency by this vector suggests that it will also be useful in somatic gene therapy experiments.
Assuntos
Fígado/enzimologia , Doença da Deficiência de Ornitina Carbomoiltransferase , Ornitina Carbamoiltransferase/genética , Retroviridae/genética , Transfecção , Animais , Southern Blotting , Células Cultivadas , DNA Recombinante , Modelos Animais de Doenças , Expressão Gênica , Terapia Genética , Vetores Genéticos , Humanos , Técnicas Imunológicas , Fígado/citologia , Camundongos , Camundongos Mutantes , Ornitina Carbamoiltransferase/biossíntese , RNA Mensageiro/biossíntese , Transdução Genética , beta-Galactosidase/genéticaRESUMO
Previously, this lab has reported the use of hepatocyte transplantation and in vivo gene therapy for the correction of a mouse model of Hereditary Tyrosinemia Type I (HT1). Here, we demonstrate repopulation of fumarylacetoacetate hydrolase (FAH)-deficient livers with cultured hepatocytes. Correction of the disease phenotype was achieved by retrovirally transducing cultured FAH- hepatocytes ex vivo, followed by transplantation and selective repopulation. Treated mice were phenotypically normal and had corrected plasma amino acid levels and liver function tests. Our results demonstrate that efficient hepatic repopulation using ex vivo genetically manipulated hepatocytes is feasible.