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1.
Am J Clin Nutr ; 117(6): 1195-1210, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36963568

RESUMO

BACKGROUND: Human breast milk has a high microRNA (miRNA) content. It remains unknown whether and how milk miRNAs might affect intestinal gene regulation and homeostasis of the developing microbiome after initiating enteral nutrition. However, this requires that relevant milk miRNA amounts survive the gastrointestinal (GI) passage, are taken up by cells, and become available to the RNA interference machinery. It seems important to dissect the fate of these miRNAs after oral ingestion and GI passage. OBJECTIVES: Our goal was to analyze the potential transmissibility of milk miRNAs via the gastrointestinal system in neonate humans and a porcine model in vivo to contribute to the discussion of whether milk miRNAs could influence gene regulation in neonates and thus might vertically transmit developmental relevant signals. METHODS: We performed cross-species profiling of miRNAs via deep sequencing and utilized dietary xenobiotic taxon-specific milk miRNA (xenomiRs) as tracers in human and porcine neonates, followed by functional studies in primary human fetal intestinal epithelial cells using adenovirus-type 5-mediated miRNA gene transfer. RESULTS: Mammals share many milk miRNAs yet exhibit taxon-specific miRNA fingerprints. We traced bovine-specific miRNAs from formula nutrition in human preterm stool and 9 d after the onset of enteral feeding in intestinal cells (ICs) of preterm piglets. Thereafter, several xenomiRs accumulated in the ICs. Moreover, a few hours after introducing enteral feeding in preterm piglets with supplemented reporter miRNAs (cel-miR-39-5p/-3p), we observed their enrichment in blood serum and in argonaute RISC catalytic component 2 (AGO2)-immunocomplexes from intestinal biopsies. CONCLUSIONS: Milk-derived miRNAs survived GI passage in human and porcine neonates. Bovine-specific miRNAs accumulated in ICs of preterm piglets after enteral feeding with bovine colostrum/formula. In piglets, colostrum supplementation with cel-miR-39-5p/-3p resulted in increased blood concentrations of cel-miR-39-3p and argonaute RISC catalytic component 2 (AGO2) loading in ICs. This suggests the possibility of vertical transmission of miRNA signaling from milk through the neonatal digestive tract.


Assuntos
Enterocolite Necrosante , MicroRNAs , Animais , Bovinos , Feminino , Humanos , Animais Recém-Nascidos , Células Epiteliais/patologia , Trato Gastrointestinal , MicroRNAs/genética , Leite , Suínos , Leite Humano
2.
Mol Cell Pediatr ; 8(1): 4, 2021 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-33893880

RESUMO

BACKGROUND: Reverse transcription of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (+)RNA genome and subgenomic RNAs (sgRNAs) and subsequent quantitative polymerase chain reaction (RT-qPCR) is the reliable diagnostic gold standard for COVID-19 diagnosis and the identification of potential spreaders. Apart from clinical relevance and containment, for specific questions, it might be of interest to (re)investigate cases with low SARS-CoV-2 load, where RT-qPCR alone can deliver conflicting results, even though these cases might neither be clinically relevant nor significant for containment measures, because they might probably not be infectious. In order to expand the diagnostic bandwidth for non-routine questions, particularly for the reliable discrimination between negative and false-negative specimens associated with high CT values, we combined the RT-qPCR workflow with subsequent pyrosequencing of a S-gene amplicon. This expansion can help to confirm SARS-CoV-2 infections without the demand of confirmative antibody testing, which requires to summon patients again for blood sampling few to several weeks after symptom onset. RESULTS: We successfully established a combined RT-qPCR and S-gene pyrosequencing method which can be optionally exploited after routine diagnostics. This allows a reliable interpretation of RT-qPCR results in specimens with relatively low viral loads and close to the detection limits of qPCR. After laboratory implementation, we tested the combined method in a large pediatric cohort from two German medical centers (n=769). Pyrosequencing after RT-qPCR enabled us to uncover 5 previously unrecognized cases of pediatric SARS-CoV-2-associated diseases, mainly exhibiting mild and heterogeneous presentation-apart from a single case of multisystem inflammatory syndrome in children (MIS-C) associated with SARS-CoV-2, who was hospitalized in the course of the study. CONCLUSIONS: The proposed protocol allows a specific and sensitive confirmation of SARS-CoV-2 infections close to the detection limits of RT-qPCR. The tested biotinylated primers do not negatively affect the RT-qPCR pipeline and thus can be optionally applied to enable deeper inspection of RT-qPCR results by subsequent pyrosequencing. Moreover, due to the incremental transmission of SARS-CoV-2 variants of concern, we note that the used strategy can uncover (Spike) P681H allowing the pre-selection of SARS-CoV-2 B.1.1.7 candidate specimens for deep sequencing.

3.
JPGN Rep ; 1(2): e025, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37206597

RESUMO

The infant was born at a gestational age of 28 + 2 weeks as second twin to a 26-year-old woman, G1/P0, due to eclampsia. The patient developed well and was on full oral feeds when he started to develop nonbilious vomiting at 5 weeks. He was diagnosed with pyloric hypertrophy and underwent pylorotomy, but the condition did not improve and the patient was referred to our hospital. Here, esophagogastroduodenoscopy showed severely inflamed esophageal and gastric mucosa which was found to be due to cytomegaly virus (CMV) infection and a nonpassable pylorus. The patient underwent pyloroplasty revealing a fibrous pyloric ring. Histology showed giant cells suggestive of CMV infection which was confirmed by polymerase chain reaction. He was started on valganciclovir and discharged 4 weeks later on full enteral feeds. To our knowledge, this is the first case of gastric outlet obstruction due to CMV infection in a premature infant.

4.
Klin Padiatr ; 231(5): 233-239, 2019 Sep.
Artigo em Alemão | MEDLINE | ID: mdl-31340405

RESUMO

BACKGROUND: Shared decision-making is indispensable when it comes to molecular genetic investigations, but data on the expectations of the parents is scarce. METHODS: Using a step-by-step approach we initially performed free in-depth-interviews with five parents on which base we developed a half standardized questionnaire. This questionnaire was then applied in interviews with 30 parents of children with intellectual disability, autism or epilepsy subject to genetic examination. RESULTS: Pre-diagnostic discussions are challenging for the parents in an intellectual as well as emotional way. The most important general aspects are diagnosis and therapy. Self-assessment of prior knowledge is very variable and many parents expressed problems in understanding. During the conversation parents rate the following specific aspects as "very important" or "important": findings of unclear relevance, incidental findings, psychic consequences, prognostic aspects, possible therapeutic interventions. 10 Parents did not have any school-degree and 20 parents were not native speakers. DISCUSSION: All parents express a high need for information covering almost all aspects of the investigation. Communicational hurdles pose additional challenges leaving a large room for improvement. Trustworthy internet-based information systems in different languages including plain language could be a first step.


Assuntos
Deficiências do Desenvolvimento/genética , Epilepsia/genética , Deficiência Intelectual/genética , Pais/psicologia , Criança , Humanos , Inquéritos e Questionários
6.
Epilepsy Behav ; 61: 192-198, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27371884

RESUMO

OBJECTIVE: Most studies on seizure detection systems focus more on the effectiveness of devices than on their practicability in and impact on everyday life. Our study investigated the impact of a technical monitoring system on subjective quality of sleep and the lives of affected families. Furthermore, we evaluated the impact of anxiety levels on seizure monitoring and vice versa. METHODS: Forty-three patients with newly diagnosed epilepsy were included. Initially, the families decided whether they did (group 1, n=27) or did not (group 2, n=16) want to use a monitoring device. In group 1, patients were randomly assigned to using Epi-Care® (group 1A, n=14) or an audio baby monitor (group 1B, n=13). Quality of life was assessed at two points (t1, at the start of the study and t2, at 5-7months of follow-up) using the SF-12, Kindl-R, and "Familien-Belastungs-Fragebogen" (German version of the "Impact on Family Scale"). In addition, parental anxiety was measured using the State-Trait Anxiety-Inventory, and subjective quality of sleep was measured using the Pittsburgh Sleep Quality Index. Statistical analysis focused on the possible differences between groups 1 and 2 that may influence parents' decisions and the effects of the presence and types of technical monitoring over time. RESULTS: Anxiety levels were not significantly different between the groups with and without monitoring (group 1 vs. group 2). We also found no statistically significant, substantial baseline differences between the Epi-Care® and audio baby monitor groups, with at least medium effect sizes (group 1A vs. group 1B). Parents' health-related mental quality of life measured via the SF-12 increased significantly over time in all groups. By tendency, the fear of further seizures as well as the frequency of cosleeping arrangements in the monitoring group decreased during the study and approached the stable values of the control group. SIGNIFICANCE: Individual parental anxiety levels are not crucial in the decision regarding the use of a monitoring device. A monitoring system may help some families in certain aspects of daily life. During the first months following a diagnosis of epilepsy, quality of life increases independently of the use of a monitoring system.


Assuntos
Epilepsia/fisiopatologia , Monitorização Fisiológica/efeitos adversos , Sono , Adolescente , Adulto , Ansiedade/psicologia , Criança , Pré-Escolar , Estudos de Coortes , Epilepsia/diagnóstico , Epilepsia/psicologia , Família , Feminino , Humanos , Lactente , Estudos Longitudinais , Masculino , Pais/psicologia , Estudos Prospectivos , Escalas de Graduação Psiquiátrica , Qualidade de Vida
7.
J Child Neurol ; 30(11): 1409-13, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25552269

RESUMO

Most studies on epileptiform discharges in young children were performed using analog electroencephalographic (EEG) recording systems and a limited numbers of electrodes that might have a lower detection rate for epileptiform discharges than modern digital recording systems. Knowing the prevalence of epileptiform discharges in healthy children is critical for a valid interpretation of findings in patients with a suspected epileptic disorder. We reviewed EEG recordings of 393 otherwise healthy children aged 12 to 60 months using digital EEG recording with respect to epileptiform discharges. We found epileptiform discharges in 3 children aged 12, 34 and 55 months resulting in a prevalence of epileptiform discharges in our cohort of 0.76% (95% confidence interval 0.0% to 1.62%). The prevalence of epileptiform discharges in children younger than 5 years is by far lower than in older children, and the digital findings are in accordance with previous data of conventional EEG.


Assuntos
Encéfalo/fisiologia , Epilepsia/epidemiologia , Epilepsia/fisiopatologia , Encéfalo/crescimento & desenvolvimento , Pré-Escolar , Eletroencefalografia , Feminino , Humanos , Lactente , Masculino , Prevalência
8.
J Child Neurol ; 28(2): 176-83, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22550090

RESUMO

There are conflicting results concerning bone metabolism in children receiving antiepileptic medication, with data concentrating on neurologically impaired patients. We performed a multicenter cross-sectional study in otherwise healthy children who received monotherapy with valproic acid, oxcarbazepine, lamotrigine, sulthiame, levetiracetam, or topiramate for at least 6 months. Data on calcium, phosphorus, alkaline phosphatase, 25-OH vitamin D, and parathormone were collected. Among 128 patients, 24.4% had hypocalcemia, 25.4% hypophosphatemia, and 13.3% (n = 17) 25-OH vitamin D levels <10 ng/mL. All patients were clinically asymptomatic. Mean calcium concentrations were found to be significantly lower among the study population (2.41 mmol/L) compared with healthy controls (2.53 mmol/L). Lowest mean concentration was observed in patients treated with sulthiame followed by oxcarbazepine and valproic acid. No influence of calcium intake or therapy on bone metabolism was noted. Effects on bone metabolism of anticonvulsive monotherapy are not restricted to neurologically impaired children but also affect otherwise healthy children.


Assuntos
Anticonvulsivantes/efeitos adversos , Osso e Ossos/metabolismo , Epilepsia/patologia , Adolescente , Análise de Variância , Antropometria , Osso e Ossos/efeitos dos fármacos , Cálcio/metabolismo , Criança , Pré-Escolar , Estudos de Coortes , Estudos Transversais , Epilepsia/sangue , Epilepsia/tratamento farmacológico , Epilepsia/epidemiologia , Feminino , Humanos , Hipocalcemia/induzido quimicamente , Hipofosfatemia/induzido quimicamente , Lactente , Masculino , Estudos Retrospectivos , Deficiência de Vitamina D/induzido quimicamente
9.
Pediatr Infect Dis J ; 31(11): 1148-52, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22863912

RESUMO

INTRODUCTION: In contrast to varicella zoster virus (VZV) primary infection, VZV vaccination does not seem to provide lifelong immunity against varicella. Because more people get vaccinated every year, the development of sensitive serological test systems for the detection of protective anti-VZV IgG will become important in the future. METHODS: We have previously developed a novel VZV line assay based on 5 different recombinant VZV antigens. In this study, we compared this novel assay with a commercially available glycoprotein enzyme immunoassay (RIDASCREEN VZV IgG) in detecting anti-VZV IgG of children with previous varicella infection and VZV vaccination. RESULTS: One hundred twenty-five children were included in this study, 72 with a history of varicella infection and 53 with VZV vaccination. Both assays detected anti-VZV IgG antibodies in both study groups with similar sensitivities. The VZV line assay revealed striking differences in the anti-VZV IgG composition against the VZV open reading frames, 4, 14 and 49, between both study groups, indicating that wild-type varicella infection causes a more diverse immune response against VZV than does vaccination. The exploitation of these results enabled the discrimination of both study groups with a sensitivity of 0.93 and a specificity of 0.83, indicating that the serologic differentiation of children with previous varicella infection and VZV vaccination might be possible. CONCLUSION: The VZV line assay enables the detection of anti-VZV IgG with sensitivities comparable to glycoprotein enzyme immunoassays and might be suitable for the serologic discrimination between children with a history of varicella infection and VZV vaccination.


Assuntos
Anticorpos Antivirais/sangue , Vacina contra Varicela/imunologia , Varicela/imunologia , Herpesvirus Humano 3/classificação , Imunoglobulina G/sangue , Varicela/sangue , Vacina contra Varicela/sangue , Criança , Humanos , Testes Sorológicos
10.
Pediatr Res ; 72(5): 513-20, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22902431

RESUMO

BACKGROUND: The aim of this study was to analyze whether the mucosal innate immune response of extremely-low-birth-weight (ELBW) infants might play a role in the development of necrotizing enterocolitis (NEC). METHODS: Between April 2008 and December 2009 antimicrobial peptides were prospectively measured in fecal samples of ELBW infants. In cases requiring abdominal surgery, full-thickness gut biopsies were analyzed for expression of human ß-defensin 2 (hBD2), interleukin-8 (IL-8), villin, MD2, and Toll-like receptor 4 (TLR4). RESULTS: Fecal hBD1 concentrations were consistently low in all patients, whereas hBD2 concentrations were high in meconium, particularly in clinical chorioamnionitis, and then dropped, followed by a steady increase after day 14. Infants with moderate NEC showed significantly increased fecal hBD2 concentrations before clinical symptoms, in contrast to infants developing severe NEC. Analysis of intestinal resection material obtained from patients with severe NEC revealed low hBD2 mRNA and protein levels, and increased expression of the inflammatory cytokine IL-8. CONCLUSION: High hBD2 concentrations, reflecting strong intestinal immune responses, were associated with moderate courses of the disease. In severe NEC, low hBD2 expression was accompanied by low TLR4/MD2 expression, suggesting an inadequate response to luminal bacteria, possibly predisposing those infants to the development of NEC.


Assuntos
Enterocolite Necrosante/metabolismo , Recém-Nascido de Peso Extremamente Baixo ao Nascer , Mucosa Intestinal/metabolismo , beta-Defensinas/metabolismo , Análise de Variância , Biomarcadores/metabolismo , Biópsia , Enterocolite Necrosante/epidemiologia , Enterocolite Necrosante/genética , Enterocolite Necrosante/imunologia , Enterocolite Necrosante/cirurgia , Fezes/química , Feminino , Regulação da Expressão Gênica , Alemanha/epidemiologia , Idade Gestacional , Humanos , Imunidade nas Mucosas , Recém-Nascido , Interleucina-8/genética , Interleucina-8/metabolismo , Mucosa Intestinal/imunologia , Antígeno 96 de Linfócito/genética , Antígeno 96 de Linfócito/metabolismo , Masculino , Mecônio/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Prevalência , Prognóstico , Estudos Prospectivos , RNA Mensageiro/metabolismo , Índice de Gravidade de Doença , Fatores de Tempo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , beta-Defensinas/genética
11.
Epilepsia ; 51(7): 1185-8, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20002145

RESUMO

PURPOSE: Data on epileptiform electroencephalography (EEG) discharges in healthy children are limited, with published studies dating back more than 20 years. Moreover, analyses have been performed exclusively using paper-recorded EEG, and reported prevalences differ significantly. With recent reports using these data as reference suggesting an increased prevalence of epileptiform EEG discharges in children with behavioral disturbances, acquisition of exact prevalence data has become even more critical. The aim of our study was to analyze the frequency of epileptiform EEG discharges in healthy children using digitally recorded EEG (DEEG) and to compare these data to those of previously published studies. METHODS: Prospective analysis of DEEG was performed in 382 healthy children (226 male, 156 female) ages 6-13 years admitted to our hospital for minor head trauma. Recording was carried out for a minimum of 20 min including hyperventilation and photic stimulation. Analysis was carried out by two board-certified clinical neurophysiologists. RESULTS: Epileptiform EEG discharges were detected in 25 of 382 children (11 of 226 male, 14 of 156 female) corresponding to an overall prevalence of 6.5%. Of these 25 children, 4 had either generalized or bifrontal spikes, 12 showed constant localized focal discharges, and 9 showed multifocal discharges. Compared to previous studies using non-DEEG recording, the prevalence of epileptiform EEG discharges in our population was significantly higher. No significant difference was found when comparing our data to prevalences recently reported in children with behavioral disturbances using DEEG. CONCLUSIONS: Our study further highlights the urgent need to reevaluate the prevalence of epileptiform EEG discharges in healthy children using DEEG recordings in a large cohort.


Assuntos
Eletroencefalografia/métodos , Epilepsia/diagnóstico , Epilepsia/epidemiologia , Saúde , Processamento de Sinais Assistido por Computador , Adolescente , Criança , Estudos de Coortes , Traumatismos Craniocerebrais/diagnóstico , Traumatismos Craniocerebrais/epidemiologia , Traumatismos Craniocerebrais/fisiopatologia , Epilepsia/fisiopatologia , Feminino , Humanos , Masculino , Estimulação Luminosa/métodos , Prevalência , Estudos Prospectivos , Estatística como Assunto/métodos
12.
Antimicrob Agents Chemother ; 52(7): 2355-9, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18474581

RESUMO

Since the emergence of viral resistance of hepatitis B virus (HBV) during treatment is becoming an important issue even with newer drugs, there is a need for alternative treatment options such as, for example, RNA interference (RNAi) technology. While short-term suppression of HBV replication is easily achieved with small interfering RNA oligonucleotides, this is not the case for long-term suppression due to the lack of an optimal vector system. Based on the nonviral scaffold/matrix attachment region (S/MAR)-based vector system pEPI-1, which is free of common side effects and is stably retained as an episome even in the absence of selection, we designed a short hairpin RNA (shRNA) expression vector called pEPI-RNAi for HBV suppression. HBV-replicating HepG2.2.15 cells were transfected with pEPI-RNAi, and the intracellular status of the plasmid was followed by PCR and Southern analysis. HBV replication was measured on the DNA, RNA, and protein level. HBV RNA expression was reduced by almost 85% 3 months posttransfection with pEPI-RNAi. At 8 months posttransfection in the absence of antibiotic selection pressure, the suppression level was still 70% and the vector was retained as an episome. The reduction of total intracellular HBV DNA at this point was 77%, showing a marked suppression of HBV DNA replication. At a comparable level, secretion of viral antigens, as well as progeny HBV virions, was inhibited. The S/MAR-based vector system pEPI-1 allows long-term suppression of HBV replication by the expression of suitable shRNAs. Due to its unique properties compared to commonly used vectors, it provides an interesting option for the treatment of chronically HBV-infected individuals.


Assuntos
Vetores Genéticos , Vírus da Hepatite B/genética , Vírus da Hepatite B/fisiologia , RNA Interferente Pequeno/genética , RNA Viral/genética , Replicação Viral/genética , Sequência de Bases , Linhagem Celular , DNA Viral/genética , DNA Viral/metabolismo , Hepatite B Crônica/terapia , Hepatite B Crônica/virologia , Humanos , Regiões de Interação com a Matriz/genética , Plasmídeos/genética , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Transfecção
13.
Hum Gene Ther ; 16(4): 533-9, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15871685

RESUMO

The inhibition of gene expression by RNA interference harbors a high potential for application in the therapy of human diseases. However, while exogenous application of siRNAs efficiently inhibits gene expression, these effects are only transient in mammalian cells. We designed a short hairpin RNA-expression cassette to target the bcr-abl oncogene that was then introduced into the nonviral vector system pEPI-1, which replicates episomally in the absence of selection in the bcr-abl-positive cell line K562. Forty-two days after transfection the bcr-abl- but not the cytokine-dependent growth rate was found to be drastically reduced in K562 cells. Western analysis revealed a more than 90% reduction in the expression of the fusion protein bcr-abl while the expression of the bcr protein remained unaffected. In addition, we show that the level of bcr-abl mRNA was specifically reduced in these cells for more than 90%. These results demonstrate that the vector system pEPI-1 allows specific and efficient long term gene suppression by using a short hairpin RNA transcription unit.


Assuntos
Proteínas de Fusão bcr-abl/genética , Vetores Genéticos/genética , Plasmídeos/genética , RNA/farmacologia , Replicação Viral/genética , Sequência de Bases , Northern Blotting , Western Blotting , Proliferação de Células , Citocinas/metabolismo , DNA Polimerase III/genética , Proteínas de Fusão bcr-abl/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/farmacologia , Humanos , Células K562 , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA/química , RNA/genética , RNA Catalítico/química
14.
Mol Biol Rep ; 31(2): 85-90, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15293783

RESUMO

Currently available vectors for mammalian cells suffer from a number of limitations which make them only partially useful for genetic modification of eukaryotic cells and organisms and for gene therapy. While integration of a vector can lead to unpredictable interactions with the host genome and silencing of the integrated transgene, most non-integrating vectors mediate only transient expression of a transgene. All available vector types can lead to transformation of the recipient cell and many of them can cause serious immunological side effects in the organism. The ideal vector has to be free of these side effects and should allow long-term expression of a transgene in the absence of selection. In this report we describe a novel non-viral episomal expression system fulfilling these criteria. The gene encoding the truncated rat NGF-receptor gene under the control of the CMV-promoter was inserted into a vector construct containing a scaffold/matrix attached region (S/MAR). This vector was then transfected into CHO cells and human HaCat cells. We show that this vector replicates episomally in these cells and is mitotically stable in the abscence of selection over more than 100 generations. Moreover, we provide the first experimental data that the CMV-promoter in an episome is not subject to silencing by cytosine methylation, thus allowing long-term expression of the transgene in the absence of selection.


Assuntos
Epigênese Genética , Inativação Gênica , Vetores Genéticos/genética , Plasmídeos/genética , Transgenes/fisiologia , Animais , Células CHO , Cricetinae , Cricetulus , Citomegalovirus/genética , Citosina/metabolismo , Metilação de DNA , Expressão Gênica/genética , Expressão Gênica/fisiologia , Vetores Genéticos/metabolismo , Humanos , Plasmídeos/metabolismo , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/fisiologia , Ratos , Receptor de Fator de Crescimento Neural/análise , Receptor de Fator de Crescimento Neural/genética , Transfecção , Transgenes/genética
15.
Proc Natl Acad Sci U S A ; 101(31): 11322-7, 2004 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-15272077

RESUMO

The activation of mammalian origins of replication depends so far on ill understood epigenetic events, such as binding of transcription factors, chromatin structure, and nuclear localization. Understanding these mechanisms is not only a scientific challenge but also represents a prerequisite for the rational design of nonviral episomal vectors for mammalian cells. In this paper, we demonstrate that a tetramer of a 155-bp minimal nuclear scaffold/matrix attached region DNA module linked to an upstream transcription unit is sufficient for replication and mitotic stability of a mammalian episome in the absence of selection. Fluorescence in situ hybridization analyses, crosslinking with cis-diammineplatinum(II)-dichloride and chromatin immunoprecipitation demonstrate that this vector associates with the nuclear matrix or scaffold in vivo by means of specific interaction of the nuclear scaffold/matrix attached region with the nuclear matrix protein SAF-A. Results presented in this paper define the minimal requirements of an episomal vector for mammalian cells on the molecular level.


Assuntos
Replicação do DNA/fisiologia , Matriz Nuclear/genética , Transcrição Gênica/fisiologia , Animais , Células CHO , Cricetinae , Escherichia coli , Mamíferos , Mitose/fisiologia , Proteínas Associadas à Matriz Nuclear/fisiologia , Plasmídeos/genética , Transgenes/genética
16.
Chromosome Res ; 11(5): 413-21, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12971718

RESUMO

In order to analyze epigenetic factors involved in the regulation of DNA replication in higher eukaryotic cells, minimal systems have to be established. We have recently constructed a non-viral episomal vector system which replicates episomally in mammalian cells and is stably maintained in the cell in the absence of selection. The potential functional elements contained in this construct are an expression cassette upstream of a chromosomal S/MAR sequence and the SV40 origin of replication. In this report we describe that an active transcription upstream of the S/MAR running into this sequence is required and probably sufficient for episomal replication. We propose a model for the activation of replication in this system which may be the basis for further analysis of replication control in other systems.


Assuntos
Replicação do DNA/genética , Epigênese Genética/genética , Modelos Genéticos , Transcrição Gênica/genética , Animais , Northern Blotting , Southern Blotting , Células CHO , Cricetinae , Cricetulus , Vetores Genéticos , Hibridização in Situ Fluorescente
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