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2.
J Virol ; 71(10): 7951-9, 1997 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9311886

RESUMO

We have developed a system for site-specific DNA integration in human cells, mediated by the adeno-associated virus (AAV) Rep proteins. In its normal lysogenic cycle, AAV integrates at a site on human chromosome 19 termed AAVS1. We describe a rapid PCR assay for the detection of integration events at AAVS1 in whole populations of cells. Using this assay, we determined that the AAV Rep proteins, delivered in cis or trans, are required for integration at AAVS1. Only the large forms of the Rep protein, Rep78 and Rep68, promoted site-specific integration. The AAV inverted terminal repeats, present in cis, were not essential for integration at AAVS1, but in cells containing Rep, they increased the efficiency of integration. In the presence of the Rep proteins, the integration of a plasmid containing AAV inverted terminal repeats occurred at high frequency, such that clones containing the plasmid could be isolated without selection. In two of the five clones analyzed by fluorescence in situ hybridization, the plasmid DNA was integrated at AAVS1. In most of the clones, at least one copy of the entire plasmid was integrated in a tandem array. Detailed analysis of the integrated plasmid structure in one clone suggested a complex mechanism producing rearrangements of the flanking genomic DNA, similar to those observed with wild-type AAV.


Assuntos
Cromossomos Humanos Par 19 , DNA Helicases/metabolismo , Proteínas de Ligação a DNA , DNA/metabolismo , Dependovirus/genética , Genoma Humano , Transativadores/metabolismo , Integração Viral , Adenovírus Humanos/genética , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular Transformada , DNA/química , Primers do DNA , Dependovirus/metabolismo , Humanos , Mamíferos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , Proteínas Recombinantes/biossíntese , Mapeamento por Restrição , Transfecção , Proteínas Virais/metabolismo , beta-Galactosidase/biossíntese
3.
J Virol ; 71(3): 1776-83, 1997 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-9032306

RESUMO

We tested the ability of a recombinant adeno-associated virus (rAAV) vector to express and integrate exogenous DNA into human hematopoietic cells in the absence of selection. We developed an rAAV vector, AAV-tNGFR, carrying a truncated rat nerve growth factor receptor (tNGFR) cDNA as a cell surface reporter under the control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat. An analogous MoMuLV-based retroviral vector (L-tNGFR) was used in parallel, and gene transfer and expression in human hematopoietic cells were assessed by flow cytometry and DNA analyses. Following gene transfer into K562 cells with AAV-tNGFR at a multiplicity of infection (MOI) of 13 infectious units (IU), 26 to 38% of cells expressed tNGFR on the surface early after transduction, but the proportion of tNGFR expressing cells steadily declined to 3.0 to 3.5% over 1 month of culture. At an MOI of 130 IU, nearly all cells expressed tNGFR immediately posttransduction, but the proportion of cells expressing tNGFR declined to 62% over 2 months of culture. The decline in the proportion of AAV-tNGFR-expressing cells was associated with ongoing losses of vector genomes. In contrast, K562 cells transduced with the retroviral vector L-tNGFR expressed tNGFR in a constant fraction. Integration analyses on clones showed that integration occurred at different sites. Integration frequencies were estimated at about 49% at an MOI of 130 and 2% at an MOI of 1.3. Transduction of primary human CD34+ progenitor cells by AAV-tNGFR was less efficient than with K562 cells and showed a declining percentage of cells expressing tNGFR over 2 weeks of culture. Thus, purified rAAV caused very high gene transfer and expression in human hematopoietic cells early after transduction, which steadily declined during cell passage in the absence of selection. Although the efficiency of integration was low, overall integration was markedly improved at a high MOI. While prolonged episomal persistence may be adequate for gene therapy of nondividing cells, a very high MOI or improvements in basic aspects of AAV-based vectors may be necessary to improve integration frequency in the rapidly dividing hematopoietic cell population.


Assuntos
Dependovirus/genética , Técnicas de Transferência de Genes , Vetores Genéticos , Receptores de Fator de Crescimento Neural/genética , Integração Viral , Animais , Antígenos CD34 , Linhagem Celular , Células Cultivadas , DNA/análise , Expressão Gênica , Genes Reporter , Genoma Viral , Humanos , Ratos , Recombinação Genética , Seleção Genética , Células Tumorais Cultivadas
4.
Proc Natl Acad Sci U S A ; 93(24): 14082-7, 1996 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-8943064

RESUMO

Somatic gene therapy has been proposed as a means to achieve systemic delivery of therapeutic proteins. However, there is limited evidence that current methods of gene delivery can practically achieve this goal. In this study, we demonstrate that, following a single intramuscular administration of a recombinant adeno-associated virus (rAAV) vector containing the beta-galactosidase (AAV-lacZ) gene into adult BALB/c mice, protein expression was detected in myofibers for at least 32 weeks. A single intramuscular administration of an AAV vector containing a gene for human erythropoietin (AAV-Epo) into mice resulted in dose-dependent secretion of erythropoietin and corresponding increases in red blood cell production that persisted for up to 40 weeks. Primary human myotubes transduced in vitro with the AAV-Epo vector also showed dose-dependent production of Epo. These results demonstrate that rAAV vectors are able to transduce skeletal muscle and are capable of achieving sustained expression and systemic delivery of a therapeutic protein following a single intramuscular administration. Gene therapy using AAV vectors may provide a practical strategy for the treatment of inherited and acquired protein deficiencies.


Assuntos
Eritropoese , Eritropoetina/biossíntese , Técnicas de Transferência de Genes , Terapia Genética/métodos , Músculo Esquelético/fisiologia , beta-Galactosidase/biossíntese , Animais , Linhagem Celular , Dependovirus , Eritropoetina/genética , Escherichia coli , Expressão Gênica , Vetores Genéticos , Histocitoquímica , Humanos , Injeções Intramusculares , Óperon Lac , Camundongos , Camundongos Endogâmicos BALB C , Fatores de Tempo , beta-Galactosidase/genética
5.
J Biol Chem ; 269(13): 9898-905, 1994 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-8144583

RESUMO

We have demonstrated that A375 melanoma cells express mRNA for both types of tumor necrosis factor (TNF) receptors and receptor proteins on their plasma membranes. Specific agonist and blocking antibodies to either 55-kDa (TNF-R1) or 75-kDa (TNF-R2) TNF receptors combined with two-dimensional gel analysis were employed to determine which receptor type is responsible for mediating the induction of individual melanoma proteins. Our results indicate that the enhanced synthesis of proteins 21/>7 (M(r)/pI), 28/5.6, and 41/5.7 is selectively induced through TNF-R1. TNF induces these proteins; antagonist antibody to TNF-R1 prevents their induction by TNF, and TNF-R1 agonist induces them in the absence of TNF. Identification of these proteins by immunoblot analysis proved that 21/>7 is manganese superoxide dismutase, protein 28/5.6 is unrelated to 27/28-kDa heat shock protein, and protein 41/5.7 is plasminogen activator inhibitor-2. Furthermore, TNF cytotoxicity for A375 cells is also mediated by TNF-R1. These studies indicate that TNF-R1 is a critical signaling receptor for TNF action on A375 cells and demonstrate the potential use of TNF-R1 antibodies to selectively block or enhance specific effects of TNF on melanoma cells.


Assuntos
Melanoma/metabolismo , Proteínas de Neoplasias/biossíntese , Inibidor 2 de Ativador de Plasminogênio/biossíntese , Receptores do Fator de Necrose Tumoral/metabolismo , Superóxido Dismutase/biossíntese , Fator de Necrose Tumoral alfa/farmacologia , Sequência de Bases , Membrana Celular/metabolismo , Primers do DNA , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/isolamento & purificação , Humanos , Interferon gama/farmacologia , Isoenzimas/biossíntese , Isoenzimas/isolamento & purificação , Cinética , Dados de Sequência Molecular , Peso Molecular , Proteínas de Neoplasias/isolamento & purificação , Inibidor 2 de Ativador de Plasminogênio/isolamento & purificação , Reação em Cadeia da Polimerase , Receptores do Fator de Necrose Tumoral/efeitos dos fármacos , Receptores do Fator de Necrose Tumoral/isolamento & purificação , Proteínas Recombinantes/farmacologia , Superóxido Dismutase/isolamento & purificação , Células Tumorais Cultivadas
6.
Child Dev ; 56(3): 573-81, 1985 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-3159553

RESUMO

The exploratory behaviors of a sample of 11 infants with Down syndrome and 11 nondelayed infants, matched on Bayley mental raw scores and gender, were analyzed. Transitional probabilities and z scores were computed for each possible behavior change as well as frequencies of each behavior. The analyses revealed significant differences in how the 2 samples distribute their exploratory activities. The significant transitional probabilities among the 6 behavioral states revealed a pattern of similarities and differences. In general, both groups of infants organized their exploratory activities in a similar manner. However, there were differences that appeared to depend on the level of exploratory sophistication. The results are discussed in the context of the similarities and differences between the samples.


Assuntos
Síndrome de Down/psicologia , Comportamento Exploratório , Atenção , Desenvolvimento Infantil , Feminino , Humanos , Lactente , Masculino , Jogos e Brinquedos
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