RESUMEN
During the past decade, lentiviral vectors based on the HIV-1 genome have been developed to become highly useful tools for efficient and stable delivery of transgenes to dividing and nondividing cells in a variety of experimental protocols. The vector system has been progressively and substantially improved,mainly to meet growing concerns over safety issues. However, the actual design and size of the lentiviral transfer vector often makes transgene cloning and DNA preparation a troublesome task. In this study, the pHR transfer vector used for lentivirus production in many laboratories was modified to contain a more versatile polylinker than the one present in the original pHR vector. In addition, the vector was significantly reduced in size from 12 to 7 kb, by replacing the original vector backbone with sequence from the multipurpose pUC18 vector. These modifications allowed for easier cloning and higher DNA yields without compromising the fundamental ability of this vector system to transduce cells in vitro and in vivo. Finally, the trimmed vector sequence was fully characterized by sequencing the vector in its entirety. In both cultured cells and directly into the rat striatum, transduction with this lentivirus, based on the modified pHsCXW vector, was as efficient and durable as with the pHR vector-based virus. In conclusion, the modified lentiviral transfer vector pHsCXW holds promise as a new valuable tool for the research community in the field of gene transfer.
Asunto(s)
Vectores Genéticos/genética , Genoma Viral/genética , VIH-1/genética , Transducción Genética , Animales , Línea Celular , Femenino , Terapia Genética/métodos , Vectores Genéticos/uso terapéutico , Humanos , Ratas , Ratas Sprague-Dawley , Análisis de Secuencia de ADN , Transducción Genética/métodos , Transgenes/genéticaRESUMEN
To clarify whether glial cell line-derived neurotrophic factor (GDNF) receptor alpha-1 (GFRalpha1), the glycosylphosphatidylinositol (GPI)-linked coreceptor for GDNF, is also a functional coreceptor for artemin (ART), we have studied receptor binding, signaling, and neuronal survival. In cell-free binding studies, GFRalpha1-Ig displayed strong preferential binding to GDNF, though in the presence of soluble RET, weak binding to ART could also be detected. However, using GFRalpha1-transfected NB41A3 cells, ART showed no detectable competition against the binding of (125)I-labeled GDNF. Moreover, ART failed to induce phosphorylation of extracellular signal-related kinase (ERK) and Akt in these cells and was >10(4)-fold less potent than GDNF in stimulating RET phosphorylation. When rat primary dorsal root ganglion (DRG) neurons were used, only the survival promoting activity of GDNF and not that of ART was blocked by an anti-GFRalpha1 antibody. These results indicate that although ART can interact weakly with soluble GFRalpha1 constructs under certain circumstances in vitro, in cell-based functional assays GFRalpha1 is at least 10 000-fold selective for GDNF over ART. The extremely high selectivity of GFRalpha1 for GDNF over ART and the low reactivity of ART for this receptor suggest that GFRalpha1 is not likely to be a functional coreceptor for ART in vivo.
Asunto(s)
Factores de Crecimiento Nervioso/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Membrana Celular/metabolismo , Supervivencia Celular/fisiología , Ganglios Espinales/citología , Ganglios Espinales/fisiología , Factor Neurotrófico Derivado de la Línea Celular Glial , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial , Humanos , Ratones , Factores de Crecimiento Nervioso/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuronas Aferentes/citología , Neuronas Aferentes/fisiología , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-ret , Ratas , Ratas Sprague-Dawley , Proteínas Tirosina Quinasas Receptoras/fisiología , Transducción de Señal/genética , Soluciones , Resonancia por Plasmón de Superficie , TransfecciónRESUMEN
NS-417 (5-(4-Chlorophenyl)-8-methyl-6-7-8-9-tetrahydro-1-H-pyrrolo[3.2-h]isoquinoline-2,3-dione-3-oxim hydrochloric acid salt) belongs to a new chemical series of compounds. NS-417 rescued differentiated PC12 cells from death induced by withdrawal of serum and nerve growth factor. Furthermore, NS-417 stimulated neurotrophic factor-induced neurite outgrowth in undifferentiated PC12 cells. In accordance with this observation, NS-417 potentiated NGF-induced signaling, such as activation of the extracellular signal-regulated kinases ERK1 and ERK2 and the Akt kinase. NS-417 also enhanced ERK activation induced by 10 minutes stimulation with NGF, bFGF or EGF in PC12 cells. In addition to the effect in PC12 cells, NS-417 increased the number of tyrosine hydroxylase (TH) positive cells in cultures established from dissociated E14 rat ventral mesencephali.
Asunto(s)
Isoquinolinas/farmacología , Factor de Crecimiento Nervioso/farmacología , Oximas/farmacología , Proteínas Serina-Treonina Quinasas , Tetrahidroisoquinolinas , Animales , Recuento de Células , Supervivencia Celular/fisiología , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Activación Enzimática/efectos de los fármacos , Isoquinolinas/química , Mesencéfalo/embriología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factor de Crecimiento Nervioso/deficiencia , Neuritas/efectos de los fármacos , Neuritas/fisiología , Neuronas/citología , Neuronas/enzimología , Oximas/química , Células PC12/efectos de los fármacos , Células PC12/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-akt , Ratas , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
NS 1231 [5-(4-chlorophenyl)-6,7,8,9-tetrahydro-1H-pyrrolo-[3.2-h]naphthalene-2,3-dione-3-oxime] belongs to a chemical series of compounds, which exhibit neurotrophic-like activities. In vitro, NS 1231 rescued nerve growth factor (NGF)-differentiated PC12 cells from death induced by withdrawal of trophic factors. In addition, NS 1231 stimulated NGF-induced neurite outgrowth of undifferentiated PC12 cells. At the molecular level, NS 1231 enhanced NGF-induced signalling events, such as TrkA phosphorylation at the Shc-binding site Tyr490 as well as ERK activation in PC12 cells. Moreover, NS 1231 reduced NMDA-induced excitotoxicity in organotypic hippocampal slice cultures. In a gerbil model of transient global ischaemia, treatment with NS 1231 reduced the delayed loss of neurons in the hippocampal CA1 layer. Furthermore, NS 1231 treatment resulted in a 43% reduction in total infarct volume in the mouse middle cerebral artery occlusion (MCAO) model. The present data thus implicate a therapeutic potential of NS 1231 or structural analogues in treatment of cerebral ischaemia.