RESUMEN
Focal molecular genetic alteration of the intact mammalian brain will be required to elucidate gene product function in cells comprising synaptic networks. To this end, a somatic mosaic approach has been developed for the mouse whereby a dormant germline transgene is activated by the somatic delivery and expression of cre recombinase. Transgenic mice harboring a recombinational substrate, the germline-transmitted nerve growth factor excision activation transgene (NGF-XAT) were generated. Somatic delivery of virus vectors expressing cre recombinase into the brain of NGF-XAT mice resulted in regional recombination and activation of the transgene as demonstrated at the DNA level by PCR and at the protein level by both immunocytochemistry and ELISA. This approach has been used to evaluate a behavioral correlate of unilateral NGF mosaicism within the dorsal hippocampal formation. NGF-XAT mice activated by expression of cre recombinase manifest increased locomotor activity compared with NGF-XAT mice transduced by a control virus expressing Escherichia coli beta-galactosidase. These data indicate that focally increased expression of NGF in one part of a synaptic network can elicit changes in behavior presumably by altering the overall function of NGF-responsive neural circuitry. This approach should have broad application to other gene products and promises to provide the unprecedented ability to create and study discrete genetic modifications in the context of an intact adult mammal.
Asunto(s)
Encéfalo/metabolismo , Herpesviridae/genética , Integrasas/biosíntesis , Mosaicismo , Factores de Crecimiento Nervioso/genética , Proteínas Virales , Células 3T3/metabolismo , Células 3T3/virología , Animales , Conducta Animal/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Femenino , Vectores Genéticos/genética , Herpesviridae/metabolismo , Hipocampo/metabolismo , Hipocampo/virología , Inmunohistoquímica , Hibridación in Situ , Integrasas/genética , Integrasas/farmacología , Ratones , Ratones Transgénicos , Actividad Motora/efectos de los fármacos , Actividad Motora/genética , Factores de Crecimiento Nervioso/biosíntesis , Factores de Crecimiento Nervioso/fisiología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Transcripción Genética , TransgenesRESUMEN
It has been suggested that the mechanism of pancreatic beta-cell death in autoimmune diabetes mellitus and in immunoisolated transplantation devices involves cytokine-induced apoptosis. To explore the feasibility of a gene transfer strategy to protect beta-cells, we evaluated the use of replication defective HSV-1 amplicon vectors as gene transfer vehicles. Post-mitotic murine and human beta-cells were efficiently transduced by a herpes simplex virus (HSV) vector that expresses the reporting gene Escherichia coli lacZ under the transcriptional control of a HSV promoter (HSVlac) both as islets and as single cells. Insulin secretion, a marker of beta-cell function, was unaffected by HSVlac transduction of a beta-cell line. A HSV amplicon vector that expressed bcl-2 (HSVbcl2) in beta-cells was constructed, and its effects on cytokine-mediated apoptosis in both a beta-cell line and primary murine beta-cells assessed by measuring internucleosomal fragmentation. beta-Cell apoptosis was blocked by transduction with HSVbcl2 but not HSVlac. The prevention of cytokine-induced apoptosis in beta-cells by bcl-2 expression has the potential both to ameliorate primary autoimmune beta-cell destruction as type I diabetes develops, and to prevent the destruction of transplanted beta-cells inside immunoisolation devices.
Asunto(s)
Apoptosis , Técnicas de Transferencia de Gen , Genes bcl-2/genética , Vectores Genéticos/genética , Islotes Pancreáticos , Simplexvirus/genética , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Citocinas/farmacología , Escherichia coli/genética , Expresión Génica , Genes Reporteros/genética , Humanos , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Operón Lac/genética , RatonesRESUMEN
Gene transfer methods can be exploited to create somatic mosaic tissues to elucidate gene product action in cells comprising a network. This article describes the evolution of approaches beginning from herpes amplicon vectors with constitutive gene expression to herpes vectors with regulated gene expression and concludes with the development of a combined germline/somatic gene transfer method that allows for highly versatile spatial and temporal control of in vivo gene expression. Where possible examples of each approach are provided. In addition, the advantages and limitations of the approaches are discussed.