RESUMO
The effectiveness of the caspase-9-based artificial "death switch" as a safety measure for gene therapy based on the erythropoietin (Epo) hormone was tested in vitro and in vivo using the chemical inducer of dimerization, AP20187. Plasmids encoding the dimeric murine Epo, the tetracycline-controlled transactivator and inducible caspase 9 (ptet-mEpoD, ptet-tTAk and pSH1/Sn-E-Fv-Fvls-casp9-E, respectively) were used in this study. AP20187 induced apoptosis of iCasp9-modified C2C12 myoblasts. In vivo, two groups of male C57BI/6 mice, 8-12 weeks old, were injected intramuscularly with 5 µg/50 g ptet-mEpoD and 0.5 µg/50 g ptet-tTAk. There were 20 animals in group 1 and 36 animals in group 2. Animals from group 2 were also injected with the 6 µg/50 g iCasp9 plasmid. Seventy percent of the animals showed an increase in hematocrit of more than 65 percent for more than 15 weeks. AP20187 administration significantly reduced hematocrit and plasma Epo levels in 30 percent of the animals belonging to group 2. TUNEL-positive cells were detected in the muscle of at least 50 percent of the animals treated with AP20187. Doxycycline administration was efficient in controlling Epo secretion in both groups. We conclude that inducible caspase 9 did not interfere with gene transfer, gene expression or tetracycline control and may be used as a safety mechanism for gene therapy. However, more studies are necessary to improve the efficacy of this technique, for example, the use of lentivirus vector.
Assuntos
Animais , Masculino , Camundongos , Anemia/terapia , Caspase 9/genética , Dimerização , Eritropoetina , Expressão Gênica/genética , Terapia Genética/métodos , Tacrolimo/análogos & derivados , Caspase 9/administração & dosagem , Eritropoetina , Vetores Genéticos/genética , Hematócrito , Injeções Intramusculares , Lentivirus/genética , Plasmídeos/uso terapêutico , Tacrolimo/uso terapêuticoRESUMO
The effectiveness of the caspase-9-based artificial "death switch" as a safety measure for gene therapy based on the erythropoietin (Epo) hormone was tested in vitro and in vivo using the chemical inducer of dimerization, AP20187. Plasmids encoding the dimeric murine Epo, the tetracycline-controlled transactivator and inducible caspase 9 (ptet-mEpoD, ptet-tTAk and pSH1/Sn-E-Fv'-Fvls-casp9-E, respectively) were used in this study. AP20187 induced apoptosis of iCasp9-modified C2C12 myoblasts. In vivo, two groups of male C57BI/6 mice, 8-12 weeks old, were injected intramuscularly with 5 microg/50 g ptet-mEpoD and 0.5 microg/50 g ptet-tTAk. There were 20 animals in group 1 and 36 animals in group 2. Animals from group 2 were also injected with the 6 microg/50 g iCasp9 plasmid. Seventy percent of the animals showed an increase in hematocrit of more than 65% for more than 15 weeks. AP20187 administration significantly reduced hematocrit and plasma Epo levels in 30% of the animals belonging to group 2. TUNEL-positive cells were detected in the muscle of at least 50% of the animals treated with AP20187. Doxycycline administration was efficient in controlling Epo secretion in both groups. We conclude that inducible caspase 9 did not interfere with gene transfer, gene expression or tetracycline control and may be used as a safety mechanism for gene therapy. However, more studies are necessary to improve the efficacy of this technique, for example, the use of lentivirus vector.
Assuntos
Anemia/terapia , Caspase 9/genética , Dimerização , Eritropoetina/administração & dosagem , Expressão Gênica/genética , Terapia Genética/métodos , Tacrolimo/análogos & derivados , Animais , Caspase 9/administração & dosagem , Eritropoetina/genética , Vetores Genéticos/genética , Hematócrito , Injeções Intramusculares , Lentivirus/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos/uso terapêutico , Proteínas Recombinantes , Tacrolimo/uso terapêuticoAssuntos
Mutagênese , Micologia/métodos , Saccharomyces cerevisiae/genética , Antibacterianos/farmacologia , Membrana Celular/metabolismo , Parede Celular/metabolismo , DNA Fúngico/efeitos dos fármacos , DNA Fúngico/genética , DNA Fúngico/efeitos da radiação , DNA Mitocondrial/genética , DNA Mitocondrial/efeitos da radiação , Resistência Microbiana a Medicamentos/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos/efeitos dos fármacos , Genes Fúngicos/efeitos da radiação , Mutagênicos/farmacologia , Consumo de Oxigênio/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos da radiação , TemperaturaAssuntos
Micologia/métodos , Saccharomyces cerevisiae/genética , Acriflavina/farmacologia , Fusão Celular/genética , DNA Fúngico/genética , DNA Mitocondrial/genética , Etídio/farmacologia , Técnicas de Transferência de Genes , Organelas/genética , Ploidias , Protoplastos , Reprodução/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/fisiologiaRESUMO
An industrial strain of Saccharomyces cerevisiae was fused with an osmotolerant yeast, Debaryomyces hansenii, to obtain hybrids having increased tolerance to elevated salt concentrations. The hybrids were intermediate to parent species in production of ethanol and polyols.
Assuntos
Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/citologia , Saccharomycetales/metabolismo , Álcoois/metabolismo , Meios de Cultura , Microbiologia Industrial , Polímeros/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomycetales/crescimento & desenvolvimento , Cloreto de Sódio/metabolismo , Equilíbrio HidroeletrolíticoRESUMO
A method has been developed for the transfer of genes from other yeast strains and species to industrial yeast strains, using a haploid, kar1-1 mutant strain of Saccharomyces cerevisiae as a vector. The sta2 gene, conferring the ability to metabolize starch was transferred from an auxotrophic haploid strain of S. cerevisiae (S. diastaticus) and the melibiose-metabolism (mel) gene(s), from S. kluyveri, to the kar1-1 mutant [K5-5A; (alpha ade2 his4 can1 gal) by normal mating and protoplast fusion. From this strain, the genes were transferred to baker's yeast and brewing yeast strains, which did not utilize starch, and to baker's yeast strains, which did not utilize melibiose, by protoplast fusion, spore-cell pairing, or rare-mating. Strains that utilized starch or melibiose were obtained by all three methods. Pulsed-field gel electrophoresis preparations showed little change in the mobility of the chromosomes of the hybrids. The most probable explanation for the results obtained is that single chromosomes were transferred, first, from the donor strains to the kar1-1 haploid mutant strain, and then from the kar1-1 vector to the recipient industrial strain of S. cerevisiae. The transfer of the genes is probably accomplished through formation of disomic strains and then, in the case of the hybrids that metabolize starch, by integration of the sta2 gene into the genome of the industrial yeast strains.