RESUMO
Preventing the replication of adenovirus could have practical uses, such as controlling infection with wild-type virus or in applications involving recombinant vectors. Mainly transient methods have been used to inhibit adenovirus replication, including siRNA or drugs. Here, we tested whether stable expression of shRNA designed to target hexon, Iva2, or pol can inhibit the replication of a recombinant adenoviral vector, Ad-LacZ (serotype 5, E1/E3 deleted), in 293T cells. Significant knockdown correlating with reduced Ad-LacZ replication was achieved only when hexon was targeted. Cell sorting and isolation of cellular clones further accentuated knockdown of the hexon transcript, reduced protein levels by more than 90%, and diminished adenovirus production. As visualized by transmission electron microscopy, the cellular clone expressing the hexon-specific shRNA yielded 89.2% fewer particles compared to the parental 293T cells. Full scale production followed by purification revealed a 90.2% reduction in Ad-LacZ biological titer. These results support the notion that stable expression of shRNA can be used as a means to control adenovirus replication.
Assuntos
Adenoviridae , Replicação Viral , Adenoviridae/genética , RNA Interferente Pequeno/genética , Vetores Genéticos/genética , Humanos , Células HEK293 , Transcrição Gênica , Células ClonaisRESUMO
Preventing the replication of adenovirus could have practical uses, such as controlling infection with wild-type virus or in applications involving recombinant vectors. Mainly transient methods have been used to inhibit adenovirus replication, including siRNA or drugs. Here, we tested whether stable expression of shRNA designed to target hexon, Iva2, or pol can inhibit the replication of a recombinant adenoviral vector, Ad-LacZ (serotype 5, E1/E3 deleted), in 293T cells. Significant knockdown correlating with reduced Ad-LacZ replication was achieved only when hexon was targeted. Cell sorting and isolation of cellular clones further accentuated knockdown of the hexon transcript, reduced protein levels by more than 90%, and diminished adenovirus production. As visualized by transmission electron microscopy, the cellular clone expressing the hexon-specific shRNA yielded 89.2% fewer particles compared to the parental 293T cells. Full scale production followed by purification revealed a 90.2% reduction in Ad-LacZ biological titer. These results support the notion that stable expression of shRNA can be used as a means to control adenovirus replication.
RESUMO
NOTCH pathway proteins, including the transcriptional factor HES1, play crucial roles in the development of the inner ear by means of the lateral inhibition mechanism, in which supporting cells have their phenotype preserved while they are prevented from becoming hair cells. Genetic manipulation of this pathway has been demonstrated to increase hair cell number. The present study aimed to investigate gene expression effects in hair cells and supporting cells after Hes1-shRNA lentivirus transduction in organotypic cultures of the organ of Corti from postnatal-day-3 mice. Forty-eight hours after in vitro knockdown, Hes1 gene expression was reduced at both mRNA and protein levels. Myo7a (hair cell marker) and Sox2 (progenitor cell marker) mRNA levels also significantly increased. The modulation of gene expression in the organ of Corti upon Hes1 knockdown is consistent with cell phenotypes related to lateral inhibition mechanism interference in the inner ear. The lentivirus-based expression of Hes1-shRNA is a valuable strategy for genetic interference in the organ of Corti and for future evaluation of its efficacy in protocols aiming at the regeneration of hair cells in vivo.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Cóclea , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular , Células Ciliadas Auditivas , Camundongos , Órgão Espiral , Receptores Notch , Fatores de Transcrição HES-1/genéticaRESUMO
NOTCH pathway proteins, including the transcriptional factor HES1, play crucial roles in the development of the inner ear by means of the lateral inhibition mechanism, in which supporting cells have their phenotype preserved while they are prevented from becoming hair cells. Genetic manipulation of this pathway has been demonstrated to increase hair cell number. The present study aimed to investigate gene expression effects in hair cells and supporting cells after Hes1-shRNA lentivirus transduction in organotypic cultures of the organ of Corti from postnatal-day-3 mice. Forty-eight hours after in vitro knockdown, Hes1 gene expression was reduced at both mRNA and protein levels. Myo7a (hair cell marker) and Sox2 (progenitor cell marker) mRNA levels also significantly increased. The modulation of gene expression in the organ of Corti upon Hes1 knockdown is consistent with cell phenotypes related to lateral inhibition mechanism interference in the inner ear. The lentivirus-based expression of Hes1-shRNA is a valuable strategy for genetic interference in the organ of Corti and for future evaluation of its efficacy in protocols aiming at the regeneration of hair cells in vivo.
Assuntos
Animais , Ratos , Cóclea , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Órgão Espiral , Diferenciação Celular , Receptores Notch , Fatores de Transcrição HES-1/genética , Células Ciliadas AuditivasRESUMO
The immune stimulatory and anti-neoplastic functions of type I interferon have long been applied for the treatment of melanoma. However, the systemic application of high levels of this recombinant protein is often met with toxicity. An approach that provides localized, yet transient, production of type I interferon may overcome this limitation. We propose that the use of mesenchymal stem cells (MSCs) as delivery vehicles for the production of interferon-ß (IFNß) may be beneficial when applied together with our cancer gene therapy approach. In our previous studies, we have shown that adenovirus-mediated gene therapy with IFNß was especially effective in combination with p19Arf gene transfer, resulting in immunogenic cell death. Here we showed that MSCs derived from mouse adipose tissue were susceptible to transduction with adenovirus, expressed the transgene reliably, and yet were not especially sensitive to IFNß production. MSCs used to produce IFNß inhibited B16 mouse melanoma cells in a co-culture assay. Moreover, the presence of p19Arf in the B16 cells sensitizes them to the IFNß produced by the MSCs. These data represent a critical demonstration of the use of MSCs as carriers of adenovirus encoding IFNß and applied as an anti-cancer strategy in combination with p19Arf gene therapy.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/administração & dosagem , Interferon beta/metabolismo , Melanoma Experimental/terapia , Células-Tronco Mesenquimais/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Terapia Genética , Masculino , Melanoma Experimental/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transdução GenéticaRESUMO
The immune stimulatory and anti-neoplastic functions of type I interferon have long been applied for the treatment of melanoma. However, the systemic application of high levels of this recombinant protein is often met with toxicity. An approach that provides localized, yet transient, production of type I interferon may overcome this limitation. We propose that the use of mesenchymal stem cells (MSCs) as delivery vehicles for the production of interferon-β (IFNβ) may be beneficial when applied together with our cancer gene therapy approach. In our previous studies, we have shown that adenovirus-mediated gene therapy with IFNβ was especially effective in combination with p19Arf gene transfer, resulting in immunogenic cell death. Here we showed that MSCs derived from mouse adipose tissue were susceptible to transduction with adenovirus, expressed the transgene reliably, and yet were not especially sensitive to IFNβ production. MSCs used to produce IFNβ inhibited B16 mouse melanoma cells in a co-culture assay. Moreover, the presence of p19Arf in the B16 cells sensitizes them to the IFNβ produced by the MSCs. These data represent a critical demonstration of the use of MSCs as carriers of adenovirus encoding IFNβ and applied as an anti-cancer strategy in combination with p19Arf gene therapy.
Assuntos
Animais , Masculino , Coelhos , Melanoma Experimental/terapia , Interferon beta/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/administração & dosagem , Células-Tronco Mesenquimais/metabolismo , Transdução Genética , Melanoma Experimental/metabolismo , Terapia Genética , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Camundongos Endogâmicos C57BLRESUMO
In mammals, damage to sensory receptor cells (hair cells) of the inner ear results in permanent sensorineural hearing loss. Here, we investigated whether postnatal mouse inner ear progenitor/stem cells (mIESCs) are viable after transplantation into the basal turns of neomycin-injured guinea pig cochleas. We also examined the effects of mIESC transplantation on auditory functions. Eight adult female Cavia porcellus guinea pigs (250-350 g) were deafened by intratympanic neomycin delivery. After 7 days, the animals were randomly divided in two groups. The study group (n=4) received transplantation of LacZ-positive mIESCs in culture medium into the scala tympani. The control group (n=4) received culture medium only. At 2 weeks after transplantation, functional analyses were performed by auditory brainstem response measurement, and the animals were sacrificed. The presence of mIESCs was evaluated by immunohistochemistry of sections of the cochlea from the study group. Non-parametric tests were used for statistical analysis of the data. Intratympanic neomycin delivery damaged hair cells and increased auditory thresholds prior to cell transplantation. There were no significant differences between auditory brainstem thresholds before and after transplantation in individual guinea pigs. Some mIESCs were observed in all scalae of the basal turns of the injured cochleas, and a proportion of these cells expressed the hair cell marker myosin VIIa. Some transplanted mIESCs engrafted in the cochlear basilar membrane. Our study demonstrates that transplanted cells survived and engrafted in the organ of Corti after cochleostomy.
Assuntos
Células Ciliadas Auditivas Internas/transplante , Perda Auditiva Neurossensorial/cirurgia , Órgão Espiral/cirurgia , Transplante de Células-Tronco/métodos , Células-Tronco , Animais , Limiar Auditivo , Sobrevivência Celular , Células Cultivadas , Potenciais Evocados Auditivos do Tronco Encefálico , Feminino , Cobaias , Imuno-Histoquímica , Camundongos Endogâmicos BALB C , Neomicina , Inibidores da Síntese de Proteínas , Reprodutibilidade dos Testes , Resultado do TratamentoRESUMO
In mammals, damage to sensory receptor cells (hair cells) of the inner ear results in permanent sensorineural hearing loss. Here, we investigated whether postnatal mouse inner ear progenitor/stem cells (mIESCs) are viable after transplantation into the basal turns of neomycin-injured guinea pig cochleas. We also examined the effects of mIESC transplantation on auditory functions. Eight adult female Cavia porcellus guinea pigs (250-350g) were deafened by intratympanic neomycin delivery. After 7 days, the animals were randomly divided in two groups. The study group (n=4) received transplantation of LacZ-positive mIESCs in culture medium into the scala tympani. The control group (n=4) received culture medium only. At 2 weeks after transplantation, functional analyses were performed by auditory brainstem response measurement, and the animals were sacrificed. The presence of mIESCs was evaluated by immunohistochemistry of sections of the cochlea from the study group. Non-parametric tests were used for statistical analysis of the data. Intratympanic neomycin delivery damaged hair cells and increased auditory thresholds prior to cell transplantation. There were no significant differences between auditory brainstem thresholds before and after transplantation in individual guinea pigs. Some mIESCs were observed in all scalae of the basal turns of the injured cochleas, and a proportion of these cells expressed the hair cell marker myosin VIIa. Some transplanted mIESCs engrafted in the cochlear basilar membrane. Our study demonstrates that transplanted cells survived and engrafted in the organ of Corti after cochleostomy.
Assuntos
Animais , Feminino , Órgão Espiral/cirurgia , Células-Tronco , Transplante de Células-Tronco/métodos , Células Ciliadas Auditivas Internas/transplante , Perda Auditiva Neurossensorial/cirurgia , Limiar Auditivo , Imuno-Histoquímica , Inibidores da Síntese de Proteínas , Neomicina , Sobrevivência Celular , Células Cultivadas , Reprodutibilidade dos Testes , Potenciais Evocados Auditivos do Tronco Encefálico , Resultado do Tratamento , Cobaias , Camundongos Endogâmicos BALB CRESUMO
Approximately 90% of melanomas retain wild-type p53, a characteristic that may help shape the development of novel treatment strategies. Here, we employed an adenoviral vector where transgene expression is controlled by p53 to deliver the p19 alternate reading frame (Arf) and interferon-ß (IFNß) complementary DNAs in the B16 mouse model of melanoma. In vitro, cell death was enhanced by combined gene transfer (63.82±15.30% sub-G0 cells); yet introduction of a single gene resulted in significantly fewer hypoploid cells (37.73±7.3% or 36.96±11.58%, p19Arf or IFNß, respectively, P<0.05). Annexin V staining and caspase-3 cleavage indicate a cell death mechanism consistent with apoptosis. Using reverse transcriptase quantitative PCR, we show that key transcriptional targets of p53 were upregulated in the presence of p19Arf, although treatment with IFNß did not alter expression of the genes studied. In situ gene therapy revealed significant inhibition of subcutaneous tumors by IFNß (571±25 mm3) or the combination of p19Arf and IFNß (489±124 mm3) as compared with the LacZ control (1875±33 mm3, P<0.001), whereas p19Arf yielded an intermediate result (1053±169 mm3, P<0.01 vs control). However, only the combination was associated with increased cell death and prolonged survival (P<0.01). As shown here, the combined transfer of p19Arf and IFNß using p53-responsive vectors enhanced cell death both in vitro and in vivo.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/genética , Interferon beta/genética , Melanoma Experimental/genética , Melanoma Experimental/terapia , Animais , Apoptose/genética , Morte Celular/genética , Linhagem Celular Tumoral , Inibidor p16 de Quinase Dependente de Ciclina/biossíntese , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Modelos Animais de Doenças , Feminino , Interferon beta/biossíntese , Interferon beta/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Transdução GenéticaRESUMO
Genetically modified animals have numerous applications, ranging from basic research to livestock production and agriculture. Recent progress in animal cloning by nuclear transfer has made possible the production of transgenic animals using previously genetically modified cell lineages. However, to produce such lineages, an additional time for in vitro culturing and great manipulation is needed. Herein, we aimed to characterize different aspects of genetically modified cells compared to control cells, and we also analyzed the development rate of embryos produced by nuclear transfer by using them as nuclei donors after short or long periods of in vitro culturing (early versus late passages). We hypothesized that the genetic material inserted in the genome of these cells, associated with the prolonged time in culture, ultimately alters cell growth physiology and cell viability, which leads to impaired nuclei reprogramming potential and consequent reduction in the production of cloned blastocysts. Fetal fibroblasts expressing the enhanced Green Fluorescent Protein gene (eGFP) cultured for different periods in vitro were analyzed with respect to chromosomal numeric abnormalities, nuclear DNA fragmentation, the ratio of BAX and BCL2 gene transcripts, and the intensity of mitochondrial membrane potential, and they were then used as nuclei donors for somatic cell nuclear transfer (SCNT). Early passages were defined as fewer than 11 passages, and late passages were 18th passage (18(th)p) to 21(st)p. No differences were observed in the percentage of cells with chromosomal abnormalities or in the mitochondrial membrane potential analysis. eGFP cells in late passages and control cells in early passages were not different regarding DNA fragmentation; however, control cells in late passages presented higher fragmentation (P < 0.05). The Bax and Bcl2 gene expression ratio in control and transgenic cells presented different patterns regarding cell conditions during culture. For SCNT experiments, no difference was observed between groups reconstructed with early or late-passage cells when fusion (63.1% and 49%), cleavage (67.7% and 69.9%), eight-cell embryo (36.4% and 44.4%) and blastocyst (21.6% and 20.8%) rates were compared. In conclusion, culture behavior was different between control and eGFP cells. However, when different in vitro culturing periods were compared, long-term cultured transgenic fetal fibroblasts remained competent for blastocyst production when used as nuclei donors in the nuclear transfer technique, a feature needed for the genetic manipulation of cell culture experiments aiming for transgenic animal production.