RESUMO
We report transgenic mouse models in which three or four reprogramming factors are expressed from a single genomic locus using a drug-inducible transgene. Multiple somatic cell types can be directly reprogrammed to generate induced pluripotent stem cells (iPSCs) by culture in doxycycline. Because reprogramming factors are carried on a single polycistronic construct, the mice can be easily maintained, and the transgene can be easily transferred into other genetic backgrounds.
Assuntos
Reprogramação Celular/genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Transgenes/genética , Animais , Desdiferenciação Celular/efeitos dos fármacos , Células Cultivadas , Reprogramação Celular/efeitos dos fármacos , Doxiciclina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Genoma , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Integrases/metabolismo , Camundongos , Camundongos Transgênicos , Mutagênese Insercional , Especificidade de Órgãos , Recombinação GenéticaRESUMO
Realizing the full potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) requires efficient methods for genetic modification. However, techniques to generate cell type-specific lineage reporters, as well as reliable tools to disrupt, repair or overexpress genes by gene targeting, are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc-finger nuclease (ZFN)-mediated genome editing. First, using ZFNs specific for the OCT4 (POU5F1) locus, we generated OCT4-eGFP reporter cells to monitor the pluripotent state of hESCs. Second, we inserted a transgene into the AAVS1 locus to generate a robust drug-inducible overexpression system in hESCs. Finally, we targeted the PITX3 gene, demonstrating that ZFNs can be used to generate reporter cells by targeting non-expressed genes in hESCs and hiPSCs.
Assuntos
Desoxirribonucleases/metabolismo , Células-Tronco Embrionárias/fisiologia , Marcação de Genes/métodos , Células-Tronco Pluripotentes/fisiologia , Dedos de Zinco/fisiologia , Linhagem Celular , Desoxirribonucleases/genética , Expressão Gênica , Inativação Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Imuno-Histoquímica , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients represent a powerful tool for biomedical research and may provide a source for replacement therapies. However, the use of viruses encoding the reprogramming factors represents a major limitation of the current technology since even low vector expression may alter the differentiation potential of the iPSCs or induce malignant transformation. Here, we show that fibroblasts from five patients with idiopathic Parkinson's disease can be efficiently reprogrammed and subsequently differentiated into dopaminergic neurons. Moreover, we derived hiPSCs free of reprogramming factors using Cre-recombinase excisable viruses. Factor-free hiPSCs maintain a pluripotent state and show a global gene expression profile, more closely related to hESCs than to hiPSCs carrying the transgenes. Our results indicate that residual transgene expression in virus-carrying hiPSCs can affect their molecular characteristics and that factor-free hiPSCs therefore represent a more suitable source of cells for modeling of human disease.
Assuntos
Doença de Parkinson/metabolismo , Células-Tronco Pluripotentes/patologia , Diferenciação Celular , Reprogramação Celular , Dopamina/metabolismo , Fibroblastos/metabolismo , Humanos , Neurônios/metabolismoRESUMO
Rett Syndrome (RTT) is a severe form of X-linked mental retardation caused by mutations in the gene coding for methyl CpG-binding protein 2 (MECP2). Mice deficient in MeCP2 have a range of physiological and neurological abnormalities that mimic the human syndrome. Here we show that systemic treatment of MeCP2 mutant mice with an active peptide fragment of Insulin-like Growth Factor 1 (IGF-1) extends the life span of the mice, improves locomotor function, ameliorates breathing patterns, and reduces irregularity in heart rate. In addition, treatment with IGF-1 peptide increases brain weight of the mutant mice. Multiple measurements support the hypothesis that RTT results from a deficit in synaptic maturation in the brain: MeCP2 mutant mice have sparse dendritic spines and reduced PSD-95 in motor cortex pyramidal neurons, reduced synaptic amplitude in the same neurons, and protracted cortical plasticity in vivo. Treatment with IGF-1 peptide partially restores spine density and synaptic amplitude, increases PSD-95, and stabilizes cortical plasticity to wild-type levels. Our results thus strongly suggest IGF-1 as a candidate for pharmacological treatment of RTT and potentially of other CNS disorders caused by delayed synapse maturation.
Assuntos
Fator de Crescimento Insulin-Like I/farmacologia , Proteína 2 de Ligação a Metil-CpG/genética , Síndrome de Rett/tratamento farmacológico , Potenciais de Ação , Animais , Encéfalo , Modelos Animais de Doenças , Frequência Cardíaca , Fator de Crescimento Insulin-Like I/uso terapêutico , Camundongos , Camundongos Mutantes , Atividade Motora , Neurônios , Tamanho do Órgão , Taxa de Sobrevida , Transmissão Sináptica , Resultado do TratamentoRESUMO
Proviruses carrying drug-inducible Oct4, Sox2, Klf4 and c-Myc used to derive 'primary' induced pluripotent stem (iPS) cells were segregated through germline transmission, generating mice and cells carrying subsets of the reprogramming factors. Drug treatment produced 'secondary' iPS cells only when the missing factor was introduced. This approach creates a defined system for studying reprogramming mechanisms and allows screening of genetically homogeneous cells for compounds that can replace any transcription factor required for iPS cell derivation.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Doxiciclina/farmacologia , Técnicas Genéticas , Fatores de Transcrição/genética , Animais , Células Cultivadas , Quimera/genética , Quimera/metabolismo , Feminino , Fibroblastos/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Provírus/genética , Provírus/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/efeitos dos fármacosRESUMO
We develop biodegradable polymeric nanoparticles to facilitate nonviral gene transfer to human embryonic stem cells (hESCs). Small (approximately 200 nm), positively charged (approximately 10 mV) particles are formed by the self assembly of cationic, hydrolytically degradable poly(beta-amino esters) and plasmid DNA. By varying the end group of the polymer, we can tune the biophysical properties of the resulting nanoparticles and their gene-delivery efficacy. We created an OCT4-driven GFP hES cell line to allow the rapid identification of nanoparticles that facilitate gene transfer while maintaining an hESC undifferentiated state. Using this cell system, we synthesized nanoparticles that have gene delivery efficacy that is up to 4 times higher than that of the leading commercially available transfection agent, Lipofectamine 2000. Importantly, these materials have minimal toxicity and do not adversely affect hESC colony morphology or cause nonspecific differentiation.
Assuntos
Células-Tronco Embrionárias/citologia , Técnicas de Transferência de Genes , Vetores Genéticos/química , Animais , Materiais Biocompatíveis/química , Cátions , Diferenciação Celular , Citometria de Fluxo , Técnicas Genéticas , Proteínas de Fluorescência Verde/metabolismo , Hidrólise , Camundongos , Nanotecnologia/métodos , Fator 3 de Transcrição de Octâmero/metabolismo , Polímeros/químicaRESUMO
Pluripotent cells can be derived from fibroblasts by ectopic expression of defined transcription factors. A fundamental unresolved question is whether terminally differentiated cells can be reprogrammed to pluripotency. We utilized transgenic and inducible expression of four transcription factors (Oct4, Sox2, Klf4, and c-Myc) to reprogram mouse B lymphocytes. These factors were sufficient to convert nonterminally differentiated B cells to a pluripotent state. However, reprogramming of mature B cells required additional interruption with the transcriptional state maintaining B cell identity by either ectopic expression of the myeloid transcription factor CCAAT/enhancer-binding-protein-alpha (C/EBPalpha) or specific knockdown of the B cell transcription factor Pax5. Multiple iPS lines were clonally derived from both nonfully and fully differentiated B lymphocytes, which gave rise to adult chimeras with germline contribution, and to late-term embryos when injected into tetraploid blastocysts. Our study provides definite proof for the direct nuclear reprogramming of terminally differentiated adult cells to pluripotency.
Assuntos
Linfócitos B/citologia , Diferenciação Celular , Células-Tronco Pluripotentes/citologia , Animais , Núcleo Celular/genética , Células-Tronco Embrionárias/citologia , Humanos , Fator 4 Semelhante a Kruppel , Camundongos , Fatores de Transcrição/metabolismoRESUMO
It has recently been demonstrated that mouse and human fibroblasts can be reprogrammed into an embryonic stem cell-like state by introducing combinations of four transcription factors. However, the therapeutic potential of such induced pluripotent stem (iPS) cells remained undefined. By using a humanized sickle cell anemia mouse model, we show that mice can be rescued after transplantation with hematopoietic progenitors obtained in vitro from autologous iPS cells. This was achieved after correction of the human sickle hemoglobin allele by gene-specific targeting. Our results provide proof of principle for using transcription factor-induced reprogramming combined with gene and cell therapy for disease treatment in mice. The problems associated with using retroviruses and oncogenes for reprogramming need to be resolved before iPS cells can be considered for human therapy.
Assuntos
Anemia Falciforme/terapia , Reprogramação Celular , Fibroblastos/citologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Pluripotentes/citologia , Anemia Falciforme/sangue , Anemia Falciforme/fisiopatologia , Animais , Diferenciação Celular , Células Cultivadas , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Células-Tronco Embrionárias/citologia , Contagem de Eritrócitos , Genes myc , Globinas/genética , Hematopoese , Hemoglobina A/análise , Hemoglobina Falciforme/análise , Humanos , Capacidade de Concentração Renal , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Masculino , Camundongos , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição SOXB1 , Transativadores/genética , Transdução GenéticaRESUMO
In humans, mutations in the X-linked MECP2 gene, are the cause of Rett syndrome (RTT), a neurodevelopmental disorder that affects mainly girls. MeCP2 binds to methylated CpGs and is thought to act as a transcriptional repressor. In male mice, deletion or targeted mutation of Mecp2 leads to lethality and causes a neuronal phenotype. Selective mutation of Mecp2 in postnatal neurons results in a similar, although delayed, phenotype, suggesting that the symptoms are caused by MeCP2 deficiency in postmitotic neurons. In agreement with this idea, expression of a Mecp2 transgene in postmitotic neurons of Mecp2-null mutant mice resulted in the phenotypical rescue of the symptoms. To assess whether postnatal activation of MeCP2 in mutant animals could also affect the progression of the disorder, we constructed a conditionally active Mecp2 "rescue transgene" that was activated between P0 and P30. The Mecp2 transgene was under the control of the CAGGS promoter and was activated by using brain specific Cre-mediated recombination. Our results indicate that postnatal, neuron-specific activation of MeCP2 as late as 2-4 weeks of age significantly prolonged the lifespan of mutant animals and delayed the onset of neurologic symptoms.
Assuntos
Animais Recém-Nascidos , Regulação da Expressão Gênica/fisiologia , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Síndrome de Rett/genética , Animais , Modelos Animais de Doenças , Feminino , Masculino , Proteína 2 de Ligação a Metil-CpG/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Camundongos Transgênicos , Síndrome de Rett/mortalidadeRESUMO
When overexpressed in primary erythroid progenitors, oncogenic Ras leads to the constitutive activation of its downstream signaling pathways, severe block of terminal erythroid differentiation, and cytokine-independent growth of primary erythroid progenitors. However, whether high-level expression of oncogenic Ras is required for these phenotypes is unknown. To address this issue, we expressed oncogenic K-ras (K-ras(G12D)) from its endogenous promoter using a tetracycline-inducible system. We show that endogenous K-ras(G12D) leads to a partial block of terminal erythroid differentiation in vivo. In contrast to results obtained when oncogenic Ras was overexpressed from retroviral vectors, endogenous levels of K-ras(G12D) fail to constitutively activate but rather hyperactivate cytokine-dependent signaling pathways, including Stat5, Akt, and p44/42 MAPK, in primary erythroid progenitors. This explains previous observations that hematopoietic progenitors expressing endogenous K-ras(G12D) display hypersensitivity to cytokine stimulation in various colony assays. Our results support efforts to modulate Ras signaling for treating hematopoietic malignancies.
Assuntos
Diferenciação Celular , Citocinas/farmacologia , Eritrócitos/citologia , Transdução de Sinais , Proteínas ras/genética , Proteínas ras/fisiologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Eritropoese , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos , Proteínas Oncogênicas , Regiões Promotoras Genéticas , Transdução de Sinais/efeitos dos fármacosRESUMO
Transcriptional silencing mediated by DNA methylation is a critical component of epigenetic regulation during early embryonic development in animals. However, the requirement for DNA methylation during activation and differentiation of mature CD8+ T cells into effector and memory cells is not clear. Using cre-mediated deletion of DNA methyltransferase 1 (Dnmt1) at the time of CD8+ T cell activation, we investigated the obligation for maintaining patterns of DNA methylation during the generation of Ag-specific effector and memory CD8+ T cells in response to acute viral infection of mice with lymphocytic choriomeningitis virus. Dnmt1-/- CD8+ T cells failed to undergo the massive CD8+ T cell expansion characteristic of lymphocytic choriomeningitis virus infection, leading to >80% reductions in Ag-specific effector CD8+ T cells at the height of the response. Despite this, Dnmt1-/- CD8+ T cells efficiently controlled the viral infection. Interestingly, the number of Ag-specific Dnmt1-/- memory CD8+ T cells was moderately reduced compared with the reductions seen at day 8 postinfection. Our data suggest that ablation of Dnmt1 and subsequent DNA methylation affect the finite proliferative potential of Ag-specific CD8+ T cells with moderate effects on their differentiation to effector and memory CD8+ T cells.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Animais , Antígenos Virais/administração & dosagem , Linfócitos T CD8-Positivos/citologia , Diferenciação Celular , Proliferação de Células , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/deficiência , DNA (Citosina-5-)-Metiltransferases/genética , Memória Imunológica , Técnicas In Vitro , Ativação Linfocitária , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/metabolismo , Coriomeningite Linfocítica/patologia , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Camundongos Knockout , Camundongos TransgênicosRESUMO
Transgenic and gene-targeted mutant mice provide powerful tools for analysis of the cellular processes involved in early development and in the pathogenesis of many diseases. Here we describe a transgene integration strategy mediated by site-specific recombination that allows establishment of multiple embryonic stem (ES) cell lines carrying tetracycline-inducible genes targeted to a specific locus to assure predictable temporal and spatial expression in ES cells and mice. Using homologous recombination we inserted an frt homing site into which tetracycline-inducible transgenes can be integrated efficiently in the presence of FLPe recombinase. This strategy and the vectors described here are generally applicable to any locus in ES cells and should allow for the rapid production of mice with transgenes efficiently targeted to a defined site.
Assuntos
Embrião de Mamíferos/citologia , Camundongos Transgênicos/genética , Células-Tronco/metabolismo , Alelos , Animais , Colágeno Tipo I/genética , Fator de Crescimento Epidérmico/genética , Citometria de Fluxo , Camundongos , Recombinação Genética , Tetraciclina/farmacologiaRESUMO
The POU-domain transcription factor Oct-4 is normally expressed in pluripotent cells of the mammalian embryo. In addition, germ-cell tumors and a few somatic tumors show detectable expression of Oct-4. While Oct-4's role during preimplantation development is to maintain embryonic cells in a pluripotent state, little is known about its potential oncogenic properties. Here we investigate the effect of ectopic Oct-4 expression on somatic tissues of adult mice using a doxycycline-dependent expression system. Activation of Oct-4 results in dysplastic growths in epithelial tissues that are dependent on continuous Oct-4 expression. Dysplastic lesions show an expansion of progenitor cells and increased beta-catenin transcriptional activity. In the intestine, Oct-4 expression causes dysplasia by inhibiting cellular differentiation in a manner similar to that in embryonic cells. These data show that certain adult progenitors remain competent to interpret key embryonic signals and support the notion that progenitor cells are a driving force in tumorigenesis.
Assuntos
Diferenciação Celular/genética , Proteínas de Ligação a DNA/genética , Epitélio/patologia , Células-Tronco/patologia , Fatores de Transcrição/genética , Animais , Linhagem da Célula/genética , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/metabolismo , Doxiciclina/administração & dosagem , Doxiciclina/toxicidade , Epitélio/metabolismo , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/patologia , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Intestinos/patologia , Camundongos , Camundongos Transgênicos , Neoplasias/etiologia , Neoplasias/genética , Neoplasias/patologia , Fator 3 de Transcrição de Octâmero , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/patologia , Células-Tronco/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , beta CateninaRESUMO
Resistance to apoptosis, often achieved by the overexpression of antiapoptotic proteins, is common and perhaps required in the genesis of cancer. However, it remains uncertain whether apoptotic defects are essential for tumor maintenance. To test this, we generated mice expressing a conditional BCL-2 gene and constitutive c-myc that develop lymphoblastic leukemia. Eliminating BCL-2 yielded rapid loss of leukemic cells and significantly prolonged survival, formally validating BCL-2 as a rational target for cancer therapy. Loss of this single molecule resulted in cell death, despite or perhaps attributable to the presence of other oncogenic events. This suggests a generalizable model in which aberrations inherent to cancer generate tonic death signals that would otherwise kill the cell if not opposed by a requisite apoptotic defect(s).
Assuntos
Genes bcl-2 , Leucemia Linfoide/genética , Proteínas Proto-Oncogênicas c-bcl-2 , Animais , Apoptose , Citocromos c/metabolismo , Doxiciclina/farmacologia , Genes myc , Humanos , Leucemia de Células B/genética , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Transplante de Neoplasias , Proteínas Proto-Oncogênicas/metabolismo , Células Tumorais Cultivadas , Proteína X Associada a bcl-2RESUMO
Mutations in MECP2 are the cause of Rett syndrome (RTT) in humans, a neurodevelopmental disorder that affects mainly girls. MeCP2 is a protein that binds CpG dinucleotides and is thought to act as a global transcriptional repressor. It is highly expressed in neurons, but not in glia, of the postnatal brain. The timing of MeCP2 activation correlates with the maturation of the central nervous system, and recent reports suggest that MeCP2 may be involved in the formation of synaptic contacts and may function in activity-dependent neuronal gene expression. Deletion or targeted mutation of Mecp2 in mice leads to a Rett-like phenotype. Selective mutation of Mecp2 in postnatal neurons leads to a similar, although delayed, phenotype, suggesting that MeCP2 plays a role in postmitotic neurons. Here we test the hypothesis that the symptoms of RTT are exclusively caused by a neuronal MeCP2 deficiency by placing Mecp2 expression under the control of a neuron-specific promoter. Expression of the Mecp2 transgene in postmitotic neurons resulted in symptoms of severe motor dysfunction. Transgene expression in Mecp2 mutant mice, however, rescued the RTT phenotype.
Assuntos
Proteínas Cromossômicas não Histona , Proteínas de Ligação a DNA/genética , Neurônios/metabolismo , Proteínas Repressoras , Síndrome de Rett/genética , Animais , Sequência de Bases , Primers do DNA , Feminino , Imuno-Histoquímica , Masculino , Proteína 2 de Ligação a Metil-CpG , Camundongos , Camundongos Transgênicos , Mitose , Neurônios/citologia , Proteínas tau/genéticaRESUMO
Regulated apoptosis is essential for both the development and the subsequent maintenance of the immune system. Interleukins, including IL-2, IL-4, IL-7 and IL-15, heavily influence lymphocyte survival during the vulnerable stages of VDJ rearrangement and later in ensuring cellular homeostasis, but the genes specifically responsible for the development and maintenance of lymphocytes have not been identified. The antiapoptotic protein MCL-1 is an attractive candidate, as it is highly regulated, appears to enhance short-term survival and functions at an apical step in genotoxic deaths. However, Mcl-1 deficiency results in peri-implantation lethality. Here we show that mice conditional for Mcl-1 display a profound reduction in B and T lymphocytes when MCL-1 is removed. Deletion of Mcl-1 during early lymphocyte differentiation increased apoptosis and arrested the development at pro-B-cell and double-negative T-cell stages. Induced deletion of Mcl-1 in peripheral B- and T-cell populations resulted in their rapid loss. Moreover, IL-7 both induced and required MCL-1 to mediate lymphocyte survival. Thus, MCL-1, which selectively inhibits the proapoptotic protein BIM, is essential both early in lymphoid development and later on in the maintenance of mature lymphocytes.