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Introduction: Human induced pluripotent stem cells (iPSCs), with their ability to generate human neural cells (astrocytes and neurons) from patients, hold great promise for understanding the pathophysiology of major neuropsychiatric diseases such as schizophrenia and bipolar disorders, which includes alterations in cerebral development. Indeed, the in vitro neurodifferentiation of iPSCs, while recapitulating certain major stages of neurodevelopment in vivo, makes it possible to obtain networks of living human neurons. The culture model presented is particularly attractive within this framework since it involves iPSC-derived neural cells, which more specifically differentiate into cortical neurons of diverse types (in particular glutamatergic and GABAergic) and astrocytes. However, these in vitro neuronal networks, which may be heterogeneous in their degree of differentiation, remain challenging to bring to an appropriate level of maturation. It is therefore necessary to develop tools capable of analyzing a large number of cells to assess this maturation process. Calcium (Ca2+) imaging, which has been extensively developed, undoubtedly offers an incredibly good approach, particularly in its versions using genetically encoded calcium indicators. However, in the context of these iPSC-derived neural cell cultures, there is a lack of studies that propose Ca2+ imaging methods that can finely characterize the evolution of neuronal maturation during the neurodifferentiation process. Methods: In this study, we propose a robust and reliable method for specifically measuring neuronal activity at two different time points of the neurodifferentiation process in such human neural cultures. To this end, we have developed a specific Ca2+ signal analysis procedure and tested a series of different AAV serotypes to obtain expression levels of GCaMP6f under the control of the neuron-specific human synapsin1 (hSyn) promoter. Results: The retro serotype has been found to be the most efficient in driving the expression of the GCaMP6f and is compatible with multi-time point neuronal Ca2+ imaging in our human iPSC-derived neural cultures. An AAV2/retro carrying GCaMP6f under the hSyn promoter (AAV2/retro-hSyn-GCaMP6f) is an efficient vector that we have identified. To establish the method, calcium measurements were carried out at two time points in the neurodifferentiation process with both hSyn and CAG promoters, the latter being known to provide high transient gene expression across various cell types. Discussion: Our results stress that this methodology involving AAV2/retro-hSyn-GCaMP6f is suitable for specifically measuring neuronal calcium activities over multiple time points and is compatible with the neurodifferentiation process in our mixed human neural cultures.
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The recombinant adenoassociated virus 8 (rAAV8) vector is a widely used tool in basic research and clinical trials. The cytomegalovirus immediateearly enhancer/chicken ßactin (CAG) promoter is a synthetic promoter used in adenoviral constructs with a wide spectrum and notable efficiency. The thyroxine binding globulin (TBG) promoter is a liverspecific promoter, which directs transgene expression in hepatocytes. However, the transduction efficiency of the rAAV vector is dependent on both the administration routes and the promoter elements. In the present study, the transduction efficiency in the liver following intraperitoneal (IP) and intravenous (IV) injections of rAAV8 with the CAG, TBG669 and TBG410 promoters was compared. Enhanced green fluorescent protein (EGFP) expression was used as the biomarker to indicate efficiency. Among the three different promoters, CAG exhibited the highest efficiency from both IV and IP injections. Following IV administration, EGFP expression, induced by the CAG promoter, was 67fold higher compared with that in the TBG410 promoter group and 26fold higher compared with that in the TBG669 promoter group. EGFP protein expression was higher with IV injection compared with that for IP injection for both the CAG and TBG669 promoters (P<0.05). With the CAG promoter, EGFP protein expression was 1.5fold higher with the use of IV injection than with IP injection. With the TBG410 promoter, no differences were observed between the two administrations. In conclusion, these findings demonstrated that the CAG promoter was much more efficient at driving gene expression in the liver compared with that for the TBG promoters in rAAV8. In addition, IP administration produced comparable efficiency for gene delivery via the rAAV8 vector, particularly with the promoter TBG410.
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Dependovirus/genética , Regiões Promotoras Genéticas/genética , Transdução Genética/métodos , Actinas/genética , Animais , Antígenos Virais/genética , China , Dependovirus/metabolismo , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Vetores Genéticos/genética , Hepatócitos/metabolismo , Proteínas Imediatamente Precoces/genética , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Globulina de Ligação a Tiroxina/genética , Transgenes/genéticaRESUMO
There is wide variability in the propensity of somatic cells to reprogram into pluripotency in response to the Yamanaka factors. How to segregate these variabilities to enrich for cells of specific traits that reprogram efficiently remains challenging. Here we report that the variability in reprogramming propensity is associated with the activity of the MKL1/SRF transcription factor and concurs with small cell size as well as rapid cell cycle. Reprogramming progressive cells can be prospectively identified by their low activity of a widely used synthetic promoter, CAG. CAGlow cells arise and expand during cell cycle acceleration in the early reprogramming culture of both mouse and human fibroblasts. Our work illustrates a molecular scenario underlying the distinct reprogramming propensities and demonstrates a convenient practical approach for their enrichment.
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Técnicas de Reprogramação Celular , Reprogramação Celular , Regiões Promotoras Genéticas , Fatores de Transcrição , Animais , Camundongos , Camundongos Transgênicos , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
Human pluripotent stem cells, including induced pluripotent stem cells (iPSCs) and embryonic stem cells, hold great promise for cell-based therapies, but safety concerns that complicate consideration for routine clinical use remain. Installing a "safety switch" based on the inducible caspase-9 (iCASP9) suicide gene system should offer added control over undesirable cell replication or activity. Previous studies utilized lentiviral vectors to integrate the iCASP9 system into T cells and iPSCs. This method results in random genomic insertion of the suicide switch and inefficient killing of the cells after the switch is "turned on" with a small molecule (eg, AP1903). To improve the safety and efficiency of the iCASP9 system for use in iPSC-based therapy, we precisely installed the system into a genomic safe harbor, the AAVS1 locus in the PPP1R12C gene. We then evaluated the efficiencies of different promoters to drive iCASP9 expression in human iPSCs. We report that the commonly used EF1α promoter is silenced in iPSCs, and that the endogenous promoter of the PPP1R12C gene is not strong enough to drive high levels of iCASP9 expression. However, the CAG promoter induces strong and stable iCASP9 expression in iPSCs, and activation of this system with AP1903 leads to rapid killing and complete elimination of iPSCs and their derivatives, including MSCs and chondrocytes, in vitro. Furthermore, iPSC-derived teratomas shrank dramatically or were completely eliminated after administration of AP1903 in mice. Our data suggest significant improvements on existing iCASP9 suicide switch technologies and may serve as a guide to other groups seeking to improve the safety of stem cell-based therapies.
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Genes Transgênicos Suicidas/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Animais , Diferenciação Celular , Humanos , CamundongosRESUMO
The loss of photoreceptors in individuals with retinal degenerative diseases leads to partial or complete blindness. Optogenetic therapy is a promising approach for restoring vision to the blind. Multiple strategies have been employed by targeting genetically encoded light sensors, particularly channelrhodopsins, to surviving retinal neurons in animal models. In particular, the strategy of targeting retinal bipolar cells has commonly been expected to result in better vision than ubiquitous expression in retinal ganglion cells. However, a direct comparison of the channelrhodopsin-restored vision between these two strategies has not been performed. Here, we compared the restored visual functions achieved by adeno-associated virus (AAV)-mediated expression of a channelrhodopsin in ON-type bipolar cells and retinal ganglion cells driven by an improved mGluR6 promoter and a CAG promoter, respectively, in a blind mouse model by performing electrophysiological recordings and behavioral assessments. Unexpectedly, the efficacy of the restored vision based on light sensitivity and visual acuity was much higher following ubiquitous retinal ganglion cell expression than that of the strategy targeting ON-type bipolar cells. Our study suggests that, at least based on currently available gene delivery techniques, the expression of genetically encoded light sensors in retinal ganglion cells is likely a practical and advantageous strategy for optogenetic vision restoration.
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BACKGROUND: Research on the biogenesis of tick-borne encephalitis virus (TBEV) would benefit gene therapy. Due to specific arrangements of genes along the TBEV genome, its viral-like particles (VLPs) could be exploited as shuttles to deliver their replicon, which carries therapeutic genes, to immune system cells. OBJECTIVE: To develop a flaviviral vector for gene delivery as a part of gene therapy research that can be expressed in secretable VLP suicidal shuttles and provide abundant unique molecular and structural data supporting this gene therapy concept. METHOD: TBEV structural gene constructs of a Swedish Torö strain were cloned into plasmids driven by the promoters CAG and CMV and then transfected into various cell lines, including COS-1 and BHK-21. Time-course sampling of the cells, culture fluid, cell lysate supernatant, and pellet specimens were performed. Western blotting and electron microscopy analyses of collected specimens were used to investigate molecular and structural processing of TBEV structural proteins. RESULTS: Western blotting analysis showed differences between promoters in directing the gene expression of the VLPs constructs. The premature flaviviral polypeptides as well as mature VLPs could be traced. Using electron microscopy, the premature and mature VLP accumulation in cellular compartments-and also endoplasmic reticulum proliferation as a virus factory platform-were observed in addition to secreted VLPs. CONCLUSIONS: The abundant virologic and cellular findings in this study show the natural processing and safety of inserting flaviviral structural genes into suicidal VLP shuttles. Thus, we propose that these VLPs are a suitable gene delivering system model in gene therapy.
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Fowl adenoviruses (FAdVs) are promising vectors for poultry vaccines and gene therapy. The commonly used human cytomegalovirus (CMV) promoter in recombinant FAdV-9 viruses (recFAdV-9s) leads to foreign gene expression that elicits an antibody response. Despite its strength, studies have shown that the CMV promoter is prone to silencing by methylation hampering the in vivo application of vectors containing this promoter. Therefore, to improve our virus vector system and circumvent potential limitations of silencing, we engineered recFAdV-9s with foreign gene expression cassettes carrying the CMV enhancer/chicken ß-actin (CAG) or the human elongation factor 1 alpha (EF1α) promoters with or without the posttranscriptional regulatory element of woodchuck hepatitis virus (WPRE). Chicken hepatoma cells (CH-SAH) infected with recFAdV-9s carrying either CAG or EF1α promoters expressed higher levels of foreign protein than those infected with recFAdV-9 carrying the CMV promoter. Incorporation of the WPRE element rendered lower gene expression regardless of promoter type. Surprisingly, most chickens inoculated with recFAdV-9 containing the CMV promoter had the highest antibody response to foreign protein compared to other promoters. Our findings suggest the importance of promoter selection for candidate virus vector vaccines based on humoral immune response rather than foreign protein expression levels in vitro.
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Adenoviridae/genética , Vetores Genéticos/genética , Regiões Promotoras Genéticas/genética , Animais , Formação de Anticorpos/genética , Formação de Anticorpos/fisiologia , GalinhasRESUMO
Carcinogenesis is etiologically associated with somatic mutations of critical genes. Recently, a number of somatic mutations and key molecules have been found to be involved in functional networks affecting cancer progression. Suitable animal models are required to validate cancer-promoting or -inhibiting capacities of these mutants and molecules. Sleeping Beauty transposon system consists of a transposon that carries gene(s) of interest and a transposase that recognizes, excises, and reinserts genes in given location of the genome. It can create both gain-of-function and loss-of-function mutations, thus being frequently chosen to investigate the etiological mechanisms and gene therapy for cancers in animal models. In this review, we summarized current advances of Sleeping Beauty transposon system in revealing molecular mechanism of cancers and improving gene therapy. Understanding molecular mechanisms by which driver mutations contribute to carcinogenesis and metastasis may pave the way for the development of innovative prophylactic and therapeutic strategies against malignant diseases.