Resumo
Cell fate specification, gene expression and spatial restriction are process finely tuned by epigenetic regulatory mechanisms. At the same time, mechanical forces have been shown to be crucial to drive cell plasticity and boost differentiation. Indeed, several studies have demonstrated that transitions along different specification states are strongly influenced by 3D rearrangement and mechanical properties of the surrounding microenvironment, that can modulate both cell potency and differentiation, through the activation of specific mechanosensing-related pathways. An overview of small molecule ability to modulate cell plasticity and define cell fate is here presented and results, showing the possibility to erase the epigenetic signature of adult dermal fibroblasts and convert them into insulin-producing cells (EpiCC) are described. The beneficial effects exerted on such processes, when cells are homed on an adequate substrate, that shows in vivo tissue-like stiffness are also discussed and the contribution of the Hippo signalling mechanotransduction pathway as one of the mechanisms involved is examined. In addition, results obtained using a genetically modified fibroblast cell line, expressing the enhanced green fluorescent protein (eGFP) under the control of the porcine insulin gene (INS) promoter (INS-eGFP transgenic pigs), are reported. This model offers the advantage to monitor the progression of cell conversion in real time mode. All these observations have a main role in order to allow a swift scale-up culture procedure, essential for cell therapy and tissue engineering applied to human regenerative medicine, and fundamental to ensure an efficient translation process from the results obtained at the laboratory bench to the patient bedside. Moreover, the creation of reliable in vitro model represents a key point to ensure the development of more physiological models that, in turn, may reduce the number of animals used, implementing non-invasive investigations and animal welfare and protection.
Assuntos
Epigênese Genética/genética , Rearranjo Gênico , Análise EspacialResumo
Cell fate specification, gene expression and spatial restriction are process finely tuned by epigenetic regulatory mechanisms. At the same time, mechanical forces have been shown to be crucial to drive cell plasticity and boost differentiation. Indeed, several studies have demonstrated that transitions along different specification states are strongly influenced by 3D rearrangement and mechanical properties of the surrounding microenvironment, that can modulate both cell potency and differentiation, through the activation of specific mechanosensing-related pathways. An overview of small molecule ability to modulate cell plasticity and define cell fate is here presented and results, showing the possibility to erase the epigenetic signature of adult dermal fibroblasts and convert them into insulin-producing cells (EpiCC) are described. The beneficial effects exerted on such processes, when cells are homed on an adequate substrate, that shows in vivo tissue-like stiffness are also discussed and the contribution of the Hippo signalling mechanotransduction pathway as one of the mechanisms involved is examined. In addition, results obtained using a genetically modified fibroblast cell line, expressing the enhanced green fluorescent protein (eGFP) under the control of the porcine insulin gene (INS) promoter (INS-eGFP transgenic pigs), are reported. This model offers the advantage to monitor the progression of cell conversion in real time mode. All these observations have a main role in order to allow a swift scale-up culture procedure, essential for cell therapy and tissue engineering applied to human regenerative medicine, and fundamental to ensure an efficient translation process from the results obtained at the laboratory bench to the patient bedside. Moreover, the creation of reliable in vitro model represents a key point to ensure the development of more physiological models that, in turn, may reduce the number of animals used, implementing non-invasive investigations and animal welfare and protection.(AU)
Assuntos
Epigênese Genética/genética , Rearranjo Gênico , Análise EspacialResumo
Enxertos cutâneos de espessura completa possuem a característica inicial de nutrição sem um aporte sanguíneo autônomo, predispondo a altas taxas de necrose. Células mesenquimais adipoderivadas (ADSC) auxiliam no desenvolvimento da angiogênese e cicatrização cutânea, contudo, os mecanismos envolvidos ainda não são completamente compreendidos. Esse trabalho teve como objetivo investigar a influência de ADSCs no desenvolvimento de novos vasos sanguíneos, seu efeito anti-inflamatório e o tempo de sobrevida das células após sua aplicação em um modelo de enxerto cutâneo de espessura completa. As ADSCs foram isoladas de ratos Wistar positivos para a proteína fluorescente verde (GFP). Os demais animais experimentais foram submetidos aos enxertos e posteriormente atribuídos de forma aleatória aos grupos ADSC, solução salina ou SHAM. Animais pertencentes ao grupo ADSC receberam 1 x 106 células ressuspendidas em 200 uL de solução salina, o grupo SS recebeu apenas 200 g de solução salina e o grupo SHAM não recebeu nenhum tratamento. Dezoito animais foram eutanasiados aos três dias, os demais aos cinco dias. A avaliação macroscópica analisou a taxa de contração, presença de sangramento ou exsudato. Os enxertos foram coletados para avaliação molecular e histopatológica, e aqueles pertencentes ao grupo ADSC foram corados com anticorpo anti-GFP para avaliar a presença e localização das células. O grupo ADSCs demonstrou menor taxa de contração e aumento da expressão de enzimas, fatores de crescimento e marcadores de superfície pró-angiogênicos. Marcadores de hipóxia, inflamação e estresse oxidativo tiveram expressão reduzida no grupo tratado com ADSCs. As células marcadas foram localizadas em ambos os dias de avaliação. Em conclusão, baseado nos dados obtidos, as células marcadas permanecem no local de aplicação ao terceiro e quinto dias de avaliação e podem promover a angiogênese precoce em enxertos cutâneos de espessura completa, reduzindo o tempo de exposição à hipóxia e podem aumentar as taxas de sucesso do enxerto.
Full-thickness skin grafts have the initial characteristic of graft nutrition without an autonomous blood supply, predicting to high necrosis rates. Adipose-derived stem cells supports angiogenesis development and wound healing, however, the mechanisms involved still not completely understood. In this study, we aimed to investigate ADSC influence in blood vessels growth, its anti-inflammatory effect and the remaining cell survival time on skin graft in full-thickness skin graft rat model. ADSCs were isolated from green-fluorescent protein positive Wistar rats. All the remaining experimental animals were submitted to skin graft and randomly assigned to ADSC, saline solution or SHAM groups. ADSC animals received 1x106 ADSCs GFP-positives resuspended in 200 g of saline solution. SS group only received 200 g of saline solution and SHAM group did not receive any treatment. Eighteen animals were euthanized at three days, the remaining at five days. Macroscopic evaluation was based on FTSG contraction rates, bleeding and exudate presence. Skin grafts were collected for molecular and histopathological assessment, and the ADSC group grafts were also immunostained with anti-GFP antibody for presence and location of cells. ADSC group have showed lower contraction rates and significant enhanced expression of proangiogenic enzymes, growth factors, surface markers. Expression of hypoxia, proinflammatory cell types and oxidative stress markers was decreased on ADSC treated group. The labeled ADSCs could be found on grafted tissue at both evaluation times. In conclusion, based on the obtained data, labeled ADSC remain on injected site at third and fifth evaluation days and can promote early angiogenesis development of FTSG, reducing hypoxia exposure and may increase the graft survival rates.
Resumo
The Green fluorescent protein (GFP) was first described after being extracted from Aequorea victoria in 1987; Since then, GFP and its derivatives have been widely used in several experiments as cell and protein marker. In the present study it was verified the genotype of the offspring from crosses between heterozygote Lewis LEW-Tg (EGFP) F455.5/Rrrc rats and analyzed the expression of the enhanced green fluorescent protein (EGFP) in different cell types and genotypes. The genotype of the offspring was assessed by PCR and analysis of EGFP expression in different cells and genotypes, including mesenchymal stem cells (MSC) derived from adipose tissue and calvarial osteoblast cells. Expression of EGFP was verified by flow cytometry, fluorescence microscopy, and immunostaining. Through these methods, it was identified the genotypes of the offspring and determined the levels of expression of EGFP in two cell types. A difference in expression between the (EGFP +/+) and (EGFP +/-) genotypes was also observed in addition to the presence of autofluorescence. Further studies on the natural fluorescence of cells with the (EGFP +/-) genotype and that induced by presence of the EGFP are necessary.(AU)
A proteína fluorescente verde (GFP) foi descrita pela primeira vez após ter sido extraída de Aequorea victoria em 1987. Desde então, a GFP e seus derivados têm sido amplamente utilizados em várias experiências como marcador celular e de proteínas. O objetivo do presente estudo foi o de verificar o genótipo dos descendentes de cruzamentos entre ratos Lewis LEW-Tg (EGFP) F455.5/Rrrc heterozigotos e de analisar a expressão da proteína fluorescente verde melhorada (EGFP) em diferentes tipos celulares e genótipos. O genótipo da descendência foi avaliado por PCR e pela análise da expressão da EGFP em diferentes células e genótipos, incluindo-se as células-tronco mesenquimais (MSC) derivadas de tecido adiposo e de osteoblastos de calvária. A expressão da EGFP foi verificada por citometria de fluxo, microscopia de fluorescência e imunocoloração. Foram, identificados os genótipos da descendência e determinados os níveis de expressão de EGFP em dois tipos de células. Foi também constatada uma diferença de expressão entre os genótipos (EGFP +/+) e (EGFP +/-) além da presença de autofluorescência. Mais estudos são necessários para esclarecer a fluorescência natural de células com o genótipo (EGFP +/-) e aquela induzida pela presença da EGFP.(AU)
Assuntos
Animais , Genótipo , Imunofluorescência/veterinária , Reação em Cadeia da Polimerase , Ratos/genética , Cruzamentos GenéticosResumo
Background: Intracytoplasmic sperm injection (ICSI) involves mechanical transfer of a single sperm cell into ooplasm. A new application has been recently found for ICSI, the production of transgenic animals. Since the birth of ''Dolly'', the first adult somatic cloned mammal, viable offspring has been produced by nuclear transfer in many species including cattle. The present review briefly summarizes our experience with ICSI and somatic cell nuclear transfer mainly to produce transgenic embryos, as well as for the generation of new micromanipulation technique. Review: We have evaluated different factors that affect SCNT and transgenesis including the chemical activator, the transfection event and the effect of recloning. Also, we included a brief description of the ICSI technique, which we used in five different species, examining its potential to produce transgenic embryos. Finally different strategies to produce transgenic animals were analyzed: ICSI- mediated gen transfer (ICSI-MGT), Injection of cumulus cell and ooplasmic vesicle incubated for 5 min with the transgene or injection of the plasmid alone. All of them were very efficient in exogenous DNA expression at embryo stages but resulted in mosaic embryos. We demonstrated that "ICSI-MGT" assisted by chemical activation is the only treatment of sperm mediated gen transfer capable to generated transgenic embryos in ovine. Besides, after ICSI-MGT, it is possible to obtain enhanced green fluorescent protein (EGFP)-expressing embryos in five diferent species: ovine, porcine, feline, bovine and equine. Our studies also established for the first time that short term transgene co-incubation with somatic cells can produce transgene-expressing mammalian SCNT embryos, and also that parthenogenic, eDNA- expressing embryos can be obtained by injection of vesicles or eDNA alone. Moreover, eDNA- -expressing embryos can be also obtained by cytoplasmic injection of vesicles in IVF zygotes, simplifying the traditional IVF pronuclear injection technique. We tried a further simplification of the technique in bovine oocytes and zygotes, by intracytoplasmically injecting them with eDNA-liposomes complexes. Approximately 70% of the cleaved embryos and 50% of the blastocysts expressed EGFP, when egfpliposome was injected 16 h post-fertilization. Different approaches were assayed to reverse the mosaicism including a novel technique of gamete cloning. Our first approach consisted of the production of transgenic IVF embryos by vesicle microinjection to generate transgenic blastomeres to be used as donor cells for cloning. A high efficiency in mosaicism reversal and multiplication of transgenic embryos was attaineded. Other technique assayed was the separation of transgenic blastomeres followed by the aggregation of two-cell fused embryos or by the asynchronous younger blastomere successfully multiplied transgenic embryos, and theoretically reduces mosaicism rates in future offspring [15]. This technology can also be used to multiply embryos from animals with high genetic value. We demonstrated that a sperm and oocyte can be efficiently cloned. Green haploid androgenic blastomeres produced with the injection of a single sperm by egfp ICSI-MGT could be used to fertilized oocytes resulting in several homogeneous expressing embryos. This approach shows great potential because it allows for determination of the sex of the sperm nucleus prior to fertilization. It is also possible to clone previously transfected oocytes followed by the reconstruction of biparental bovine embryos to generate homogeneous transgene-expressing embryos. This review summarizes recent experiments in micromanipulation and gene transfer in domestic animals. The objective is not to exhaustedly describe the research done in this field but to present the promising methods recently developed or evaluated in our lab. Conclusion: Significant advancements have been made in the course of the recent years in micromanipulation and transgenesis techniques. In our lab we have been evaluating ICSI and Nuclear transfer mainly to produce transgenic embryos. We used also transgensis to apply or developed new micromanipulation technique in domestic animals linke sperm and oocyte cloning.
Assuntos
Animais , Transgenes , Injeções de Esperma Intracitoplásmicas/veterinária , Micromanipulação/tendências , Micromanipulação/veterinária , Técnicas de Transferência Nuclear/veterináriaResumo
A produção de animais transgênicos possui aplicações que envolvem desde a pesquisa básica à produção agropecuária. O recente progresso na clonagem animal por transferência nuclear (TN) possibilitou a produção de animais transgênicos utilizando linhagens de células doadoras de núcleo previamente modificadas geneticamente. A possibilidade de manipulação genética, estudo da expressão gênica e adequada seleção da célula doadora de núcleo na TN não somente pode garantir a presença da construção gênica em toda a prole, como também pode evitar a produção de animais portadores de modificações indesejáveis resultantes da inserção do inserto em regiões codificantes do genoma, em decorrência da inserção aleatória das técnicas de transferência gênica mais comuns. Este trabalho teve como objetivo geral produzir animais transgênicos a partir de transferência nuclear utilizando como células doadoras de núcleo fibroblastos modificados geneticamente por transdução lentiviral. Objetivos específicos foram a produção e caracterização de linhagens de fibroblasto fetal portadores do gene da Proteína Fluorescente Verde (eGFP) quanto à seleção da expressão do transgene, passagem celular e posição da inserção do transgene e sua utilização na técnica de transferência de núcleos para a análise da competência de desenvolvimento a blastocisto e estabelecimento de gestações. Para tal, fibroblastos fetais bovinos foram transduzidos pelo sistema lentiviral. Células expressando o gene da eGFP foram selecionadas por citometria de fluxo e utilizadas como doadoras de núcleo na TN. Foram analisados o efeito do período de cultivo dos fibroblastos, assim como o efeito da reclonagem na competência de desenvolvimento a blastocisto e estabelecimento de gestações. O cultivo celular submetido à reclonagem foi analisado quanto à posição de inserção do transgene, sendo constatado nos fetos produzidos neste experimento a presença de uma inserção única em região não transcrita do cromossomo 14. Não houve efeito neste experimento do tempo de cultivo na competência de desenvolvimento a blastocisto, mas houve efeito benéfico da reclonagem celular. Além disso, foram obtidos 4 fetos, sendo 3 transgênicos, dos embriões transferidos provenientes das TNs que utilizaram células transgênicas de inserção aleatória em baixas e altas passagens e 6 fetos dos 37 embriões transferidos provenientes das TN que utilizaram células reclonadas, sendo todos transgênicos. Conclui-se que a produção de células transgênicas mediante mecanismo de transdução lentiviral, pode resultar, após TNCS, em embriões geneticamente idênticos à células doadora capazes de sustentar o desenvolvimento in vitro a blastocisto e o estabelecimento de gestações. Finalmente, são discutidos fatores ligados ao processo de seleção e reclonagem que aumentam a eficiência da produção de gestações por TNCS
Genetically modified animals have numerous applications ranging from basic research to agriculture production. Recent progress in animal cloning by nuclear transfer (NT) has made possible the production of transgenic animals using previously genetically modified cell lineages. The possibility of genetic manipulation, gene expression studies and adequate selection of the nuclei donor cell for NT not only can guarantee the presence of the gene construction in the offspring, but also can avoid the production of animals that carries undesirable characteristics, often as a result of the random insertion of transgenes in transcripted areas of the genome. General objective of this study was to produce transgenic animals by nuclear transfer using lentivirus-genetically modified nuclei donor fibroblasts. Specifically, objectives were the production and characterization of fetal fibroblasts lineages expressing eGFP (enhanced Green Fluorescent Protein, eGFP) gene in different cell passages regarding transgene insertion position and its use for the nuclear transfer procedure. The potential of blastocyst development and pregnancy establishment were analyzed in embryos reconstructed with late and early passages and with clonal or random insertion of transgenes (recloning). For that, bovine fetal fibroblasts were transduced with lentiviruses. eGFP expressing cells were selected by flow citometry sorting and used as nuclei donor cells for NT. Transgene integration site of the cell culture submitted to recloning was analised. It was observed that an unique insertion in a non-transcribed área of chromossome 14 was present in the fetuses recovered in this recloning experiment. No effect of culture time on development of blastocysts was observed, however, there was a beneficial effect of the cell recloning Besides, 4 fetuses (3 of them were transgenic) were obtained when 64 embryos reconstructed with random transgene position cells in late and early passages were transferred and 6 fetuses were obtained when 37 embryos reconstructed with recloned cells were transferred (all of them were transgenic). In conclusion, lentivirus transduction was able to produce transgenic cells with a stable expression of the transgene. These cells, when used for SCNT, can be reprogrammed and genetically identical embryos able to sustain in vitro culture, pregnancy establishment and recognition. Finally, recloning and cell selection procedures are discussed as a possible approach to increase pregnancy efficiency after TNCS