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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Lentiviral mediated gene delivery to human antigen presenting cells

Neil, Stuart John Douglas January 2001 (has links)
No description available.
2

Etude de l'étape d'entrée des vecteurs lentiviraux dérivés du VIH-1 dans les cellules hématopoïétiques humaines / Study of the entry step of HIV-1-derived lentiviral vectors into human hematopoietic cells

Ingrao, Dina 29 November 2013 (has links)
Les vecteurs lentiviraux (LV) sont des outils efficaces de transfert de gène, largement utilisés en thérapie génique, en particulier pour la transduction ex vivo de cellules souches et progénitrices hématopoïétiques (CSPH). Afin d’étudier simultanément la fusion et la transduction dans les CSPH avec les LV, nous avons adapté une méthode basée sur latechnologie du transfert d’énergie entre deux molécules fluorescentes (FRET). Pour mettre en place cette technique, des LV capables d’incorporer spécifiquement une enzyme, la bétalactamase (BLAM-LV) et de coder une forme tronquée du récepteur au facteur de croissance nerveuse (DELTA-NGFR), sont produits. Nos résultats montrent que les LV sont soumis à une restriction post-entrée forte dans les cellules hématopoïétiques, que ce soit dans des lymphocytes T immortalisés ou bien des CSH CD34+. Nous montrons également que cette inhibition post-entrée peut être partiellement saturée après une forte augmentation de la multiplicité d’infection ou en présence d’additifs de culture, comme la Vectofusin-1® ou laRetronectin®. De plus, nous avons montré lors de la transduction de CSPH avec des vecteurs BLAM-LV que la Vectofusin-1® agit sur l’étape d’entrée en augmentant l’adhésion et la fusion entre les membranes virale et cellulaire. Cette technique représente donc un nouvel outil sensible et efficace pour étudier de façon concomitante l’étape de fusion et le niveau de transduction dans les cellules cibles. A terme, ce travail permettra une meilleure compréhension de la biologie des LV mais pourra également conduire à l’élaboration de protocoles de transduction lentivirale plus efficaces. / Lentiviral vectors (LV) are used for various gene transfer applications, notably for hematopoietic gene therapy, but methods are lacking to precisely evaluate parameters that control the efficiency of transduction in relation with the entry of vectors into target cells. We adapted a fluorescence resonance energy transfer (FRET)-based HIV-1 fusion assay to measure the entry of non-replicative recombinant LV in various cell types, including primary human hematopoietic stem and progenitor cells, and to quantify the level of transduction of he same initially-infected cells. The assay utilizes recombinant LV containing betalactamase (BLAM)-Vpr chimeric proteins (BLAM-LV) and encoding a truncated form of thelow affinity nerve growth factor receptor (DELTA-NGFR). This LV-based fusion/transduction assay is a dynamic and versatile tool, revealing for instance the extent of lentiviral post-entry restrictions occuring in cells of hematopoietic origin. The assay also shows that transduction enhancers like Vectofusin®-1 or Retronectin® can partially relieve this post-entry block but their effects differ in the way to promote LV entry. Furthermore, our results show that Vectofusin®-1 acts at the entry step by promoting the adhesion and the fusion between lentiviral and cellular membranes. In conclusion, one such assay should be useful to study hematopoietic post-entry restrictions directed against LV and should allow improvements in various LV-based gene therapy protocols.
3

Knockdown of HCN1 channels in the dorsal hippocampal CA1 region

Kim, Chung Sub 15 January 2013 (has links)
The hippocampus is an integral brain region for affective disorders. HCN1 protein shows age-dependent increase in expression resulting in an increase in I[subscript h] in the dorsal hippocampal CA1 region. TRIP8b knockout mice lacking functional HCN channels as well as both HCN1 and HCN2 knockout mice have been shown to display antidepressant-like behaviors. The mechanisms or brain regions involved in these alterations in behavior, however, are not clear. We developed a lentiviral shRNA system to examine whether knockdown of HCN1 protein, and therefore h-channels, in the dorsal hippocampal CA1 region is sufficient to produce antidepressant-like effects. We found that silencing of HCN1 gene resulted in physiological changes consistent with a reduction of I[subscript h] and increased cellular excitability of CA1 pyramidal neurons. Rats infused with lentiviral-shRNA-HCN1 in the dorsal hippocampal CA1 region displayed antidepressant- and anxiolytic-like behaviors. Using voltage-sensitive dye imaging, we found that knockdown of HCN1 in the dorsal hippocampal CA1 region led to enhancement of hippocampal activity in large regions of the dorsal hippocampus. Our results demonstrate that changed hippocampal network activity by local manipulation of HCN1 channels in dorsal hippocampus led to anxiolytic- and antidepressant-like behaviors and suggest that HCN1 channels could be a potential target for treatment of anxiety and depression. / text
4

Geração de células de pluripotência induzida (iPS) humanas utilizando vetores lentivirais e determinação do perfil de integração lentiviral / Generation of human induced pluripotent stem (iPS) cell using lentiviral vector and determination of the lentiviral integration profile

Reis, Luiza Cunha Junqueira 28 November 2012 (has links)
As células iPS surgiram com a promessa de contornar as limitações das células-tronco embrionárias, como questões éticas, segurança, compatibilidade e disponibilidade. Essas células podem ser obtidas a partir de células somáticas de indivíduos normais ou de pacientes com doenças genéticas, fazendo destas uma importante ferramenta para o screening de drogas, modelos de doenças e testes toxicológicos. Grandes avanços ocorreram na reprogramação de células diferenciadas pela expressão forçada de fatores de transcrição (FT), principalmente, através de vetores lentivirais (VL), que proporcionam uma reprogramação eficiente. Entretanto, a inserção lentiviral no genoma humano e sua influência na reprogramação é pouco conhecida. Neste trabalho, avaliamos o perfil de inserção dos VL utilizados na geração de iPS. As iPS foram geradas e caracterizadas por nosso grupo a partir de fibroblastos humanos transduzidos com VL contendo 3 FT [SOX2, TCL-1A e C-MYC (célula TSM)], e de células mesenquimais derivadas de tecido adiposo com um vetor lentiviral policistrônico contendo 4 FT [OCT4, SOX2, KLF4 e C-MYC (iPS 4FT)]. Cinco colônias isoladas de cada iPS foram mapeadas e analisadas quanto aos sítios de inserção pela técnica de LM-PCR. O DNA genômico digerido foi amplificado com um primer específico para o LTR viral e outro para um linker sintético. Os produtos foram clonados, sequenciados, e analisados em bancos de dados para identificar similaridades com o genoma humano, entre outras análises. Na célula TSM, 176 sequências, obtidas com a técnica de LM-PCR, apresentaram identidade com o genoma humano, sendo que cerca de 50% ocorreram em regiões gênicas com 94% destas em introns. Já nas iPS 4FT, 251 sequências apresentaram identidade, com cerca de 45% atingindo genes, 92% destas em introns. As inserções distribuíram-se por todos os cromossomos, com preferência pelos cromossomos 16, 17 e 20 para a TSM e pelos cromossomos 11, 15 e 17 para a iPS 4FT. Analisamos a distância da inserção ao sítio de início de transcrição (TSS), e inserções próximas a ilhas CpG, que em geral correspondem a regiões regulatórias. A maior proporção de inserção ocorreu a partir de ±30Kb de distância desses sítios. Os sítios frágeis e as regiões repetitivas do genoma foram atingidas, mas com uma frequência baixa. Os resultados mostraram uma preferência de inserção lentiviral por regiões gênicas nas iPS, indicando a possível participação de proteínas como LEDGF/p75 na integração nas células estudadas. Este trabalho mostrou que o local da integração pode contribuir para a reprogramação e, apesar de possíveis efeitos negativos das integrações, estas as células iPS ainda são uma ferramenta importante para estudos in vitro. E identificar fatores que influenciem a seleção do sítio de inserção é importante para determinar regiões cromossômicas \"seguras\" para a integração, aumentando a segurança no uso clínico. / The induced pluripotent stem (iPS) cells came with the promise of circumvent some of the limitations in the use of embryonic stem cells, like ethical issues, biological safety, immune compatibility and availability. This cells can be generated from somatic cells of normal individuals or from patients with some genetic disease, making then an important tool for drug screening, construction of disease models and toxicological trials. Great advances have happened in reprogramming differentiated cells through the forced exogenous expression of transcription factors (TF), mostly by lentiviral vectors (LV), which provide an efficient reprogramming. However, the lentiviral insertion in the human genome and its influence in reprogramming is not well known. In this work, we evaluate the insertion profile of LV used to generate human iPS cells. The iPS cells were generated, by our group, from human fibroblasts transduced by LV containing 3 TF [SOX2, TCL-1A and C-MYC (TSM reprogrammed cell)], and from mesenchymal cells derived from human adipose tissue transduced by a polycistronic LV containing 4 TF [OCT4, SOX2, KLF4 and C-MYC (iPS 4TF)]. Five isolated colonies of each iPS cell were mapped and analyzed for the insertion sites through LM-PCR technique. The digested genomic DNA was amplified with a primer for the viral LTR e another for a synthetic linker. The products were cloned, sequenced and analyzed in database to identify similarities with the human genome, among other analyzes. In TSM cell, 176 sequences, derived from the LM-PCR technique, presented identity with the human genome, and about 50% of those occurred in genic regions with 94% in introns. In iPS 4TF, 251 sequences showed identity, with about 45% reaching genes, 92% of these in introns. The insertions were distributed on all chromosomes, with preference for the 16, 17 and 20 for the TSM cell, and for the 11, 15 and 17 for the iPS 4TF. We analyzed the distance of the insertion from de transcription start site, and insertions near CpG islands, which, overall, correspond to regulatory regions. The highest proportion of insertion occurred starting ±30Kb distance from these sites. The fragile sites and the repetitive regions of the genome were also reached, but with low frequency. The results showed a preference of lentiviral insertion for genic regions in iPS, indicating the potential participation of proteins like LEDGF/p75 in integration in the cells of this work. This work shows that the integration site may contribute to the reprogramming, and, despite possible negative effects of integration, these iPS cells are still an important tool for in vitro studies. Identify factors that influence the selection of insertion site is important for determination of \"safe\" chromosomal regions for the integration, increasing the safe in clinical use.
5

Geração de células de pluripotência induzida (iPS) humanas utilizando vetores lentivirais e determinação do perfil de integração lentiviral / Generation of human induced pluripotent stem (iPS) cell using lentiviral vector and determination of the lentiviral integration profile

Luiza Cunha Junqueira Reis 28 November 2012 (has links)
As células iPS surgiram com a promessa de contornar as limitações das células-tronco embrionárias, como questões éticas, segurança, compatibilidade e disponibilidade. Essas células podem ser obtidas a partir de células somáticas de indivíduos normais ou de pacientes com doenças genéticas, fazendo destas uma importante ferramenta para o screening de drogas, modelos de doenças e testes toxicológicos. Grandes avanços ocorreram na reprogramação de células diferenciadas pela expressão forçada de fatores de transcrição (FT), principalmente, através de vetores lentivirais (VL), que proporcionam uma reprogramação eficiente. Entretanto, a inserção lentiviral no genoma humano e sua influência na reprogramação é pouco conhecida. Neste trabalho, avaliamos o perfil de inserção dos VL utilizados na geração de iPS. As iPS foram geradas e caracterizadas por nosso grupo a partir de fibroblastos humanos transduzidos com VL contendo 3 FT [SOX2, TCL-1A e C-MYC (célula TSM)], e de células mesenquimais derivadas de tecido adiposo com um vetor lentiviral policistrônico contendo 4 FT [OCT4, SOX2, KLF4 e C-MYC (iPS 4FT)]. Cinco colônias isoladas de cada iPS foram mapeadas e analisadas quanto aos sítios de inserção pela técnica de LM-PCR. O DNA genômico digerido foi amplificado com um primer específico para o LTR viral e outro para um linker sintético. Os produtos foram clonados, sequenciados, e analisados em bancos de dados para identificar similaridades com o genoma humano, entre outras análises. Na célula TSM, 176 sequências, obtidas com a técnica de LM-PCR, apresentaram identidade com o genoma humano, sendo que cerca de 50% ocorreram em regiões gênicas com 94% destas em introns. Já nas iPS 4FT, 251 sequências apresentaram identidade, com cerca de 45% atingindo genes, 92% destas em introns. As inserções distribuíram-se por todos os cromossomos, com preferência pelos cromossomos 16, 17 e 20 para a TSM e pelos cromossomos 11, 15 e 17 para a iPS 4FT. Analisamos a distância da inserção ao sítio de início de transcrição (TSS), e inserções próximas a ilhas CpG, que em geral correspondem a regiões regulatórias. A maior proporção de inserção ocorreu a partir de ±30Kb de distância desses sítios. Os sítios frágeis e as regiões repetitivas do genoma foram atingidas, mas com uma frequência baixa. Os resultados mostraram uma preferência de inserção lentiviral por regiões gênicas nas iPS, indicando a possível participação de proteínas como LEDGF/p75 na integração nas células estudadas. Este trabalho mostrou que o local da integração pode contribuir para a reprogramação e, apesar de possíveis efeitos negativos das integrações, estas as células iPS ainda são uma ferramenta importante para estudos in vitro. E identificar fatores que influenciem a seleção do sítio de inserção é importante para determinar regiões cromossômicas \"seguras\" para a integração, aumentando a segurança no uso clínico. / The induced pluripotent stem (iPS) cells came with the promise of circumvent some of the limitations in the use of embryonic stem cells, like ethical issues, biological safety, immune compatibility and availability. This cells can be generated from somatic cells of normal individuals or from patients with some genetic disease, making then an important tool for drug screening, construction of disease models and toxicological trials. Great advances have happened in reprogramming differentiated cells through the forced exogenous expression of transcription factors (TF), mostly by lentiviral vectors (LV), which provide an efficient reprogramming. However, the lentiviral insertion in the human genome and its influence in reprogramming is not well known. In this work, we evaluate the insertion profile of LV used to generate human iPS cells. The iPS cells were generated, by our group, from human fibroblasts transduced by LV containing 3 TF [SOX2, TCL-1A and C-MYC (TSM reprogrammed cell)], and from mesenchymal cells derived from human adipose tissue transduced by a polycistronic LV containing 4 TF [OCT4, SOX2, KLF4 and C-MYC (iPS 4TF)]. Five isolated colonies of each iPS cell were mapped and analyzed for the insertion sites through LM-PCR technique. The digested genomic DNA was amplified with a primer for the viral LTR e another for a synthetic linker. The products were cloned, sequenced and analyzed in database to identify similarities with the human genome, among other analyzes. In TSM cell, 176 sequences, derived from the LM-PCR technique, presented identity with the human genome, and about 50% of those occurred in genic regions with 94% in introns. In iPS 4TF, 251 sequences showed identity, with about 45% reaching genes, 92% of these in introns. The insertions were distributed on all chromosomes, with preference for the 16, 17 and 20 for the TSM cell, and for the 11, 15 and 17 for the iPS 4TF. We analyzed the distance of the insertion from de transcription start site, and insertions near CpG islands, which, overall, correspond to regulatory regions. The highest proportion of insertion occurred starting ±30Kb distance from these sites. The fragile sites and the repetitive regions of the genome were also reached, but with low frequency. The results showed a preference of lentiviral insertion for genic regions in iPS, indicating the potential participation of proteins like LEDGF/p75 in integration in the cells of this work. This work shows that the integration site may contribute to the reprogramming, and, despite possible negative effects of integration, these iPS cells are still an important tool for in vitro studies. Identify factors that influence the selection of insertion site is important for determination of \"safe\" chromosomal regions for the integration, increasing the safe in clinical use.
6

Identification of ebola glycoprotein mutants that exhibit increased transduction efficiency

Sandersfeld, Lindsay Marie 01 December 2009 (has links)
Gene delivery via lentiviruses can yield long term expression of transgenes. Specificity of host cell targeting by viral vectors occurs primarily through viral glycoprotein (GP)/cellular receptor interactions. Ebola virus (EBOV) GP has broad tropism for a variety of cell types making this viral GP a potentially useful reagent for delivery of gene therapy. However, titers of EBOV GP pseudotyped lentiviruses are insufficient for practical use in clinical applications. Enhancement of EBOV-GP pseudotyped titers by as little as half a log might yield clinically applicable titers. In an alanine scanning study, we identified 19 residues in EBOV-GP1 that increased transduction efficiency two to three fold. When mapped onto the crystal structure of EBOV GP, these residues were primarily located at the interface of GP1/GP2 suggesting these residue substitutions may confer conformational changes in the protein structure thereby enhancing transduction efficiency. To determine if combinations of these alanine substitutions might further enhance transduction, we have introduced the changes into EBOV GP in a stepwise manner. To date, introduction of some combinations of alanine substitutions resulted in as much as an eight-fold increase in transduction over WT GP, this being our super mutant combination, whereas other combinations eliminated transduction. Identification of 5 additional mutations via 3D modeling of the glycoprotein uncovered an additional mutation in GP2, located at the GP1/GP2 interface, which also enhances EBOV GP transduction. Transduction of cell lines important for gene therapy including hepatocytes and porcine airway cells confirmed an enhancement in transduction as well. Other cell populations, specifically fibroblasts and renal cells, were also transduced but enhanced transduction was not observed indicating this phenomenon may be cell type specific. The in vivo studies were inconclusive because no expression was detected from any of the EBOV GP pseudovirions. Even expression of the positive control, GP64 particles, waned after 3 weeks post inoculation indicating insufficient quantities or poor quality pseudovirions were used. These EBOV GPs should prove useful for future gene therapy studies by providing an alternate glycoprotein that is as effective as GP64 at producing high titer lentiviral vectors.
7

Lentivirus-mediated gene expression in corneal endothelium

Parker, Douglas George Anthony, park0290@flinders.edu.au January 2008 (has links)
Modulation of corneal transplant rejection using gene therapy shows promise in experimental models but the most appropriate vector for gene transfer is yet to be determined. The overarching aim of the thesis was to evaluate the potential of a lentiviral vector for use in human corneal transplantation. Specific aims were: (i) to assess the ability of an HIV-1-based lentiviral vector to mediate expression of the enhanced yellow fluorescent protein (eYFP), and a model secreted protein interleukin-10 (IL10), in ovine and human corneal endothelium; and (ii) to examine the influence of lentivirus-mediated IL10 expression on the survival of ovine corneal allografts. Four lentiviral vectors expressing eYFP under the control of different promoters, were tested: the simian virus type-40 (SV40) early promoter, the phosphoglycerate kinase (PGK) promoter, the elongation factor-1alpha (EF) promoter, and the cytomegalovirus (CMV) promoter. Two lentiviral vectors expressing IL10 were tested: one containing the SV40 promoter and another containing a steroid-inducible promoter (GRE5). Lentivirus-mediated expression in transduced ovine and human corneal endothelium was assessed by fluorescence microscopy, real-time quantitative RT-PCR and ELISA, following alterations of transduction period duration (2–24 hr) and vector dose, as well as in the presence or absence of polybrene or dexamethasone (GRE5 vector). It was also compared to expression mediated by adenoviral vectors. Orthotopic transplantation of ex vivo transduced donor corneas was performed in outbred sheep. Allografts were reviewed daily for vascularisation and signs of immunological rejection. Lentivirus-mediated eYFP expression was delayed in ovine corneal endothelium compared to human. However, in both species the final transduction rate was greater than 80% and expression was stable for at least 14 d in vitro. Lentivirus-mediated expression in ovine and human corneal endothelium was higher with the viral promoters in comparison to the mammalian promoters. A 24 h transduction of ovine corneal endothelium with the lentiviral vector encoding IL10 resulted in expression levels which were increasing after 15 d of organ culture but logarithmically lower than those achieved by adenovirus. Shortening the lentiviral transduction period to 2 h led to a reduction in expression, but the addition of polybrene (40 micrograms / ml) to the transduction mixture restored expression to levels comparable to those attained after a 24 h transduction period. Lentivirus-mediated IL10 expression was higher and more rapid in human corneal endothelium compared to ovine corneas. Dexamethasone-responsive transgene expression was observed in both ovine and human corneal endothelium using the lentiviral vector containing the GRE5 promoter. Lentivirus-mediated expression in ovine corneal endothelium was stable for 28 d in vivo. A modest prolongation of ovine corneal allograft survival (median of 7 d) was achieved by transduction of donor corneas for 2–3 h with the lentivirus expressing IL10. Attempts to increase the expression of IL10 by the addition of polybrene (40 micrograms / ml) to the transduction mixture, resulted in a toxic effect on corneal allografts which abrogated the beneficial effect of IL10. The lentiviral vector shows potential for the stable expression of therapeutic transgenes in human corneal transplantation. However, the mechanisms underlying the species-specific differences in HIV-1-mediated transgene expression will need to be elucidated and overcome if the ovine preclinical model is to provide justification for a clinical trial.
8

Étude de l'implication potentielle des marqueurs du striatum dans la maladie de Huntington / Study of potential involvement of striatal markers in Huntington's disease

Galvan, Laurie 15 June 2011 (has links)
La maladie de Huntington (MH) est une maladie neurodégénérative héréditaire, incurable.Elle est due à une mutation dans le gène HD codant l'huntingtine (htt). Cette mutation setraduit dans la protéine par une augmentation de l'expansion polyglutamine (polyGln) qui larend toxique. Bien que la htt soit ubiquitaire dans le système nerveux central, ladégénérescence touche préférentiellement le striatum. Un patron d'expression de gènesspécifiques du striatum pourrait expliquer cette vulnérabilité préférentielle. Nous avons étudiéles effets "modificateurs" de 5 gènes préférentiellement exprimés dans le striatum vis-à-visde la toxicité de la htt mutée par une approche lentivirale chez la souris. Nous avonscaractérisé les effets de ces marqueurs striataux sur la toxicité induite par la htt mutée pardifférentes approches histologiques. Les "modificateurs" de la MH ont été étudiés plus endétail. Nous avons examiné leur localisation et les mécanismes sous-jacents à leurs effetsneuroprotecteurs. Outre une meilleure compréhension du striatum, cette étude a permis ladécouverte de candidat neuroprotecteur qui pourrait permettre de développer de nouvellesthérapies. / Huntington's disease (HD) is an incurable inherited neurodegenerative disease. HD iscaused by a mutation in the HD gene coding huntingtin (htt). This mutation leads to anexpanded polyglutamine tract (polyQ) in the protein which is toxic to neurons. Although thehtt is ubiquitously expressed in the central nervous system, the first area which degeneratesis the striatum. A pattern of genes selectively expressed into the striatum may confer itsvulnerability to mutated htt. We have studied the modifying effects of five newly identifiedstriatal markers against the toxicity induced by mutated htt using lentiviral strategy in miceand histological approaches. For one of these markers, Double Cortin Kinase Like 3(DCLK3), we have further determined their cellular localization and the potential mechanismsunderlying their neuroprotector effects. The present work led to a better understanding of thefunction of the newly identified markers in the striatum and their potential roles in thepreferential vulnerability of the striatum in HD.
9

Geração de animais transgênicos por inoculação de vetor viral em meio de cultura de óvulos

Ravache, Thaís Terpins, Simões, Renata, Goissis, Marcelo Demarchi January 2014 (has links)
Orientador: Prof. Dr. Marcelo Augusto Christoffolete / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biotecnociência, 2014. / Desde o século XV, animais fazem parte da rotina na área da pesquisa, principalmente para estudos de doenças, e hoje em dia o modelo animal mais utilizado para estes estudos é o camundongo, tendo uma participação em mais de 90% das pesquisas em todo o mundo, sendo considerado como uma primeira via para definir funções de genes em mamíferos. Os camundongos são considerados os principais modelos nas técnicas de transgenia animal, porém estas técnicas ainda estão em desenvolvimento, uma vez que as metodologias hoje utilizadas para a geração de animais transgênicos ainda se encontram com uma taxa de sucesso considerada baixa e são dispendiosas, necessitando de muitas etapas. Uma das dificuldades é o contato com a membrana do óvulo devido a zona pelúcida, que é considerada uma barreira física. Vetores virais estão em evidência nas técnicas de transgenia animal, sendo o lentivírus o mais utilizado. Portanto, o objetivo deste projeto é estabelecer um protocolo para a integração de DNA exógeno em óvulos por infecção lentiviral, anteriormente a fertilização in vitro juntamente com a técnica de dissecção parcial da zona pelúcida. Como vetor foi utilizado um lentivírus com GFP em sua construção. Para ocorrer a fertilização in vitro, foram feitas coletas de óvulos em camundongos fêmeas da linhagem C57BL/6, tratadas com injeções hormonais, e coletas de espermatozoides em machos desta mesma linhagem. Os óvulos obtidos foram divididos em grupos controle e com dissecção parcial da zona pelúcida, e estes foram subdivididos em grupos com e sem infecção lentiviral. Entre os grupos houve variação de 20% a 56,25% de embriões em estágio de duas células, e em alguns grupos foi possível alcançar o estágio de blastocisto eclodido. Porém não foi possível visualizar a emissão de fluorescência para confirmar a infecção lentiviral. Em conclusão as metodologias utilizadas tanto para a fertilização in vitro como para a dissecção parcial da zona pelúcida foram de sucesso. Porém a integração do DNA exógeno mostrou resultados não conclusivos, necessitando de estudos futuros. / Since the XV century, animals are used routinely in research, mainly for diseases studies, and nowadays the most used animal model is the mouse, which one has more than 90% of participation in researches around the world and it is considered the first track to define gene function in mammals. Mouse is the main model in transgenic techniques, however the methods available to generate transgenic animals still have a considerable low rate, and also it is expensive, requiring many degrees. An ordinary issue is the contact with the membrane of oocyte due zona pellucida that is considered a physical barrier. In transgenic animals technique, it is in evidence the utilization of viral vectors, and the most used are the lentiviruses. Therefore, the objective of this project is to establish a protocol for the integration of exogenous DNA by lentiviral infection into oocytes, before the in vitro fertilization, using the technique of partial dissection of the zona pellucida. It was used as a vector a lentivirus with GFP in your construction. For in vitro fertilization, were collected oocytes from C57Bl/6 mice, treated with hormones, and sperm from males of the same strain. The obtained oocytes were divided in control group and partial dissection of the zona pellucida group, and then subdivided in groups with and without lentiviral infection. Between the groups, was achieved 20% to 56,25% of two cells stage embryo, and hatched blastocysts stage were obtained at some groups. Therefore it was not possible to visualize florescence emission to confirm the lentiviral infection. In conclusion we have a practicable protocol for in vitro fertilization and partial dissection of the zona pellucida, reaching blastocysts stages in two groups. However the integration of exogenous DNA results were inconclusive, requiring further studies.
10

Targeting and repair of adult testicular somatic cells through viral gene therapy

Darbey, Annalucia Leigh January 2018 (has links)
Androgens are essential for the maintenance of male health and wellbeing. A disturbance in androgen signalling has been associated with a number of clinically relevant disorders such as cardiovascular disease, diabetes and metabolic disorders as well as infertility. Primarily produced in the testis in males, the actions of androgens are mediated through binding to androgen receptor (AR), a member of the nuclear receptor superfamily of ligand-activated transcription factors. The somatic cells of the testis are known to have a number of key roles in both testis function and development and the Sertoli, Leydig and Peritubular Myoid cells are known to express AR in adulthood. It is through AR that some testicular functions are mediated; for example, the Sertoli cells support of complete spermatogenesis with Sertoli cell androgen receptor knockout (SCARKO) testis demonstrating a halt of spermatogenesis before meiosis. However, how androgen signalling is impacting testicular function through each of the somatic cell types is not yet fully understood. Currently, treatments for male reproductive disorders such as hypogonadism (low androgens) and infertility are limited to treatment of the symptoms; using androgen replacement therapy and in vitro fertilisation techniques. This has been, up until recently, a result of a lack of understanding of the causes of these conditions and a lack of resources able to treat them, with research suggesting that a genetic component may be responsible in a number of cases. However, due to the limited genetic investigation diagnosis of men with male reproductive disorders, the wider understanding of the genetics underpinning male hypogonadism and infertility is incomplete. Developments in technology for the investigation and editing of the genetic code are triggering a surge in the exploration of genetic disorders and, in parallel, into the fields of gene delivery vectors and editing technologies. These technologies will allow an expansion into the knowledge and understanding of genetic disorders whilst simultaneously affording the opportunity to exploit this understanding for the development of therapeutics. There have been a small handful of previous studies using technologies such as viral vectors to target the testicular somatic cells and deliver exogenous transgenes with the purpose of both gene editing and repair, all with varying degrees of success. Here, techniques to introduce and target the Leydig and Sertoli cells were investigated to determine the most appropriate methodology for gene delivery to and manipulation of the testis. Refinement of injections into the interstitial compartment were carried out before introducing lentiviral vectors and targeting of Leydig cells was validated and optimised. Lentiviral vectors are able to permanently integrate into the host cell. Surprisingly, analysis of testis post lentiviral injection determined that the lentiviral targeted Leydig cells began to undergo apoptosis one week post injection and were subsequently cleared from the testis after ten days. Contrastingly, this was not the case when adenoviral vectors were introduced into the interstitial compartment, with Leydig cells continuing to express the delivered reporter transgene and, importantly, not expressing markers of apoptosis, ten days post injection. This would suggest that using adenoviral vectors to target the Leydig cell population in the adult testis would be more appropriate than using lentiviral vectors. Previous studies have successfully used lentiviral vectors to target the Sertoli cells in the adult testis via the introduction of the particles through the efferent duct. However, this can result in damage to efferent duct, resulting in blockages and subsequently the seminiferous tubules. To circumvent this, introduction of the lentiviral particles through the rete compartment of the testis at a range of lower injection pressures was examined and injecting at a lower pressure through the rete testis was found to reduce the likelihood of introducing negative impacts on testicular histology when targeting the seminiferous tubules. Using these refined methods of introducing lentiviral vectors, targeted Sertoli cells stably expressed the delivered transgene for up to one year post injection. Using viral vector delivered transgenes for both the investigation of testicular genetic disorders and for the development of therapeutics has great potential. To explore this potential, we first generated a mouse model in which AR was ablated from both the Leydig and Sertoli cells using Cre/LoxP technology, termed the SC-LC-ARKO. Alongside providing a potential model to 'repair' with viral vectors, the SC-LC-ARKO model also provided an additional model for comparison with other models exhibiting ablation of AR from both single somatic cell types and double somatic cell types. This further enabled a characterisation of the roles of AR in adult testicular function, with results suggesting that loss of AR from more than one cell type results in an additive phenotype when compared to single cell knock outs. Despite providing further insight into the roles of AR in the testis, further analysis of the Cre line used to generate the SC-LC-ARKO model indicated that a small number of Leydig cells were expressing the Cre recombinase, resulting in only a small population of Leydig cells with ablated AR. Considering this, to explore the potential of rescuing Sertoli cell AR using lentiviral vectors, we then utilised an already well characterised Sertoli Cell AR knockout (SCARKO) model. Lentiviral vectors expressing mouse AR and monomeric GFP (moeGFP) downstream of a CMV promoter were generated and injected into the rete testis of WT and SCARKO adult (day 100) males at low pressure. The contralateral testis was injected with a lentiviral vector expressing moeGFP alone (also downstream of a CMV promoter) using the same technique. Analysis of testis sections revealed a reintroduction of AR to Sertoli cells in 100% of SCARKO testis injected with lentivirus expressing mouse AR. As a result of this re-expression of AR in Sertoli cells, 66% of the testis injected with lentivirus expressing mouse AR had evidence of morphologically mature elongated spermatids, indicative of ongoing spermatogenesis. These results suggest that a rescue of the infertility phenotype reported in previous studies of SCARKO testis. Also demonstrated is the reversal of the SCARKO testicular phenotype in tubules targeted by the mAR expressing lentiviral vector. This suggests that absence Sertoli cell AR throughout development does not have a permanent impact on the Sertoli cells capacity to support spermatogenesis in adulthood following rescue of SC AR expression in adulthood. In summary, the results of these studies have provided a refinement in the methodologies for targeting the Sertoli and Leydig cells of the adult testis with viral vectors as well as demonstrating successful rescue of a previously reported mouse model exhibiting infertility through reintroduction of a functional gene. Alongside this, comparisons of AR knockout models have afforded insight into maintenance of testis function through AR.

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