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Reverse genetics systems that are based on either viral transcripts or cDNA genome segments cloned in plasmids have recently been reported for some of the dsRNA viruses of the Reoviridae family, namely African horsesickness virus, bluetongue virus and orthoreovirus. For rotaviruses, three reverse genetics systems which only allow the manipulation of a single genome segment have been described. These rotavirus single genome segment reverse genetics systems are not true stand-alone systems because they require a helper virus and a recombinant virus selection step. A true selection-free, plasmid- only or transcript-based reverse genetics system for rotaviruses is lacking. This study sought to identify and characterise the factors that need to be understood and overcome for the development of a rotavirus reverse genetics system using mRNA derived from the in vitro transcription of a consensus nucleotide sequence as well as from double-layered particles. The consensus whole genome sequence of the prototype rotavirus DS-1 and SA11 strains was determined using sequenceindependent whole genome amplification and 454® pyrosequencing. For the rotavirus DS-1 strain, a novel isoleucine in a minor population variant was found at position 397 in a hydrophobic region of VP4. NSP1 contained seven additional amino acids MKSLVEA at the N-terminal end due to an insertion in the consensus nucleotide sequence of genome segment 5. The first 34 nucleotides at the 5'- terminus and last 30 nucleotides at the 3'-terminal end of genome segment 10 (NSP4) of the DS-1 strain were determined in this study. The consensus genome segment 11 (NSP5/6) sequence was 821 bp in length, 148 bp longer than previously reported. The 454® pyrosequence data for a rotavirus SA11 sample with no known passage history revealed a mixed infection with two SA11 strains. One of the strains was a reassortant which contained genome segment 8 (NSP2) from the bovine rotavirus O agent. The other ten consensus genome segments of the two strains could not be differentiated. Novel minor population variants of genome segments 4 (VP4), 9 (VP7) and 10 (NSP4) were identified. Molecular clock phylogenetic analyses of the rotavirus SA11 genomes showed that the two SA11 strains were closely related to the original SA11-H96 strain isolated in 1958. Plasmids containing inserts of the consensus cDNA of the rotavirus DS-1 strain were purchased and used to generate exact capped transcripts by in vitro transcription with a T7 polymerase. Wild-type transcripts of rotavirus SA11 were obtained from in vitro transcription using purified rotavirus SA11 double-layered particles. The purified rotavirus DS-1 and SA11 transcripts were transfected into BSR, COS-7 and MA104 cells. Work on MA104 cells was discontinued due their very low transfection efficacy. In BSR and COS-7 cells, rotavirus DS-1 and SA11 transcripts induced cell death. However, no viable rotavirus was recovered following attempts to infect MA104 cells with the BSR and COS-7 transfected cell lysates. The cell death was determined to be due to apoptotic cell death mechanisms. Immunostaining showed that the DS-1 genome segment 6 (VP6) and SA11 transcripts were translated in transfected BSR and COS-7 cells. Based on visual inspection, the translation seemed to be higher in the retinoic acid-inducible gene-I (RIG-I) deficient BSR cells than in COS-7 cells. This suggested that the transfection of rotavirus transcripts induced an innate immune response which could lead to the development of an antiviral state. Therefore, the innate immune response to rotavirus transcripts was investigated in HEK 293H cells using qRT-PCR and western blot analyses. Results of this investigation showed that RIG-I, but not MDA5 sensed rotavirus transcripts in transfected HEK 293H cells. Furthermore, rotavirus transcripts induced high levels of cellular mRNA encoding the cytokines IFN-1β, IFN-λ1, CXCL10 and TNF-α. Other cytokines namely, IFN-α, IL-10, IL-12 p40 and the kinase RIP1 were not significantly induced. Inhibiting the RNA-dependent protein kinase R (PKR) reduced the induction of cytokines IFN-1β, IFN-λ1, CXCL10 and TNF-α, but the expression levels were not abrogated. The importance of a consensus sequence and the insights gained in the current study regarding the role of the innate immune response after transfection of rotavirus transcripts into cells in culture, should aid the development of a true rotavirus reverse genetics system. / Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2013

Reverse genetics systems that are based on either viral transcripts or cDNA genome segments cloned in plasmids have recently been reported for some of the dsRNA viruses of the Reoviridae family, namely African horsesickness virus, bluetongue virus and orthoreovirus. For rotaviruses, three reverse genetics systems which only allow the manipulation of a single genome segment have been described. These rotavirus single genome segment reverse genetics systems are not true stand-alone systems because they require a helper virus and a recombinant virus selection step. A true selection-free, plasmid- only or transcript-based reverse genetics system for rotaviruses is lacking. This study sought to identify and characterise the factors that need to be understood and overcome for the development of a rotavirus reverse genetics system using mRNA derived from the in vitro transcription of a consensus nucleotide sequence as well as from double-layered particles. The consensus whole genome sequence of the prototype rotavirus DS-1 and SA11 strains was determined using sequenceindependent whole genome amplification and 454® pyrosequencing. For the rotavirus DS-1 strain, a novel isoleucine in a minor population variant was found at position 397 in a hydrophobic region of VP4. NSP1 contained seven additional amino acids MKSLVEA at the N-terminal end due to an insertion in the consensus nucleotide sequence of genome segment 5. The first 34 nucleotides at the 5'- terminus and last 30 nucleotides at the 3'-terminal end of genome segment 10 (NSP4) of the DS-1 strain were determined in this study. The consensus genome segment 11 (NSP5/6) sequence was 821 bp in length, 148 bp longer than previously reported. The 454® pyrosequence data for a rotavirus SA11 sample with no known passage history revealed a mixed infection with two SA11 strains. One of the strains was a reassortant which contained genome segment 8 (NSP2) from the bovine rotavirus O agent. The other ten consensus genome segments of the two strains could not be differentiated. Novel minor population variants of genome segments 4 (VP4), 9 (VP7) and 10 (NSP4) were identified. Molecular clock phylogenetic analyses of the rotavirus SA11 genomes showed that the two SA11 strains were closely related to the original SA11-H96 strain isolated in 1958. Plasmids containing inserts of the consensus cDNA of the rotavirus DS-1 strain were purchased and used to generate exact capped transcripts by in vitro transcription with a T7 polymerase. Wild-type transcripts of rotavirus SA11 were obtained from in vitro transcription using purified rotavirus SA11 double-layered particles. The purified rotavirus DS-1 and SA11 transcripts were transfected into BSR, COS-7 and MA104 cells. Work on MA104 cells was discontinued due their very low transfection efficacy. In BSR and COS-7 cells, rotavirus DS-1 and SA11 transcripts induced cell death. However, no viable rotavirus was recovered following attempts to infect MA104 cells with the BSR and COS-7 transfected cell lysates. The cell death was determined to be due to apoptotic cell death mechanisms. Immunostaining showed that the DS-1 genome segment 6 (VP6) and SA11 transcripts were translated in transfected BSR and COS-7 cells. Based on visual inspection, the translation seemed to be higher in the retinoic acid-inducible gene-I (RIG-I) deficient BSR cells than in COS-7 cells. This suggested that the transfection of rotavirus transcripts induced an innate immune response which could lead to the development of an antiviral state. Therefore, the innate immune response to rotavirus transcripts was investigated in HEK 293H cells using qRT-PCR and western blot analyses. Results of this investigation showed that RIG-I, but not MDA5 sensed rotavirus transcripts in transfected HEK 293H cells. Furthermore, rotavirus transcripts induced high levels of cellular mRNA encoding the cytokines IFN-1β, IFN-λ1, CXCL10 and TNF-α. Other cytokines namely, IFN-α, IL-10, IL-12 p40 and the kinase RIP1 were not significantly induced. Inhibiting the RNA-dependent protein kinase R (PKR) reduced the induction of cytokines IFN-1β, IFN-λ1, CXCL10 and TNF-α, but the expression levels were not abrogated. The importance of a consensus sequence and the insights gained in the current study regarding the role of the innate immune response after transfection of rotavirus transcripts into cells in culture, should aid the development of a true rotavirus reverse genetics system. / Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2013

Gene delivery for therapeutic purposes is quickly emerging as the best potential treatment option for inherited genetic diseases and cancer. Viral gene carriers have been the choice for this purpose due to their high efficiency, but harmful immunogenic and oncogenic host reactions have limited their in vivo use. Cationic polymers provide a safe alternative to viral carriers as they can be engineered to reduce immunogenic and toxic responses and serve therapeutic purposes in the body. Due to their strong positive charge, they are able to compact the negatively charged nucleotides to small nano-sized particles appropriate for cellular uptake. Additionally, they efficiently encapsulate the highly sensitive nucleotides, and protect them against degradation by the nucleases present at the physiological milieu. In this thesis work, I have used a novel approach for gene delivery by combining the critical properties of a cationic polymer (i.e., nucleotide condensing ability) with that of a fatty acid (i.e., lipid membrane compatibility). The resulting lipid modified polymer increased delivery of our gene of interest into target cells and resulted in increased siRNA delivery for cancer gene therapy. / Biomedical Sciences

Das Harnblasenkarzinom (BCa) stellt in Deutschland die vierthäufigste Tumorneuerkrankung und die zehnthäufigste krebsbedingte Todesursache bei Männern dar. Nichtmuskelinvasive BCa werden organerhaltend aus der Blasenwand entfernt und zur Rezidiv- und Progressionsprophylaxe mittels intravesikaler Chemo- oder Immuntherapien behandelt. Trotz dieser adjuvanten Therapien, die mit starken Nebenwirkungen verbunden sein können, ist nur eine bedingte Minimierung des Rezidivrisikos möglich. Besonders im fortgeschrittenen Stadium weisen Harnblasenkarzinome eine schlechte Prognose auf. Obwohl das BCa eine chemosensitive Erkrankung darstellt, wird das Ansprechen auf lokale oder systemische Chemotherapien häufig durch auftretende Resistenzmechanismen limitiert. Daher stehen sowohl die Verbesserung konventioneller Chemotherapien als auch die Suche nach neuartigen Behandlungsstrategien im Fokus der experimentellen BCa-Forschung. Die Apoptose, eine Form des programmierten Zelltodes, ist ein essenzieller, streng regulierter biologischer Prozess, welcher der Aufrechterhaltung der Gewebshomöostase und der gezielten, entzündungsfreien Eliminierung geschädigter Zellen dient. Fehlregulationen in den Apoptosesignalwegen stellen ein zentrales Ereignis in der Tumorgenese dar und tragen außerdem zur Entstehung von Chemo- und Radiotherapieresistenzen bei. Eine wichtige Rolle in der Apoptoseregulation spielen die Mitglieder der BCL2- und der Inhibitor of Apoptosis Protein (IAP)-Familien, deren wichtigste antiapoptotische Vertreter BCL2, BCL-XL, XIAP und Survivin häufig in Tumoren, einschließlich des BCa, überexprimiert sind. Unter Verwendung von small interfering RNAs (siRNAs), synthetischen Nukleinsäurekonstrukten zur selektiven Geninhibition, wurde im Rahmen der Arbeit in vitro und in vivo untersucht, ob die Hemmung der Apoptoseinhibitoren BCL2, BCL-XL, XIAP und Survivin – allein und in Kombination mit Chemotherapie – eine Therapieoption zur Behandlung des BCa darstellen könnte. Da zur Tumorentstehung und -progression eine Vielzahl von genetischen Veränderungen beitragen, erscheint der Angriff eines einzelnen Zielgens unzureichend für eine effektive Tumortherapie. Aufgrund dessen wurde untersucht, ob durch simultane Reduktion der ausgewählten Apoptoseinhibitoren in BCa-Zellen stärkere wachstumsinhibitorische Effekte erzielt werden können. In der vorliegenden Arbeit wurde gezeigt, dass insbesondere die siRNA-vermittelte Hemmung von BCL-XL und Survivin in den BCa-Zelllinien EJ28 und J82 antiproliferative Effekte hervorruft und diese Tumorzellen gegenüber einer nachgeschalteten Chemotherapie mit Mitomycin C oder Cisplatin sensitiviert. Hingegen bewirkte sowohl die transiente als auch die stabile RNAi-induzierte Hemmung von BCL2 und XIAP in den untersuchten BCa-Monolayerzellkulturen, möglicherweise infolge kontinuierlicher Versorgung der Tumorzellen mit Sauerstoff und Nährstoffen, keine Reduktion des Tumorwachstums. Eine gegenüber den Einzelbehandlungen deutliche Verstärkung der antitumoralen und insbesondere der chemosensitivierenden Effekte in den BCa-Zelllinien wurde durch simultane Hemmung von BCL-XL und Survivin erzielt. Beispielsweise stieg der Anteil apoptotischer Zellen von 64 % nach Survivin-siRNA+Cisplatin-Behandlung auf 94 % nach gleichzeitiger BCL-XL+Survivin-Inhibition in Kombination mit Cisplatin. Folglich stellt die simultane Inhibition von BCL-XL und Survivin in Kombination mit Chemotherapeutika eine äußert viel versprechende BCa-Therapieoption dar. Tierexperimentelle Studien belegen die wachstumsinhibitorische Wirkung der Survivin-Reduktion und der kombinierten BCL-XL-siRNA+Chemotherapie-Behandlung, so wurde das Tumorendvolumen im Vergleich zur Kontrollbehandlung um 43 % bzw. um 48 % reduziert.

Main purpose of this study was to increase protein expression in Chinese hamster ovary (CHO) cells by improving protein secretion of translated proteins. The goal was to find signal peptides from the screening of signal peptide libraries for improvement of protein secretion using a CHO-cell express selection system. Biopharmaceutical products, proteins such as monoclonal antibodies (mAbs), are most commonly produced using mammalian expression systems such as the expression in CHO cells. The posttranslational modifications of the proteins being expressed in CHO cells are similar to the expressional modifications in human cells, why the CHO cells are suitable for production of proteins used for human therapy. The expression of proteins in the cell is a complex mechanism, fundamentally depending on the DNA sequences in the cell nucleus. Secretion of translated proteins has been showed to be a bottleneck when improving expression. Secretion is initiated by the signal peptide, a n-terminal prolongation of the protein that is recognized by a signal recognition particle (SRP) when being translated by the ribosome. The sequence and structure of the signal peptide has been proved to affect secretion and altering the signal peptide could improve secretion even when changing signal peptide between different species. Designing variants of the signal peptides and analyzing protein expression might lead to improvements of the construct design and more protein produced from the cells, which would save time, money and material for the producer. To construct plasmids containing the gene of interest (GOI) and different signal peptides, several gene cloning methods were used. The plasmids were amplified using Escherichia coli (E. coli) transformation. The constructs were expressed by transfection into the CHO cell genome, and expression were analyzed using flow cytometry. When analyzing expression of a Fc-fusion protein with 5 different signal peptides, the signal peptide Azurocidin is the one showing highest expression levels in this study. In addition, IgG kapa and Albumin signal peptides did not show as high protein expression levels, even if they were better than the L1d and H5b signal peptides. Since signal peptides are exchangeable between proteins and species, it might be that Azurocidin is improving secretion and protein expression with other proteins than Fc-fusion proteins which would be an interesting aspect for further studies. When altering signal peptides with library sequences, the experimental challenges were crucial for the protein expression results and due to these issues, no library sequence could be seen to conquer others when it comes to protein expression levels. Transfection and cultivation procedures needs to be studied and improved before being able to draw conclusions about which signal peptide library sequences that might improve secretion and increase the protein expression.

Made available in DSpace on 2014-06-11T19:35:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-05Bitstream added on 2014-06-13T20:26:13Z : No. of bitstreams: 1 goncalves_fs_dr_jabo.pdf: 3109118 bytes, checksum: 5a4b81536e1b9bd9d62a0e42796b675b (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O fator de transcrição Oct-4 é bem conservado entre as espécies e é conhecido por ser expresso em embriões e células-tronco embrionárias (CTE), sendo um importante marcador da pluripotência. Recentemente, foi relatado que a combinação de Oct-4 com três outros fatores de transcrição Klf-4, c-Myc e Sox2 foram capazes de reprogramar células somáticas a um estado indiferenciado pluripotente, chamadas células-tronco pluripotentes induzidas (“células iPS”), as quais apresentam várias das mesmas propriedades das CTE incluindo a pluripotência, auto-renovação e proliferação. O objetivo desse estudo foi avaliar a funcionalidade do promotor Oct-4 de eqüino em CTE de murinos. Três vetores plasmidiais expressando GFP (“green fluorescent protein”) sob o controle do promotor Oct-4 de equinos, camundongo e quatro vetores lentivirais, também contendo o gene reporter GFP e os promotores Oct-4 de equinos, camundongo e humanos, pLZ2-ecOCT-EGFP (meq) (sequência equivalente de camundongos), pLZ2-ecOCT-EGFP (heq) (sequência equivalente de humanos), pLZ2-mOCT-EGFP e pLZ2-hOCT-EGFP, respectivamente, foram construídos. Todos os vetores também contêm um sítio de resistência à blasticidina que permite a seleção das células estáveis e das células transduzidas. Essas construções plasmidiais foram verificadas se funcionavam eficientemente, bem como o efeito do promotor Oct-4 em transfectar transientes e estáveis CTE. As construções com promotor Oct-4 de camundongo, humano e eqüino (sequência análoga à de camundongo) produziram somente 6% de células GFP positivas com intensidade de fluorescência (IF) >1000 pela análise em citômetro de fluxo, enquanto que o plasmídeo contendo o promotor Oct-4 de eqüino (sequência equivalente à de humanos) produziu menos células GFP positivas (>3%) com IF >1000, quando... / The pluripotency transcription factor Oct-4 is well conserved among species and is known to be expressed in embryos and embryonic stem (ES) cells; it is being an important pluripotency marker. It was recently demonstrated that the combination of Oct-4 with three other factors Klf-4, c-myc and Sox2 were able to reprogram somatic cells to a pluripotent and undifferentiated state. These cells known as induced pluripotent stem (iPS) cells share several properties with ES cells including self-renewal, proliferation and pluripotency. The aim of this study was to assess the functionality of the horse Oct-4 promoter in mouse ES cells. Three plasmids vectors expressing GFP (green fluorescent protein) under the control of the horse, mouse and four lentivirus vectors also containing reporter gene GFP and horse, mouse and human promoters, pLZ2-ecOCT-EGFP (mouse sequence equivalent), pLZ2-ecOCT-EGFP (human sequence equivalent), pLZ2-mOCT-EGFP and pLZ2-hOCT-EGFP, respectively, were built. All these vectors also contain a blasticidin resistance cassette to allow selection of transfected stable cells and transduced cells. Afterwards, to assess the functionality of the Oct-4 promoter all plasmids were tranfected the into transient and stable mouse ES cells. Constructs with mouse, human and horse (mouse analog sequence) Oct-4 promoter produced only 6% GFP positive cells with fluorescence intensity (FI)>1000 by 20 FACs assay, while plasmid horse (human analog sequence) Oct-4 promoter produced less GFP positive cells (>3%) with FI>1000, when compared with the positive control and among groups. However, GFP expression was not present in stable cells, whereas there were Blasticidin-resistant colonies-forming from 6 days post-transfection. To optimize the system in mouse ES cells, pLZ2-mOCT-EGFP and pLZ2-hOCT-EGFP lentivectors, were tested as controls. It was used HIV-1-derived... (Complete abstract click electronic access below)

Made available in DSpace on 2016-08-17T18:39:44Z (GMT). No. of bitstreams: 1 4615.pdf: 2688296 bytes, checksum: 18c8fbe6fb83004856a32c02827d70f5 (MD5) Previous issue date: 2011-11-22 / Universidade Federal de Sao Carlos / Rabies is a zoonotic viral disease caused by a virus of the genus Lyssavirus that affects several species of mammals. Rabies remains a global public health threat that kills more than 55,000 people per year mainly in developing countries, this disease once established do not have a specific treatment. The RV envelope is composed of a glycoprotein, known as a unique antigen capable of conferring immune response against the rabies, and therefore, is the focus of research for development an efficient and safe recombinant vaccine based on this viral antigen. Cell line stably transfected S2 Drosophila melanogaster have been used in the production of many heterologous proteins and has been studied for the production of the rabies virus glycoprotein (RVGP) in our laboratory. This approach involves the selection of high producing cell populations; procedure that requires considerable periods of time (months), increasing management and costs of production. In this sense, in recent decades, many systems focused on the expression of heterologous proteins by transient expression of genes, were analyzed because they allow obtaining significant quantities of recombinant protein in a short period of time (weeks). For the use of transient transfection technology can be found a variety of methods and available agents, such as electroporation, cationic lipids, cationic polymers and calcium phosphate precipitated. The choice and optimization of each of them depends mainly on the cell type and protein being expressed. Thus, the objective of this study was to evaluate the transient expression of the glycoprotein gene of rabies virus (RVGP) in insect cells of Drosophila melanogaster S2 lineage, evaluating the vehicles transfection: calcium phosphate, cationic lipid (Cellfectin) and cationic polymer (ExGen500 and JetPEI). In order to determine the most efficient transfection agent, experiments were performed in 6 well plate and bottle of 100 mL of culture, which analyzed the influence of cell density, the concentration of DNA and transfection reagent volume on the expression of RVGP assessed by ELISA and fluorescence microscopy. Yields ranging from 50-90 ng/107cel were obtained in different experiments on multiwell plate, suggesting strong effect of ratio DNA: transfection agent used. Comparison of transfection agents showed no significant differences. In transfections made in suspension culture was analyzed the effect of the plasmid (whether or not the signal of BiP cell secretion) on the expression RVGP. When we used the plasmid containing the signal BiP (pMTiRVGP) were obtained 160 ng/107cel of RVGP production, and 200 ng/mL of volumetric production without significant differences between the different transfection agents. However, significant differences were found when we used the plasmid not containing the signal BiP (pMTRVGP), with the RVGP production was 60 ng/107cells in cells transfected with Cellfectin, ExGen500 and calcium phosphate, except in cells transfected with JetPEI was reached a production of 120 ng/107cells. In preliminary experiment bottle type "spinner" with a working volume of 60 mL were achieved expressions of 140 ng/107cel of RVGP in cells transfected with JetPEI and calcium phosphate. This suggests that optimization of culture conditions and transfection are possible to increase recombinant protein expression in cultured on a large scale. / A raiva é uma enfermidade causada por um vírus do gênero Lyssavirus que afeta várias espécies de mamíferos. Esta doença apresenta um alto custo social e econômico principalmente em países em desenvolvimento. Na superfície do vírus da raiva está localizada a glicoproteína do vírus, reconhecida como antígeno capaz de conferir resposta imunológica contra a raiva, sendo, o foco de pesquisas no desenvolvimento de uma vacina recombinante. Células da linhagem Drosophila melanogaster S2 estavelmente transfectadas têm sido usadas na produção de muitas proteínas heterólogas e tem sido estudada para a produção da glicoproteína do vírus da raiva (RVGP) em nosso laboratório. A abordagem para a obtenção de linhagens recombinantes estáveis envolve a seleção de populações celulares altamente produtoras; sendo um processo que requer consideráveis períodos de tempo (meses), uma elevada manipulação e altos custos de produção. Neste sentido, nas últimas décadas, muitos sistemas focados na expressão de proteínas heterólogas através da expressão transiente de genes foram analisados, porque eles permitem a obtenção de quantidades consideráveis de proteína recombinante em um curto período de tempo (semanas). Para o uso da tecnologia de transfecção transiente pode ser encontrada uma variedade de métodos e agentes disponíveis, tais como eletroporação, lipídeos catiônicos, polímeros catiônicos e fosfato de cálcio. Assim, o objetivo deste trabalho foi avaliar a expressão transiente do gene da glicoproteína do vírus da raiva (RVGP) em células de inseto da linhagem Drosophila melanogaster S2, avaliando os veículos de transfecção fosfato de cálcio, lipídeo catiônico (Cellfectin) e polímero catiônico (ExGen500 e JetPEI). A fim de determinar o agente de transfecção mais eficiente, foram feitos experimentos em placa de 6 poços e frasco de cultivo de 100mL, onde foram analisados a influência da densidade celular; a concentração de DNA, o volume do reagente de transfecção sobre a expressão da RVGP analisada através do método de ELISA. Quantidades de RVGP que variaram entre 50-90 ng/107cel foram obtidas nos diferentes experimentos feitos em placa, sugerindo um efeito da relação DNA: agente de transfecção. A comparação entre os agentes de transfecção não mostrou diferenças significativas. Nas transfecções feitas em cultura em suspensão foi analisado o efeito de transfectar o plasmídeo para expressão de RVGP contendo ou não o sinal de secreção celular BiP. Quando foi usado o plasmídeo contendo o sinal BiP (pMTiRVGP) foram atingidas valores de RVGP de 160 ng/107cel e produções volumétricas de 200 ng/mL, porém sem diferenças significativas entre os diferentes agentes de transfecção. Entretanto, foram encontradas diferenças quando foi usado o plasmídeo não contendo o sinal BiP (pMTRVGP), onde a produção de RVGP foi de 60 ng/107cells nas células transfectadas com Cellfectin, ExGen500 e fosfato de cálcio, porém as células transfectadas com JetPEI obtiveram uma produção de 120 ng/107cels de RVGP. Em experimento em frasco de cultivo tipo spinner com volume de trabalho de 60 mL, foram atingidas expressões de RVGP de 140 ng/107cel para células transfectadas com JetPEI e fosfato de cálcio, sugerindo que otimizações nas condições de cultivo e transfecção ainda podem ser testadas visando aumentar a expressão da proteína recombinante em cultivos em larga escala.

Modelos humanizados de porco são muito importantes para pesquisa biomédica e desenvolvimento de novas drogas e tratamentos. Além de ser um melhor modelo para doenças humanas do que animais de menor porte devido sua maior semelhança fisiológica, anatômica, de metabolismo e tempo de vida, o modelo suíno ainda permite suprimento ilimitado de órgãos para transplante. Apesar dessas vantagens, a expressão gênica inconsistente de animais transgênicos tornam a criação e avaliação desses animais muito dispendiosas, imprevisível e não permite a comparação de resultados de animais diferentes de maneira apropriada. Nesse estudo descrevemos uma nova técnica utilizando o promoter endógeno para a geração de um protocolo de substituição de genes com padrão clonal (transplante clonal de genes) sem clonagem de células, preservando a expressão genética e sua regulação intactas. Esse protocolo é reprodutível e pode ser aplicado para mais de um alvo genético, permitindo geração rápida de linhas transgênicas de animais (14-20 dias) com potencial de se tornar o novo padrão para geração de animais transgênicos de grande porte Suínos / Humanized pig models are very important for biomedical research, and drugs and treatment development. Not only it is a better model for diseases than smaller animals because of its closer physiology, anatomy, metabolism and life span, it also may provide unlimited organs for transplantation. In spite of all this advantages, inconsistent gene expression in transgenic animals make its generation and evaluation expensive, unpredictable and do not allow proper outcome comparison between different animals. In this report we describe a reproducible technique utilizing the endogenous promoter for generation of a clonal pattern gene replacement protocol (clonal gene transplant) without cell cloning, maintaining the normal gene expression and its regulation. This protocol is reproducible and applicable to more than one gene target, allowing fast generation of transgenic animals cell lines (as low as 14-20 days) and could become the new standard for transgenic large animal generation

O desenvolvimento da imunoterapia do câncer baseada em células dendríticas (DCs) é alvo de vários estudos. Para tumores sólidos, a abordagem baseada no uso de DCs alogenêicas fundidas com células tumorais tem se mostrado relativamente eficaz. Por outro lado, esta estratégia necessita uma massa tumoral considerável de cada paciente para a geração das células híbridas. Para contornar este problema o uso de DCs transfectadas com mRNA tumoral, o qual pode ser amplificado in vitro a partir de uma pequena amostra inicial do tumor, tem sido investigado. Para isto, uma transfecção eficiente e tradução correta do mRNA tumoral nas DCs são etapas críticas. Sabendo-se que as DCs de pacientes com câncer possuem atividade aloestimuladora defeituosa, DCs derivadas de doadores saudáveis poderiam ser uma alternativa para induzir uma resposta imune mais eficiente. Assim, este trabalho pretendeu aprimorar a metodologia de transfecção de DCs alogenêicas, derivadas de monócitos de doadores saudáveis, com mRNA de antígenos tumorais (survivina e RPSA) super-expressos na leucemia linfóide crônica (LLC) e avaliar sua capacidade em estimular a resposta linfocitária. Ao mesmo tempo, foram estabelecidas as metodologias para amplificação e síntese do RNA destes antígenos tumorais específicos, assim como do RNA mensageiro total, contidos nas células tumorais de pacientes com LLC. Os resultados mostraram ser possível a amplificação do mRNA total extraído das células leucêmicas com manutenção da expressão dos antígenos tumorais. Ainda, várias condições de transfecção com mRNA da survivina, transcrito in vitro, foram testadas, encontrando-se na lipofecção, a melhor maneira de transfectar as DCs. A lipofecção mostrou-se com baixa toxicidade quando comparada à técnica de eletroporação. Observou-se uma eficiência em torno de 40% de células transfectadas num intervalo de tempo entre 12 e 48 horas. Estas células foram usadas como estimuladoras em ensaios de proliferação usando-se linfócitos T alogenêicos como células respondedoras. As células transfectadas com mRNA da survivina foram capazes de estimular resposta linfoproliferativa com maior produção de IFN-gama, avaliado por ELISA. Além disso, a transfecção não alterou o padrão de expressão dos marcadores de superfície característicos das DCs. Estes dados mostram que a transfecção das DCs com mRNA pode afetar a resposta imune induzida por estas APCs. Nossos resultados suportam o uso de DCs transfectadas com mRNA para produção de vacinas anti-tumorais e mostram a survivina como um potente antígeno indutor da resposta linfocitária. / The development of dendritic cell (DC)-based cancer immunotherapy has been the target of many studies. For solid tumors, a promising approach based in allogeneic DCs fused to tumor cells has been relatively effective. On the other hand, this approach needs large tumor samples to generate enough DC-tumor cell hybrids. To overcome this problem, tumor mRNA-transfected DCs can be used, since mRNA can be amplified in vitro and allow unlimited vaccine production. For this, efficient transfection and optimal translation of tumor mRNA in DCs are critical. Moreover, DC derived from cancer patients has defective alostimulatory activity. In this case, DC derived from healthy donors may be an alternative to induce immune response more efficiently. Here, we established the methodology of allogeneic DC transfection with mRNA for tumor antigens (survivin and RPSA) overexpressed in chronic lymphocytic leukemia (CLL), and evaluated their ability for T cell stimulation. At the same time, we established mRNA amplification and mRNA in vitro transcription methodology for specific tumor antigens and total messenger RNA, present in CLL tumor cells. Our results showed it to be possible to amplify total mRNA derived from leukemic cells maintaining tumor antigen expression. Moreover, several transfection conditions using survivin mRNA obtained from in vitro transcript reactions were evaluated, defining lipofection as the better way to transfect DC. We obtained nearly 40% of transfected DCs between 12 and 48 hours. Transfected DCs were used as stimulator cells in proliferation assays using allogeneic T cells as responder cells. Survivin mRNA transfected DCs were able to stimulate T cell proliferation with increased IFN-gama production, measured by ELISA. Furthermore, the transfection did not change the pattern of surface molecules expression characteristic of DC. These data show that mRNA DC transfection can affect immune responses induced by these APCs. These findings support the use of tumor mRNA transfected DCs for anti-cancer vaccine production and show survivin as a potent antigen to induce T cell responses.