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1.
Mem. Inst. Oswaldo Cruz ; 108(supl.1): 74-79, 2013. tab, graf
Artigo em Inglês | LILACS | ID: lil-697834

RESUMO

In this review, we analyse the impact of a population and evolutionary genetics approach on the study of insect behaviour. Our attention is focused on the model organism Drosophila melanogaster and several other insect species. In particular, we explore the relationship between rhythmic behaviours and the molecular evolution of clock and ion channel genes.


Assuntos
Animais , Comportamento Animal/fisiologia , Relógios Circadianos/genética , Drosophila melanogaster/genética , Evolução Molecular , Genética Populacional , Proteínas CLOCK/genética , Drosophila/genética , Especiação Genética , Canais Iônicos/genética , Proteínas Circadianas Period/genética , Psychodidae/genética , Comportamento Sexual Animal , Temperatura , Transgenes/genética
3.
Electron. j. biotechnol ; 15(4): 2-2, July 2012. ilus, tab
Artigo em Inglês | LILACS | ID: lil-646952

RESUMO

Background: For successful in vitro plant regeneration, plant cell lines with multiple transgene integration and low transgene expression levels need to be ruled out. Although real-time polymerase chain reaction (RT-PCR) is a rapid way to accomplish this, it is also expensive and typically limits the size of the target sequence. Quantitative competitive PCR (QC-PCR) is proven to be a safe and accurate method for determination of both copy number and quantification of transcript levels of synthetic transgenes in transformed plants. Results: The glyphosate oxidoreductase genewas chemically synthesized and used to transform Brassica napus L. via Agrobactrium-mediated transformation. A construct containing the mutated form of a synthetic glyphosate oxidoreductase (gox) gene (internal standard) was prepared. Gene copy number was estimated in nine independent transgenic lines using QC-PCR as well as the standard method of Southern blot analysis. By quantitative RT-PCR, transcript levels were also determined in these lines. High (> 3), medium to high (2.2-3), medium to low (1-2.2), and low (< 1) levels of transcript were detected. Conclusions: No direct relationship was found between copy number and transgene expression levels. QC-PCR method could be implemented to screen putative transgenic plants and quickly select single T-DNA inserts. QC-PCR methods and the prepared competitor construct may be useful for future quantification of commercial transgenic food and feed.


Assuntos
Brassica napus/enzimologia , Brassica napus/genética , Glicina/genética , Oxirredutases/genética , Reação em Cadeia da Polimerase/métodos , Glicina/análogos & derivados , Glicina/fisiologia , Oxirredutases/fisiologia , Transcrição Genética , Transgenes
4.
Electron. j. biotechnol ; 14(4): 9-9, July 2011. ilus, tab
Artigo em Inglês | LILACS | ID: lil-640504

RESUMO

Background: The genetic diversity of maize in Peru includes several landraces (within race clusters) and modern open pollinated and hybrid cultivars that are grown by farmers across various regions, thereby making this country a secondary center of diversity for this crop. A main topic of controversy in recent years refers to the unintended presence of transgenic events in locally grown cultivars at main centers of crop diversity. Peru does not yet have biosafety regulations to control or permit the growing of genetically modified crops. Hence, the aim of this research was to undertake a survey in the valley of Barranca, where there were recent claims of authorized transgenic maize grown in farmers fields as well as in samples taken from feed storage and grain or seed trade centers. Results: A total of 162 maize samples (134 from fields, 15 from local markets, eight from the collecting centers of poultry companies, from the local trading center and four samples from seed markets) were included for a qualitative detection by the polymerase chain reaction (PCR) of Cauliflower Mosaic Virus (CaMV) 35S promoter (P35S) and nopaline synthase terminator (Tnos) sequences, as well as for six transgenic events, namely BT11, NK603, T25, 176, TC1507 and MON810. The 134 maize samples from farmers fields were negative for Cry1Ab delta-endotoxin insecticidal protein and enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) using lateral flow strips. The PCR analysis did not detect any of the six transgenic events in samples from farmers fields, local markets, seed trading shops and the local collecting center. There were four transgenic events (T25, NK603, MON810 and TC1507) in grain samples from the barns of poultry companies. Conclusions: This research could not detect, at the 95 percent probability level, transgenes in farmers' fields in the valley of Barranca. The four transgenic events in grain samples from barns of poultry companies...


Assuntos
Variação Genética , Segurança , Transgenes , Zea mays/genética , Alimentos Geneticamente Modificados , Peru
5.
Biol. Res ; 44(3): 229-234, 2011. ilus, tab
Artigo em Inglês | LILACS | ID: lil-608618

RESUMO

Testis-mediated gene transfer (TMGT) has been used as in vivo gene transfer technology to introduce foreign DNA directly into testes, allowing mass gene transfer to offspring via mating. In this study, we used plasmid DNA (pEGFP-N1) mixed with dimethylsulfoxide (DMSO), N,N-dimethylacetamide (DMA) or liposome (Lipofectin) in an attempt to improve TMGT. Males receiving consecutive DNA complex injections were mated to normal females to obtain F0 progeny. In vivo evaluation of EGFP expression, RT-PCR and PCR were used to detect the expression and the presence of exogenous DNA in the progeny. We also evaluated possible testicular damage by histological procedures. PC R and RT-PCR analyses revealed that liposome and DMSO increased the rate of TMGT. Histological analyses demonstrated that repeated (4 times) injections of DNA complexes can affect spermatogenesis. DMSO was the most deleterious among the reagents tested. In this study, we detected the presence of transgene in the progeny, and its expression in blood cells. Consecutive injections of DNA complexes were associated with impaired spermatogenesis, suggesting requirement of optimal conditions for DNA delivery through TMGT.


Assuntos
Animais , Feminino , Camundongos , Dimetil Sulfóxido/farmacologia , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/administração & dosagem , Camundongos Transgênicos/genética , Testículo , Transgenes , Animais Geneticamente Modificados , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Lipossomos/farmacologia , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase , Testículo/efeitos dos fármacos , Testículo/patologia , Transfecção/métodos
6.
Mem. Inst. Oswaldo Cruz ; 104(3): 399-410, May 2009. ilus
Artigo em Inglês | LILACS | ID: lil-517020

RESUMO

Amplicons are defective and non-integrative vectors derived from herpes simplex virus type 1. As the vector genome carries no virus genes, amplicons are both non-toxic for the infected cells and non-pathogenic for the inoculated organisms. In addition, the large transgenic capacity of amplicons, which allow delivery of up to 150 Kbp of foreign DNA, makes these vectors one of the most powerful, interesting and versatile gene delivery platforms. We present here recent technological developments that have significantly improved and extended the use of amplicons, both in cultured cells and in living organisms. In addition, this review also discusses the many difficulties still pending to be solved, in order to achieve stable and physiologically regulated transgene expression.


Assuntos
Animais , Humanos , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Herpesvirus Humano 1/genética , Transgenes/genética , Engenharia Genética , Herpesvirus Humano 1/fisiologia
7.
Electron. j. biotechnol ; 11(5): 2-3, Dec. 2008. ilus, tab
Artigo em Inglês | LILACS | ID: lil-538014

RESUMO

Total number of cells in cloned embryos is generally lower than that of in vivo derived embryos and in bovines cell allocation at the blastocyst stage, has been observed to be affected in a large proportion of cloned embryos. The current embryo staining procedures are toxic for mammalian cells and thus can not be used to determine the developmental potential of a stained embryo. Therefore, in the present study we sought to assess the feasibility to develop a noninvasive embryo model that would be suitable for the evaluation of cloned embryos subjected to different nuclear transfer and embryo culture procedures. For doing this, we stably transfected a bovine embryonic fibroblast cell line and generated a number of clones that constitutively expressed a red fluorescent protein (HcRed) in the nuclear compartment of the cell. Those clones with normal chromosomal content were further used as nuclear donor in nuclear transfer procedures (SCNT) to generate transgenic cloned embryos. These embryos expressed the red fluorescent protein in each blastomere, allowing their in vivo evaluation during development, thus demonstrating the potential of this model as a noninvasive tool for the assessment of the quality of cloned embryos.


Assuntos
Animais , Bovinos , Clonagem de Organismos/métodos , Clonagem de Organismos , Transferência Embrionária , Fibroblastos , Corantes Fluorescentes , Marcadores Genéticos , Técnicas Genéticas , Transgenes
9.
São Paulo; s.n; 2008. 106 p. ilus, tab.
Tese em Português | LILACS | ID: lil-500934

RESUMO

O mosquito Culex quinquefasciatus é considerado praga urbana e tem a capacidade de se desenvolver em águas altamente poluídas atingindo elevada densidade . Mosquitos desta espécie possuem importância vetorial na transmissão de parasitas e arboviroses. Medidas de controle químico têm se mostrado ineficazes, além de serem altamente prejudiciais ao meio ambiente. Desse modo, novas tecnologias de controle foram desenvolvidas, entre elas o SIT (Sterile Insect Technique) que utiliza radiação para esterilizar Machos e liberá-los no ambiente para copular com fêmeas selvagens. Posteriormente métodos genéticos baseados nessa técnica têm sido proposto. O sistema RIDL (Liberação de Insetos Carregando Gene Letal Dominante), consiste na integração de um gene letal dominante associado a um promotor específico de fêmea, dispensando a etapa de esterilização por radiação. Nesse processo os insetos recebem dieta suplementada com um repressor químico. A expressão do gene letal dominante é mantida desligada enquanto este repressor é adicionado ao meio das larvas. Para as amostras que estariam sendo preparadas para a liberação, o repressor é retirado, e o gene letal dominante é ativado, causando a morte de todas as fêmeas, restando apenas machos para liberação. Os machos homozigotos para gene letal seriam liberados para copular com fêmeas selvagens. A progênie seria heterozigota para o gene letal, porém semente os machos sobreviveriam. Parte crucial para o sucesso deste projeto foi à adaptação do método de microinjeção de embriões para a espécie Culex quinquefasciatus tornando possível à injeção dos transgenes LA513, LA882 e LA3653 com objetivo de obtermos linhagens transgênicas. A obtenção de linhagens transgênicas com estas construções se mostraram mais laboriosa do que o previsto, dificultando a transgenia. Porém, as aplicações práticas em controle de vetores utilizando a técnica do RIDL são imensas e pode se tornar uma importante ferramenta do Manejo Integrado de vetores.


Assuntos
Animais Geneticamente Modificados , Culex/genética , Técnicas de Transferência de Genes , Transgenes , Insetos Vetores , Controle de Vetores
10.
Genet. mol. res. (Online) ; 6(2): 445-452, 2007. ilus, tab, graf
Artigo em Inglês | LILACS | ID: lil-482025

RESUMO

Evaluation of transgenic crops under field conditions is a fundamental step for the production of genetically engineered varieties. In order to determine if there is pollen dispersal from transgenic to nontransgenic soybean plants, a field release experiment was conducted in the Cerrado region of Brazil. Nontransgenic plants were cultivated in plots surrounding Roundup Ready transgenic plants carrying the cp4 epsps gene, which confers herbicide tolerance against glyphosate herbicide, and pollen dispersal was evaluated by checking for the dominant gene. The percentage of cross-pollination was calculated as a fraction of herbicide-tolerant and -nontolerant plants. The greatest amount of transgenic pollen dispersion was observed in the first row, located at one meter from the central (transgenic) plot, with a 0.52% average frequency. The frequency of pollen dispersion decreased to 0.12% in row 2, reaching 0% when the plants were up to 10 m distance from the central plot. Under these conditions pollen flow was higher for a short distance. This fact suggests that the management necessary to avoid cross-pollination from transgenic to nontransgenic plants in the seed production fields should be similar to the procedures currently utilized to produce commercial seeds.


Assuntos
Soja/genética , Fluxo Gênico , Plantas Geneticamente Modificadas/genética , Brasil , Cruzamentos Genéticos , Genes Dominantes , Genes de Plantas , Engenharia Genética , Modelos Genéticos , Plantas/genética , Pólen/metabolismo , Análise de Regressão , Sementes/metabolismo , Transgenes
11.
Genet. mol. res. (Online) ; 4(2): 177-184, 30 jun. 2005. ilus, graf
Artigo em Inglês | LILACS | ID: lil-445294

RESUMO

Transgene elimination is a poorly studied phenomenon in plants. We made genetic and molecular studies of a transgenic dry bean line immune to bean golden mosaic geminivirus and a soybean line. In both lines, the transgenes were stable during the vegetative phase but were eliminated during meiosis. Due to its potential biotechnological value, this transgenic line was micropropagated by grafting and the vegetative copies were studied for more than two years. More than 300 plants of progeny were obtained during this period, demonstrating that the phenomenon of elimination was consistently repeated and offering an opportunity for detailed study of transgene elimination, including the characterization of the integration sites. Cloning and sequencing of the transgenic loci, reciprocal crosses to untransformed plants, genomic DNA blots, and GUS assays were performed in the transgenic lines. Based on the molecular and genetic characterization, possible mechanisms involved in transgene elimination include intrachromosomal recombination, genetic instability resulting from the tissue culture manipulations, and co-elimination of transgenes, triggered by a process of genome defense.


Assuntos
Soja/genética , Vírus do Mosaico , Phaseolus/genética , Plantas Geneticamente Modificadas/genética , Transgenes/genética , DNA de Plantas , Soja/virologia , Deleção de Genes , Reação em Cadeia da Polimerase , Phaseolus/virologia , Vetores Genéticos/genética
12.
Genet. mol. res. (Online) ; 4(4): 812-821, 2005. tab, ilus
Artigo em Inglês | LILACS | ID: lil-444840

RESUMO

Transgenesis in cattle has provided numerous opportunities for livestock production. The development of nuclear transfer (NT) technology has improved the production of transgenic livestock. However, the isolation of pure colonies from a single transfection event remains laborious and can be a constraint in the production of transgenic livestock. We used 96-well cell culture plates to isolate cell lineages obtained from a single fibroblast transfected with the pCi-Neo plasmid. Since single mammalian cells do not grow well in fresh medium, we evaluated the use of conditioned medium. The neomycin phosphotransferase gene was detected in isolated colonies and NT embryos were produced from these cells. Multiplex-PCR assays were performed to detect the transfected fragment as well as autosomal satellite DNA in single NT embryos. This approach provided a reliable method for isolating transfected mammalian cells and for diagnosing the incorporation of desirable vectors in NT embryos. This method can reduce the time and cost of transgenic livestock production.


Assuntos
Animais , Animais Geneticamente Modificados/genética , Bovinos/genética , Técnicas de Transferência Nuclear , Transgenes/genética , Animais Geneticamente Modificados/embriologia , Bovinos/embriologia , Fibroblastos/citologia , Núcleo Celular/genética
14.
Genet. mol. res. (Online) ; 3(4): 456-462, 2004. tab, graf
Artigo em Inglês | LILACS | ID: lil-410890

RESUMO

Inducible transgenic mouse models that impose a constraint on both temporal and spatial expression of a given transgene are invaluable. These animals facilitate experiments that can address the role of a specific cell or group of cells within an animal or in a particular window of time. A common approach to achieve inducibility involves the site-specific recombinase ‘Cre’, which is linked to a modified version of one of various steroid hormone-binding domains. Thus, the expression of Cre is regulated such that a functional nuclear transgene product can only be generated with the addition of an exogenous ligand. However, critical requirements of this system are that the nuclear localization of the transgene product be tightly regulated, that the dosage of the inducing agent remains consistent among experimental animals and that the transgene cassette cannot express in the absence of the inducing agent. We used the Cre ER(T2) cassette, which is regulated by the addition of the estrogen antagonist tamoxifen to determine whether cross-contamination of tamoxifen between animals housed together can be a significant source of spurious results. We found that cross-contamination of exogenous tamoxifen does occur. It occurred in all animals tested. We suggest that the mechanism of contamination is through exposure to tamoxifen in the general environment and/or to coprophagous behavior. These results have important implications for the interpretation and design of experiments that use ‘inducible’ transgenic animals.


Assuntos
Animais , Camundongos , Regulação Enzimológica da Expressão Gênica , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Tamoxifeno/farmacologia , Transgenes/efeitos dos fármacos , Proteínas Virais/genética , Citometria de Fluxo , Camundongos Transgênicos , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Cauda/efeitos dos fármacos , Cauda/patologia , Transgenes/genética , Proteínas Virais/efeitos dos fármacos
15.
An. acad. bras. ciênc ; 72(3): 381-8, Sept. 2000. ilus, graf
Artigo em Inglês | LILACS | ID: lil-269389

RESUMO

The cell adhesion molecule Rst-irreC is a transmembrane glycoprotein of the immunoglobulin superfamily involved in several important developmental processes in Drosophila, including axonal pathfinding in the optic lobe and programmed cell death and pigment cell differentiation in the pupal retina. As an initial step towards the "in vivo" functional analysis of this protein we have generated transgenic fly stocks carrying a truncated cDNA construct encoding only the extracellular domain of Rst-IrreC under the transcriptional control of the heat shock inducible promoter hsp70. We show that heat-shocking embryos bearing the transgene during the first 8hs of development lead to a 3-4 fold reduction in their viability compared to wild type controls. The embryonic lethality can already be produced by applying the heat pulse in the first 3hs of embryonic development, does not seem to be suppressed in the absence of wildtype product and is progressively reduced as the heat treatment is applied later in embryogenesis. These results are compatible with the hypothesis of the lethal phenotype being primarily due to heterophilic interactions between Rst-IrreC extracellular domain and an yet unknown ligand.


Assuntos
Animais , Masculino , Feminino , Moléculas de Adesão Celular Neuronais/genética , Drosophila melanogaster/genética , Embrião não Mamífero/fisiologia , Expressão Gênica , Genes Letais/fisiologia , Transgenes/fisiologia , Moléculas de Adesão Celular Neuronais/fisiologia , Genes de Insetos/genética , Temperatura Alta , Fenótipo , Choque
16.
Arq. bras. endocrinol. metab ; 44(4): 300-5, ago. 2000.
Artigo em Português | LILACS | ID: lil-268990

RESUMO

A resistência aos hormônios tireóideos (RTH) é uma síndrome de herança dominante, decorrente da hipossensibilidade dos tecidos aos hormônios tireóideos e caracterizada pela elevação dos hormônios tireóideos séricos com TSH normal ou aumentado e bócio. Tem sido atribuída a mutações na isoforma ß do receptor para hormônios tireóideos (TR ). Modelos de transgênese têm contribuído para a compreensão da RTH. A ausência da expressão do TR em camundongos TR knockout tornou os tireotrofos parcialmente resistentes aos hormônios tireóideos, o que não ocorreu nos animais knockout para a isoforma a do TR. Entretanto, camundongos que não expressam as duas formas de TR apresentam completa resistência aos hormônios tireóideos, sendo os hormônios tireóideos e TSH séricos elevadíssimos. Mutantes de TR humano expressos em tecidos de camundongo reproduziram várias manifestações da RTH. A expressão de TR mutado apenas no coração ou apenas na hipófise induziu diminuição dos efeitos de hormônios tireóideos e resistência à administração dos mesmos nestes tecidos. Modelos transgênicos evidenciaram que, além da resistência hipofisária, a resistência nos neurônios hipotalâmicos, de TRH, é imprescindível para que haja aumento de produção de hormônios tireóideos. Camundongos knockout para o coativador SRC-1 também se mostraram parcialmente resistentes aos hormônios tireóideos, sugerindo que outras proteínas envolvidas no mecanismo de ação dos hormônios tireóideos possam causar RTH. Assim, os modelos transgênicos forneceram provas que o mutante TRb, in vivo, interfere com a ação das diferentes isoformas do TR selvagem e causa RHT. Modelos transgênicos são uma valiosa ferramenta para a compreensão da heterogeneidade de apresentação clínica da RTH.


Assuntos
Humanos , Animais , Camundongos , Síndrome da Resistência aos Hormônios Tireóideos/genética , Transgenes/fisiologia , Hormônios Tireóideos/sangue , Camundongos Knockout , Camundongos Transgênicos , Mutação/fisiologia , Síndrome da Resistência aos Hormônios Tireóideos/fisiopatologia , Tireotropina/sangue
18.
Braz. j. med. biol. res ; 32(7): 867-75, July 1999.
Artigo em Inglês | LILACS | ID: lil-234893

RESUMO

A large number of DNA sequences corresponding to human and animal transcripts have been filed in data banks, as cDNAs or ESTs (expression sequence tags). However, the actual function of their corresponding gene products is still largely unknown. Several of these genes may play a role in regulation of important biological processes such as cell division, differentiation, malignant transformation and oncogenesis. Elucidation of gene function is based on 2 main approaches, namely, overexpression and expression interference, which respectively mimick or suppress a given phenotype. The currently available tools and experimental approaches to gene functional analysis and the most recent advances in mass cDNA screening by functional analysis are discussed


Assuntos
Animais , Humanos , Elementos Antissenso (Genética) , Expressão Gênica , RNA Catalítico , Transgenes , DNA Recombinante
19.
Braz. j. med. biol. res ; 32(2): 155-62, feb. 1999. tab, graf
Artigo em Inglês | LILACS | ID: lil-228257

RESUMO

The use of mammalian gene expression vectors has become increasingly important for genetic immunization and gene therapy as well as basic research. Essential for the success of these vectors in genetic immunization is the proper choice of a promoter linked to the antigen of interest. Many genetic immunization vectors use promoter elements from pathogenic viruses including SV40 and CMV. Lymphokines produced by the immune response to proteins expressed by these vectors could inhibit further transcription initiation by viral promoters. Our objective was to determine the effect of IFN-g on transgene expression driven by viral SV40 or CMV promoter/enhancer and the mammalian promoter/enhancer for the major histocompatibility complex class I (MHC I) gene. We transfected the luciferase gene driven by these three promoters into 14 cell lines of many tissues and several species. Luciferase assays of transfected cells untreated or treated with IFN-g indicated that although the viral promoters could drive luciferase production in all cell lines tested to higher or lower levels than the MHC I promoter, treatment with IFN-g inhibited transgene expression in most of the cell lines and amplification of the MHC I promoter-driven transgene expression in all cell lines. These data indicate that the SV40 and CMV promoter/enhancers may not be a suitable choice for gene delivery especially for genetic immunization or cancer cytokine gene therapy. The MHC I promoter/enhancer, on the other hand, may be an ideal transgene promoter for applications involving the immune system


Assuntos
Regulação da Expressão Gênica , Vetores Genéticos , Transgenes , Vacinas de DNA , Citomegalovirus , Elementos Facilitadores Genéticos , Genes MHC Classe I , Luciferases , Regiões Promotoras Genéticas , Vírus 40 dos Símios
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