Construction of fluorescent transgenic zebrafish Tg (ttn.2: EGFP) / 生物工程学报

In order to develop a transgenic zebrafish line with green fluorescent protein (enhanced green fluorescent protein, EGFP) expressed specifically in muscle and heart, the recombinant expression vector constructed using the zebrafish ttn.2 gene promoter fragment and EGFP gene coding sequence and the capped mRNA of Tol2 transposase were co-injected into the zebrafish 1-cell stage embryos. The stable genetic Tg (ttn.2: EGFP) transgenic zebrafish line was successfully developed by fluorescence detection, followed by genetic hybridization screening and molecular identification. Fluorescence signals and whole-mount in situ hybridization showed that EGFP expression was located in muscle and heart, the specificity of which was consistent with the expression of ttn.2 mRNA. Inverse PCR showed that EGFP was integrated into chromosomes 4 and 11 of zebrafish in No. 33 transgenic line, while integrated into chromosome 1 in No. 34 transgenic line. The successful construction of this fluorescent transgenic zebrafish line, Tg (ttn.2: EGFP), laid a foundation for the research of muscle and heart development and related diseases. In addition, the transgenic zebrafish lines with strong green fluorescence can also be used as a new ornamental fish.

Lentivirus vectors fail to deliver transgenes into bovine zygotes after co-incubation with sperm during in vitro fertilization / [Lentivírus falham na entrega de transgenes em zigotos bovinos após coincubação com espermatozoide, durante fertilização in vitro]

As vantagens dos animais transgênicos têm sido demonstradas em diferentes aplicações, entretanto muitas metodologias usadas para gerar animais geneticamente modificados (GM) apresentam baixas taxas de eficiência. O objetivo deste estudo foi avaliar a entrega dos vetores lentivirais (VLs) em zigotos durante a fertilização in vitro (FIV), para gerar embriões GM, com o gene da proteína verde fluorescente (GFP) ou do fator IX de coagulação humana (FIX). Vetores lentivirais com os genes GFP (pLGW-GFP-LV) ou FIX (pLWE2-FIX-LV) foram utilizados na FIV ou na cultura de embriões in vitro (CIV). A coincubação de pLWE2-FIX-LV com espermatozoides e complexos oócitos-células do cumulus (COCs) durante a FIV diminuiu (P<0,05) as taxas de clivagem e de blastocistos, enquanto com pLGW-GFP-LV diminuiu (P<0,05) a taxa de blastocisto quando se comparou ao controle sem VL. A coincubação de pLWE2-FIX-LV e pLGW-GFP-LV com presumíveis zigotos durante a CIV não afetou (P>0,05) o desenvolvimento embrionário. A expressão da proteína GFP não foi detectada em embriões após a coincubação de FIV ou CIV, embora as células do cumulus expressassem a proteína até o dia oito de cultivo in vitro. Reações em cadeia da polimerase (PCR) não detectaram os genes GFP ou FIX em embriões, mas ambos foram detectados em células do cumulus. Assim, a coincubação de VL com espermatozoide bovino e COCs não é eficaz para produzir embriões geneticamente modificados por meio de FIV.(AU)

Identification of abnormal gene expression in bovine transgenic somatic cell nuclear transfer embryos

This study was conducted to investigate the expression of three genes related to early embryonic development in bovine transgenic cloned embryos. To accomplish this, development of bovine transgenic somatic cell nuclear transfer (SCNT) embryos was compared with non-transgenic embryos. Next, mRNA transcription of three specific genes (DNMT1, Hsp 70.1, and Mash2) related to early embryo development in transgenic SCNT embryos was compared between transgenic and non-transgenic SCNTs, parthenogenetic embryos, and in vitro fertilization (IVF) embryos. Transgenic SCNT embryos showed significantly lower rates of development to the blastocyst stage than non-transgenic ones. To investigate normal gene expression, RNA was extracted from ten blastocysts derived from parthenogenesis, IVF, non-transgenic, and transgenic SCNT embryos and reverse-transcribed to synthesize cDNA. The cDNA was then subjected to PCR amplification and semi-quantified. More DNMT1 mRNA was detected in the transgenic SCNT group than the other three groups. Hsp 70.1 mRNA was detected in the IVF embryos, while lower levels were found in SCNT and parthenogenetic embryos. Mash2 mRNA was present at the highest levels in transgenic SCNT embryos. In conclusion, the higher levels of methylation and lower protein synthesis after heat shock in the transgenic SCNT embryos expected based on our results may cause lower embryonic development.

Insights on bovine genetic engineering and cloning / Experiências em clonagem e transgenia em bovinos

Transgenic technology has become an essential tool for the development of animal biotechnologies, and animal cloning through somatic cell nuclear transfer (SCNT) enabled the generation of genetically modified animals utilizing previously modified and selected cell lineages as nuclei donors, assuring therefore the generation of homogeneous herds expressing the desired modification. The present study aimed to discuss the use of SCNT as an important methodology for the production of transgenic herds, and also some recent insights on genetic modification of nuclei donors and possible effects of gene induction of pluripotency on SCNT.

Tecnologias de modificação genética têm se tornado ferramentas essenciais para o desenvolvimento de biotecnologias animais. A clonagem animal mediante transferência nuclear de célula somática (TNCS) possibilitou a geração de animais geneticamente modificados através da utilização de linhagens celulares previamente modificadas e selecionadas como doadoras de núcleo, garantindo desta maneira a geração de rebanhos homogênoes expressando a modificação desejada. O presente estudo objetivou discutir o uso da TNCS como importante metodologia para a produção de rebanhos transgênicos, assim como experiências recentes na manipulação genética de células doadoras de núcleo e possíveis efeitos da indução gênica à pluripotência na TNCS.

Culex quinquefasciatus (Diptera: Culicidae): aspectos da manipulação genética e estudos populacionais utilizando marcadores microssatélites / Culex quinquefasciatus (Diptera: Culicidae) aspects of genetic manipulation and population Characterization using microsatellites

O avanço na distribuição geográfica de mosquitos vetores é seguido pela emergência de vírus e doenças em novas áreas para as quais não há disponibilidade de vacinas efetivas e drogas terapêuticas específicas são insuficientes. Métodos de controle de mosquitos tradicionais perderam efetividade, devido principalmente a grande capacidade reprodutiva e flexibilidade genômica dos mosquitos. Controle químico cada vez mais tornase restrito, acarretando na urgente necessidade de novas formas de controle. A liberação de machos carregando um gene letal dominante (RIDL) oferece novas abordagens aplicáveis ao controle de mosquitos e ainda assim ecológicas e espécie específica. Mosquitos Culex quinquefasciatus foram transformados com sucesso apenas uma vez, apesar do esforço de diversos laboratórios em obter uma linhagem transgênica estável. Foi desenvolvido um método de expressão transiente em mosquitos Culex, que insere plasmídeos contendo genes efetores na hemolinfa e tecidos subjacentes do mosquito. Foi observada a expressão da proteína fluorescente DsRed2, em mosquitos Culex quinquefasciatus adultos mediada por plasmídeos. Esta expressão pode ser considerada um importante passo na transformação de mosquitos Culex, além de potencial uso em estratégias de controle genético e interações gênicas. Para que novas formas de controle sejam realmente efetivas é vital que se conheça a estrutura genética da população alvo. Marcadores moleculares têm sido extensivamente utilizados em estudos filogenéticos e taxonômicos de diversas espécies de insetos. Microssatélites são de grande utilidade para observar estruturas populacionais, tanto em âmbito geográfico, quanto na escala evolucionária. Foi possível observar a formação de clusters e de padrões genéticos distintos entre as populações analisadas, criando um panorama genético dos mosquitos Culex quinquefasciatus coletados no Brasil.

Novedades Científicas: “utilidad de animales transgénicos en la investigación” / Scientific novelties: utility of transgenic animals in reserch

La patología comparada resurge como el punto de encuentro entre el patrón de estudio morfológico humano y el animal. Su aplicación desde sus inicios se apoyó en los biomodelos animales, muchos de los cuales se lograron por manipulación genética. Estos métodos de estudio fueron generados a partir del comportamiento de la enfermedad en el humano. La ingeniería genética se refiere a tecnología desarrollada por el manejo del ADN recombinante, definida por ser una secuencia “nueva” de ADN creada en los laboratorios por la unión de porciones de ADN con orígenes diferentes. A un organismo cuyo material genético ha sido modificado artificialmente mediante la supresión de expresiones génicas o la incorporación de fracciones o secuencias de ADN ajeno a su especie, se le llama organismo genéticamente modificado (OGM); organismo modificado genéticamente (OMG) o simplemente “transgénico” (antes, “transgenético”). La ingeniería genética se define como el conjunto de técnicas y métodos que se utilizan para “construir” moléculas de ADN recombinante y luego introducirlas en moléculas receptoras. En Venezuela existe una prohibición para la manipulación en animales. Se aplican la Ley de Biodiversidad, el Convenio de Diversidad biológica y el Protocolo Internacional de Biodiversidad. Con la investigación animal, los científicos han descubierto las maneras de salvar y prolongar la vida humana. Vacunaciones tales como la poliomielitis trasplantes de órganos perfeccionados así como el desarrollo de técnicas quirúrgicas y de traumatología reconstructivas. De manera activa se ha generado un debate por el uso de animales en la investigación y en los ensayos biotecnológica. Se plantea la bioética en la aplicación de los biomodelos animales. Es importante el conocimiento de estos métodos de investigación genética en animales de investigación, más aún en instituciones generadoras de productos biológicos. En poco tiempo se ha generado un fenómeno transgénico de la...

Diversidad genética de la raza criollo limonero utilizando marcadores de ADN microsatélites / Genetic diversity of limonero creole breed using microsatellites molecular markers

Con la finalidad de estudiar la variabilidad genética de la raza Criollo Limonero considerada patrimonio Nacional y orientada a la producción de leche, se analizaron 95 animales puros utilizando 14 marcadores moleculares de ADN del tipo microsatélites. Los animales pertenecen al rebaño de la estación local Carrasquero, adscrita al Instituto Nacional de Investigaciones Agrícolas del estado Zulia (INIA-Zulia) y ubicado al noroeste del Estado. Para medir la diversidad genética se estimaron y discutieron los valores de heterocigosis observada (HO) y esperada (HE) total y entre familias (HMO, HME), probabilidad de exclusión (PE), Índice de Contenido Polimórfico (PIC) y número de alelos por locus. El número promedio de alelos por locus, HE y PIC fueron: 8,7; 0,689 y 0,651, respectivamente. Las HE variaron desde 0,355 hasta 0,787 y el PIC fluctuó de 0,302 a 0,757. El locus menos polimórfico fué el ILST5 y el más polimórfico fue el CSSM66. La PE con los 14 marcadores fue de 0,9962 y 0,9999 para uno y dos padres conocidos, respectivamente. Los resultados indicaron que este grupo de marcadores resultaron ser eficientes para realizar pruebas de paternidad en esta raza, así mismo se muestran que los niveles de heterocigosis indican la existencia de una alta diversidad molecular en la población estudiada, la cual deberá mantenerse como estrategia para la conservación del Criollo Limonero como recurso genético bovino de Venezuela para la producción animal en la región tropical.

In order to study the genetic variability of the Criollo Limonero Breed, a dairy breed considered National Patrimony of Venezuela, 95 purebred animals were analyzed using 14 DNA microsatellites. The animals belonged to the local Carrasquero station, assigned to the National Institute of Agriculture Research of Zulia (INIA-Zulia) and located in the northwest of Zulia State. Average values of observed and expected heterocigocities (HO, HE), between families (HMO, HME) respectively, exclusion probability (PE), Polymorphic Information Content (PIC) and number of alelles by locus were considered and discussed to measure the genetic diversity. The average of alleles by locus, HE and PIC were: 8.7; 0.689 and 0.651, respectively. HE ranged from 0.355 to 0.787 and PIC fluctuated from 0.302 to 0.757. The least polymorphic locus was ILST5 and the most polymorphic was the CSSM66. PE with the 14 markers was of 0.9962 and 0.9999 for one and both known parents, respectively. The results indicate that this set of markers are efficient to make paternity tests in this breed, it is also evident that there are heterozigosity levels indicating the existence of a high molecular diversity in this population, which should be maintained as a strategy for the conservation of the Criollo Limonero breed, a bovine genetic resource of Venezuela for animal production in the tropical regions.

Development and evaluation of an in vivo assay in Caenorhabditis elegans for screening of compounds for their effect on cytochrome P450 expression.

Most drugs and xenobiotics induce the expression of cytochrome P450 (CYP) enzymes, which reduce the bioavailability of the inducer and/or co-administered drugs. Therefore, evaluation of new drug candidates for their effect on CYP expression is an essential step in drug development. The available methods for this purpose are expensive and not amenable to high-throughput screening. We developed a fluorescence-based in vivo assay using transgenic Caenorhabditis elegans worms that express the green fluorescent protein (GFP) under the control of various CYP promoters. Using this assay, we found striking similarities between the worm CYPs and their human orthologs in their response to treatment with various drugs. For example,the antibiotic rifampicin, one of the strongest inducers of the human gene CYP3A4, was the strongest inducer of the worm ortholog CYP13A7. Since worms can be easily grown in liquid medium in microtitre plates, the assay described in this paper is suitable for the screening of a large number of potential lead compounds in the drug discovery process.

Isolation of transfected fibroblast clones for use in nuclear transfer and transgene detection in cattle embryos

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.

Expression of active human erythropoietin in the mammary gland of lactating transgenic mice and rabbits

Transgenic mice and rabbits were generated using a chimeric gene comprising the human erythropoietin (hEPO) cDNA under the 5' and 3' regulatory sequences of the rabbit whey acidic protein gene. Transgenic mice expressed hEPO at levels of 0.01 mg/l in the milk of lactating females showing that the genetic construct was functional. Reverse transcriptase polymerase chain reaction with RNA from various tissues showed that this transgene was expressed mainly in the ovary and mammary gland. In rabbits, we demonstrated the germ line transmission of the transgene. The hEPO was obtained in the milk of lactating females at levels of up to 0.0003 mg/l. Although the expression levels were low, biologically active hEPO was obtained in the milk of transgenic rabbits without any apparent detrimental effect for the animals. In vitro, the specific activity of the rabbit-derived hEPO was higher than that reported for the natural hEPO, thus suggesting differences in the glycosylation pattern in at least part of the molecules secreted by the mammary gland of transgenic rabbits