Transgenic Rodent Models in Toxicological and Environmental Research: Future Perspectives

The coexistence of humans and animals has existed for centuries. Over the past decade, animal research has played a critical role in drug development and discovery. More and more diverse animals, including transgenic animals, are used in basic research than in applied research. Transgenic animals are generated using molecular genetic techniques to add functional genes, alter gene products, delete genes, insert reporter genes into regulatory sequences, replace or repair genes, and make changes in gene expression. These genetically engineered animals are unique tools for studying a wide range of biomedical issues, allowing the exhibition of specific genetic alterations in various biological systems. Over the past two decades, transgenic animal models have played a critical role in improving our understanding of gene regulation and function in biological systems and human disease. This review article aims to highlight the role of transgenic animals in pharmacological, toxicological, and environmental research. The review accounts for various types of transgenic animals and their appropriateness in multiple types of studies.

Research on regulatory effect of lncRNA-ZFASl on myocardial fibrosis / 中国药理学通报

Aim To explore the regulatory effect of lncRNA-ZFASl on myocardial fibrosis. Methods The mouse model of myocardial fibrosis was established by ligating the left anterior descending coronary artery of heart; the lncRNA-ZFASl cardiac-specific transgenic mice were constructed by CRISPR/Cas9 technology. Masson staining was used to detect the fibrosis of heart tissues ; angiotensin E ( Ang H ) was added to cardiac fibroblasts to establish an in vitro myocardial fibrosis model, and siRNA was transfected to knock down lncRNA-ZFASl ; MTT method was used to detect the proliferation of cardiac fibroblasts; real-time quantitative PCR ( qRT-PCR ) was used to detect the expression of lncRNA-ZFASl, transforming growth factor ßl (TGFßl), type I collagen (Collai) and type M col¬lagen (Col3al). Western blot was used to detect a-SMA expression. Results In cardiac tissues of myo¬cardial fibrosis model mice, the expression of lncRNA-ZFASl significantly increased. Knocking down of lncRNA-ZFASl significantly reduced the expression of CoBal and TGFßl in Ang II treated cardiac fibroblasts. Collagen deposition appeared in cardiac tissues of lncRNA-ZFASl transgenic mice, and the expression of a-SMA, Collai and CoBal markedly increased. Conclusions The high expression of lncRNA-ZFASl in heart tissues could induce myocardial fibrosis, and knocking down lncRNA-ZFASl could alleviate myocardial fibrosis.

Genetic background of human lactoferrin transgenic goats / 生物工程学报

The genetic background such as copy number, integration site and chromosome karyotype of exogenous genes of transgenic animals obtained by random integration is still unclear. There may be some problems such as silent integration, invalid integration, toxic integration and unpredictable expression level of exogenous genes. In this study, six primary (F0) and their corresponding offspring (F1) of human lactoferrin (hLF) transgenic goats were selected as the research objects, and blood samples were collected from jugular vein and DNA were extracted. The genetic background and expression level of exogenous genes were studied by chromosome karyotype analysis, real-time quantitative PCR (qPCR), ELISA and Western blotting. The chromosomes of six F0 transgenic goats had no obvious morphological variation, number change and other abnormalities. The relative copy number was different (2-16) and could be steadily inherited to the next generation. The copy number of F0 and F1 hLF gene was the same. The highest expression level of hLF was 1.12 g/L in F1 transgenic goats (L3-1, 8 copies). The results proved that the integrated exogenous genes could steadily inherit the next generation, and did not cause obstacles to the growth and development of transgenic goat individuals. Moreover, there was no obvious correlation between the number of copies and the expression level of hLF. This laid a foundation for the new varieties cultivation of transgenic goats and other transgenic animals, and analysis of genetic background.

Relevance of Small Laboratory Animals as Models in Translational Research: Challenges and Road Ahead.

Translational research using small laboratory animals is being done to demonstrate proof of concept, to study pharmacokinetics as well as to understand efficacy and safety of new drug molecules. During the evaluation of a drug candidate, the assessment of efficacy and safety is normally performed in different experiments using various animal models. In such experiments, efficacy is assessed by mimicking the disease state in animal model while safety is investigated in healthy animals. Inventing new drugs using biotechnological and nanotechnological approaches is becoming a major thrust area in drug research. Apart from this, the development of medicine from traditional knowledge like Ayurveda has emerged as major area for drug industry. Use of conventional in-vivo approaches may not prove useful to answer many questions. Transgenic/knock-out/knock-in animals are now getting space in pharmaceutical research for target identification and validation. Predictability of in-vivo research depends on scientific protocols and methods adopted for model selection and development. Various alternative approaches for in-vivo research are being followed. It is a fact that no animal model is 100 % capable of mimicking the complex human body but still, researchers have not yet found any alternative model which can completely replace in-vivo models. This review is a holistic approach explaining the various animal models being used for translational research, animal ethical issues, alternative approaches available and provides a critical analysis of major issues/challenges faced in translational research using in-vivo approaches.

Animales transgénicos: usos y limitaciones en la medicina del siglo XXI / Transgenic animals: uses and limitations in the 21st century medicine

Uno de los avances biotecnológicos más importantes de las últimas décadas fue el desarrollo de los animales transgénicos. En este artículo seanaliza por qué los animales transgénicos son excelentes modelos para estudiar la función y regulación de los genes y para buscar nuevas estrategias terapéuticas para las enfermedades humanas. Se discute su uso como biorreactores para producir productos farmacológicos para el tratamiento de enfermedades y la posibilidad de generar cerdos transgénicos como fuente alternativa a la donación de órganos.

One of the most important advances in biotechnology during the last decades was the development of transgenic animals. In this article, I discuss why transgenic animals are excellentmodels to analyze gen function and regulation, and to look for new therapeutic strategies for human diseases. Moreover, their use as bioreactors to produce pharmaceutical products for the treatment of human diseases, and the possibility of generating transgenic pigs as analternative source of organ donors for humans is also discussed.

?C31 Integrase and Transgenic Animals Research / 中国生物工程杂志

Streptomyces phage ?C31 integrase is a site-specific recombinase and introduce foreign gene to unidirectional recombination.Because of its integration mediated by ?C31 integrase without any cofactors and especially directed integration,it possesses high efficiency and stable expression of foreign gene.These advantages make it an attractive tool for genetic engineering.It has been widely used to transgene integration in mammalians and provides an opportunity to overcome the bottlenecks in the generation of transgenetic animals,such as random integration,low integral rate,low level of expression and so on.The ?C31 integrase will have a wide application in the near future.

Transfection of Gametes: A Method to Generate Transgenic Animals / Transfección de Gametos: Un Método para Generar Animales Transgénicos

The production of transgenic animals (TA) using transfected spermatozoa or eggs is commented. Different methods have been employed to introduce transgenes into the gametes of several vertebrates and invertebrates. Methods for the transfection of gametes have employed naked DNA, viral vectors, DNA/Liposome complexes, electroporation or high velocity microprojectiles. Spermatozoa and oocytes or eggs have showed a good transfection efficience (80% in some cases), and microscopical observations demonstrated that the transgenes appeared localized in the nucleus. Gametes have shown to be naturally protected against the entrance of foreign genes because some semino plasma or plasma membrane proteins block the entrance of foreign genes in spermatozoa. In the female this blockage is undertaken by the egg covers (the zona pellucida in mammals and the perivitelline coat in mollusks). In several cases the production of TA has been described after using the transfected gametes for in vitro fertilization or inseminations. Sometimes, larger percentages of TA were observed (85% in salmon). Nevertheless, these TA were mainly chimeric for the transgene and they were not capable to establish transgenic lines. It seems probable that TA produced by gamete transfections are different from those originated by the conventional microinjection procedures. Furthermore, gametes would develop some kind of mechanisms that modify the integration/expression of transgenes, or that block the integration of transgenes in the germinal line.

Se comentan algunos aspectos sobre la transfección de gametos y su empleo para la producción de animales transgénicos (AT). Para la transfección de gametos han sido empleados ADN desnudo, vectores virales, complejos ADN-Liposomas, electroporación, o microproyectiles de alta velocidad. En general, se han obtenido altos porcentajes de transfección (hasta del 80% en espermatozoides) y se ha observado que los transgenes se localizan en el interior del núcleo. Se ha constatado que los gametos están naturalmente protegidos frente a la entrada de genes extraños: en espermatozoides esta función la cumplen diversas proteínas presentes en los fluidos seminales o en su membrana plasmática; mientras que en los gametos femeninos son las envolturas del huevo (zona pelúcida en mamíferos o membrana perivitelina en moluscos) las que desarrollan esta labor. En muchos casos, los gametos transfectados se han utilizado en fecundaciones in vitro o en inseminaciones a fin de crear AT. En algunos casos, los porcentajes de estos AT han sido altos, como en el salmón (85%). Pero los AT, así creados, han sido en su mayoría quimeras y no han sido capaces de producir líneas transgénicas. Se sugiere que los AT producidos por gametos transfectados, son diferentes a los producidos por el método convencional de microinyección. Es probable que los gametos posean mecanismos, aún no descritos, capaces de modificar la integración/expresión de los transgenes, o que impedirían la integración de los transgenes en la línea germinal.