Progress and biotechnological prospects in fish transgenesis.

The history of transgenesis is marked by milestones such as the development of cellular transdifferentiation, recombinant DNA, genetic modification of target cells, and finally, the generation of simpler genetically modified organisms (e.g. bacteria and mice). The first transgenic fish was developed in 1984, and since then, continuing technological advancements to improve gene transfer have led to more rapid, accurate, and efficient generation of transgenic animals. Among the established methods are microinjection, electroporation, lipofection, viral vectors, and gene targeting. Here, we review the history of animal transgenesis, with an emphasis on fish, in conjunction with major developments in genetic engineering over the past few decades. Importantly, spermatogonial stem cell modification and transplantation are two common techniques capable of revolutionizing the generation of transgenic fish. Furthermore, we discuss recent progress and future biotechnological prospects of fish transgenesis, which has strong applications for the aquaculture industry. Indeed, some transgenic fish are already available in the current market, validating continued efforts to improve economically important species with biotechnological advancements.

The transgenic animal platform for biopharmaceutical production.

The recombinant production of therapeutic proteins for human diseases is currently the largest source of innovation in the pharmaceutical industry. The market growth has been the driving force on efforts for the development of new therapeutic proteins, in which transgenesis emerges as key component. The use of the transgenic animal platform offers attractive possibilities, residing on the low production costs allied to high productivity and quality of the recombinant proteins. Although many strategies have evolved over the past decades for the generation of transgenic founders, transgenesis in livestock animals generally faces some challenges, mainly due to random transgene integration and control over transgene copy number. But new developments in gene editing with CRISPR/Cas system promises to revolutionize the field for its simplicity and high efficiency. In addition, for the final approval of any given recombinant protein for animal or human use, the production and characterization of bioreactor founders and expression patterns and functionality of the proteins are technical part of the process, which also requires regulatory and administrative decisions, with a large emphasis on biosafety. The approval of two mammary gland-derived recombinant proteins for commercial and clinical use has boosted the interest for more efficient, safer and economic ways to generate transgenic founders to meet the increasing demand for biomedical proteins worldwide.

Emerging RNA-based drugs: siRNAs, microRNAs and derivates.

An emerging new category of therapeutic agents based on ribonucleic acid has emerged and shown very promising in vitro, animal and pre-clinical results, known as small interfering RNAs (siRNAs), microRNAs mimics (miRNA mimics) and their derivates. siRNAs are small RNA molecules that promote potent and specific silencing of mutant, exogenous or aberrant genes through a mechanism known as RNA interference. These agents have called special attention to medicine since they have been used to experimentally treat a series of neurological conditions with distinct etiologies such as prion, viral, bacterial, fungal, genetic disorders and others. siRNAs have also been tested in other scenarios such as: control of anxiety, alcohol consumption, drug-receptor blockage and inhibition of pain signaling. Although in a much earlier stage, miRNAs mimics, anti-miRs and small activating RNAs (saRNAs) also promise novel therapeutic approaches to control gene expression. In this review we intend to introduce clinicians and medical researchers to the most recent advances in the world of siRNA- and miRNA-mediated gene control, its history, applications in cells, animals and humans, delivery methods (an yet unsolved hurdle), current status and possible applications in future clinical practice.

The state of the art of adeno-associated virus-based vectors in gene therapy.

The adeno-associated virus (AAV) has rapidly gained popularity in gene therapy since the establishment of the first AAV2 infectious clone, in 1982, due to some of their distinguishing characteristics such as lack of pathogenicity, wide range of infectivity, and ability to establish long-term transgene expression. Notably over the past decade, this virus has attracted considerable interest as a gene therapy vector, and about 85% of the currently available 2,041 PubMed references on adeno-associated viruses have been published during this time. The exponential progress of AAV-based vectors has been made possible by the advances in the knowledge of the virology and biology of this virus, which allows great improvement in AAV vectors construction and a better comprehension of their operation. Moreover, with the recent discovery of novel AAV serotypes, there is virtually one preferred serotype for nearly every organ or tissue to target. Thus, AAV-based vectors have been successfully overcoming the main gene therapy challenges such as transgene maintenance, safety and host immune response, and meeting the desirable vector system features of high level of safety combined with clinical efficacy and versatility in terms of potential applications. Consequently, AAV is increasingly becoming the vector of choice for a wide range of gene therapy approaches. This report will highlight the state of the art of AAV-based vectors studies and the advances on the use of AAV vectors for several gene therapy approaches.

Queratinocitos modificados geneticamente por vectores retrovirales / Genetic modifying Keratinocytes for retroviral vectors

Objetivo: revisar la literatura relacionada con la utilización de queratinocitos como células blanco de transferencia de genes en general y utilizando vectores retrovirales como vehículo de transferencia en particular. Fuente de los datos: se describen resultados obtenidos acerca del tema mencionado, publicados en las principales revistas de investigación en dermatología y terapia génica; además información obtenida de revisiones recientes publicadas por expertos en el área y algunas páginas web. Selección de los datos: se seleccionaron los datos más relevantes en relación con el tema propuesto, de trabajos publicados en los últimos 10 años, en los cuales se abordara experimentalmente el tema. Extracción de los datos: se incluyeron datos obtenidos de algunas páginas web especializadas en el tema de queratinocitos, transferencia de genes y vectores retrovirales (http://info.med.yale.edu/). (www.interscience.wiley.com). entre otros. Adicionalmente, datos de publicaciones y revisiones que tuvieran relevancia en áreas como: principios generales de transferencia de genes, tipos de vectores más utilizados, aplicaciones más importantes de los queratinocitos, importancia, ventajas y desventajas de estas células como células blanco de transferencia. Las bases de datos utilizadas fueron: Entrez Home.http:// www.ncbi.nim.nih.gov/Entrez/. SRS:http://srs.ebi.ac.uk/, DBGET: www.genome.ad.jp/dbget/ dbget2.html/ Síntesis de los datos: las bases de la terapia génica fueron cimentadas a principios de la década de los 60; este progreso científico continuó en los años ochenta con el desarrollo de las técnicas de clonación de genes, los vectores virales, los cultivos celulares y las técnicas de transfección in vitro. La comprensión de los procesos moleculares y celulares de muchas enfermedades, permitió pensar en la transferencia de genes como una herramienta terapéutica para el tratamiento tanto de enfermedades heredadas como adquiridas. Igualmente la transferencia de genes ha hecho posible introducir y expresar material genético exógeno en las células somáticas de mamíferos, proporcionando una poderosa herramienta para el estudio de la función y la regulación genética. Los queratinocitos han sido utilizados como células blanco para transferencia de genes...