RESUMEN

Entamoeba histolytica genetic organization and genome structure is complex and under intense research. The genome is fully sequenced, and several tools have been developed for the molecular study of this organism. Nevertheless, good protein tracking tags that are easy to measure and image, like the fluorescent proteins are lacking. In this report, we codon-optimized the red fluorescent protein from the coral Discosoma striata (DsRFP) for its use in E. histolytica and demonstrated functionality in vivo. We envision that this protein can be widely used for the development of transcriptional reporter systems and protein-tagging applications.

Asunto(s)

Entamoeba histolytica/metabolismo , Sustancias Luminiscentes/metabolismo , Proteínas Luminiscentes/metabolismo , Animales , Antozoos/química , Clonación Molecular , Codón/fisiología , Entamoeba histolytica/genética , Entamoeba histolytica/patogenicidad , Citometría de Flujo , Expresión Génica , Proteínas Luminiscentes/genética , Microscopía Confocal , Plásmidos/genética , Plásmidos/metabolismo , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , Esfingomielina Fosfodiesterasa/genética , Virulencia , Proteína Fluorescente Roja

RESUMEN

ABSTRACT Lentiviral vector-mediated gene transfer offers several advantages over other gene delivery vectors when considering gene and cell therapy applications. However, using these therapies in clinical applications involves large-scale vector production in an efficient and cost-effective manner. Here we describe a high yield production of a lentivirus encoding recombinant factor VIII in a scalable and GMP-compliant culture system, based on serum free suspension cultures and transient transfection with an inexpensive reagent, polyethylenimine (PEI), reaching a total viral yield of 2.48x108 particles.