Study of the quantitative dinamics of heterologous gene transcription in transfected animals cells for bioprocess optimization

Abstract

The quantitative RT-PCR (qRT-PCR) is an important tool for gene quantification and few studies have focused its use to study, monitor and control of heterologous gene expression dynamics in eukaryotic systems used for recombinant protein production. Most of the studies, in this area, use the qRT-PCR just as an indirect method for evaluating gene expression. Our aim, on the other hand, is to extend the use of qRT-PCR as a tool for a molecular kinetic detailed study of gene transcription dynamics and bioprocess development in view of better productivity.The aim of this work is to analyze by qRT-PCR the expression of the messenger RNA (mRNA) coding for rabies virus glycoprotein (RVGP) using Drosophila melanogaster S2 cells stably transfected and by BHK-21 cells infected with a recombinant Semliki Forest Virus (SFV) carrying the RVGP mRNA.So, the main purpose of this project is to use the qRT-PCR together with ELISA and flow cytometry for: 1. kinetic studies of mRNA synthesis in several conditions of bioprocess in transfected or infected cells looking for an optimization of recombinant protein synthesis bioprocess in different metabolic conditions, establishing parameters for a bioprocess with higher and better production; 2. Standardization of an original methodology for recombinant no infectious viral particles (rSFV) titration which are today important candidates to viral vaccines. (AU)