An Internal Ribosome Entry Site (IRES) Mutant Library for Tuning Expression Level of Multiple Genes in Mammalian Cells

Esther Y C Koh,Mariati Mariati,Zhiwei Song,Steven C L Ho,Xuezhi Bi,Yuansheng Yang,Muriel Bardor,Linda M Hendershot

doi:10.1371/journal.pone.0082100

Abstract

A set of mutated Encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) elements with varying strengths is generated by mutating the translation initiation codons of 10th, 11th, and 12th AUG to non-AUG triplets. They are able to control the relative expression of multiple genes over a wide range in mammalian cells in both transient and stable transfections. The relative strength of each IRES mutant remains similar in different mammalian cell lines and is not gene specific. The expressed proteins have correct molecular weights. Optimization of light chain over heavy chain expression by these IRES mutants enhances monoclonal antibody expression level and quality in stable transfections. Uses of this set of IRES mutants can be extended to other applications such as synthetic biology, investigating interactions between proteins and its complexes, cell engineering, multi-subunit protein production, gene therapy, and reprogramming of somatic cells into stem cells.

Highlights

Simultaneous expression of multiple genes in mammalian cells at finely controlled amounts or ratios is required for applications such as synthetic biology, investigating interactions between proteins and its complexes, cell engineering, multi-subunit protein production, gene therapy, and reprogramming of somatic cells into stem cells [1,2,3,4]
renilla luciferase (Rluc) was used as an internal standard to normalize the transfection efficiency for accurate determination of firefly luciferase (Fluc) expression to reflect the strength of internal ribosome entry site (IRES) variants
Rluc reading variations should be due to variations in transfection efficiency and Fluc reading variations could be due to both varied transfection efficiency and different strengths of IRES variants

Summary

Introduction

Simultaneous expression of multiple genes in mammalian cells at finely controlled amounts or ratios is required for applications such as synthetic biology, investigating interactions between proteins and its complexes, cell engineering, multi-subunit protein production, gene therapy, and reprogramming of somatic cells into stem cells [1,2,3,4]. Three common strategies for controlling multiple gene expression in mammalian cells are (i) co-transfection of multiple vectors at different relative amounts [5,6,7], (ii) single vector containing multiple promoters or polyadenylation signals with different strengths [8,9,10], and (iii) insertion of splicing signals with varied splicing efficiencies between genes [11]. The use of splicing signals allows stricter control of relative gene expression as all genes are expressed in one transcript [11]. This method requires elimination of cryptic splicing sites in protein coding sequences to prevent incorrect RNA splicing

Methods

Results

Conclusion