Insert sequence length determines transfection efficiency and gene expression levels in bicistronic mammalian...

Area of Science:

• Molecular Biology
• Gene Expression
• Biotechnology

Background:

• Bicistronic expression vectors utilize internal ribosome entry site (IRES) sequences for co-expression.
• IRES enables cap-independent translation initiation for multiple genes on a single mRNA.

Purpose of the Study:

• Investigate the impact of 5' gene insert length on IRES-mediated expression.
• Evaluate the influence of gene insert length on transfection efficiency.
• Assess the effect of co-expressed gene levels on each other.

Main Methods:

• Utilized a commercial mammalian bicistronic expression vector with an IRES and a green fluorescent protein marker.
• Constructed a novel bicistronic vector with two distinct fluorescent markers.
• Analyzed the relationship between 5' gene insert length and 3' marker expression/transfection efficiency.

Main Results:

• The length of the gene of interest upstream of the IRES significantly impacts 3' fluorescent marker expression and overall transfection efficiency.
• In a dual-marker system, high expression of one gene can inversely affect the expression of the other.
• Short 5' gene sequences (<300 bp) require particular attention.

Conclusions:

• Caution is advised when designing IRES-based experiments, especially with short 5' gene inserts.
• Cell selection based solely on a single 3' marker's expression profile may be unreliable.
• Assumptions in data analysis for IRES systems need critical evaluation.