Comparison of IRES and F2A-based locus-specific multicistronic expression in stable mouse lines

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Co-expression strategies are crucial for gene therapy, vaccine development, and basic biological research, enabling simultaneous expression of multiple genes from a single vector.
• Internal ribosome entry site (IRES) and 2A peptide (F2A) are popular methods for achieving stoichiometric gene expression.
• Understanding the efficiency and limitations of these strategies is vital for optimizing gene delivery and expression systems.

Purpose of the Study:

• To compare the in vivo effectiveness of IRES and F2A co-expression strategies.
• To analyze the impact of reporter genes, specifically enhanced green fluorescent protein (EGFP), on F2A-mediated gene expression.
• To evaluate locus-dependency and potential inhibitory effects on the F2A 'skipping' mechanism.

Main Methods:

• Generation and analysis of locus-specific knock-in mouse lines: Sox9(IRES-EGFP) and Sox9(F2A-EGFP).
• Further analysis of FLAG(3)-Bapx1(F2A-EGFP) and Bapx1(F2A-Cre-F2A-EGFP) mouse lines to assess locus-dependency and reporter gene effects.
• Quantitative assessment of protein expression levels and fusion protein formation.

Main Results:

• Both IRES and F2A constructs successfully expressed Sox9 and EGFP in appropriate domains.
• F2A strategy resulted in higher EGFP levels compared to IRES, but showed ~42.2% Sox9-EGFP fusion protein, indicating inefficient 'skipping'.
• EGFP significantly inhibited F2A 'skipping' efficiency when placed downstream of Cre recombinase in the Bapx1(F2A-Cre-F2A-EGFP) line.

Conclusions:

• The F2A peptide enables efficient and near-stoichiometric gene expression in stable mouse lines.
• Enhanced green fluorescent protein (EGFP) can undesirably inhibit the F2A 'skipping' mechanism.
• Alternative reporter genes should be considered when using F2A peptides to ensure optimal gene expression.