Transient gene expression levels from multigene expression vectors

Area of Science:

• Molecular Biology
• Gene Expression Systems

Background:

• Multigene expression vectors are widely used for simultaneous, equivalent gene (over)expression in eukaryotic cells.
• Optimizing coordinated expression of multiple genes requires robust vector design and characterization.

Purpose of the Study:

• To characterize dual-gene expression plasmids with a c-myc Internal Ribosome Entry Site (IRES) for coordinated gene expression.
• To evaluate the expression levels and limitations of multigene expression systems in Chinese Hamster Ovary (CHO) cells.

Main Methods:

• Construction and testing of dual-gene and pEE14.4 RlucMFluc expression plasmids in CHO cells.
• Utilized renilla and firefly luciferase reporter genes to assess expression from different positions within the vectors.
• Quantified reporter gene mRNA levels using qRT-PCR to correlate with protein expression.

Main Results:

• Dual-gene constructs showed enhanced expression of the second gene (4- to 50-fold increase).
• The pEE14.4 RlucMFluc plasmid exhibited enhanced first cistron expression (approx. 19-fold increase).
• Transcriptional limitation, not translational, was identified as the primary factor affecting first cistron expression.

Conclusions:

• Relative protein expression from multigene vectors cannot be solely predicted by copy number.
• Characterization of multigene or oligocistronic expression systems is essential before application.
• Co-transfection of single-gene plasmids can achieve more equivalent transient expression levels.