Optimization of transcriptional regulatory elements for constructing plasmid vectors.

Area of Science:

• Molecular Biology
• Gene Therapy Vector Development

Background:

• High-level transgene expression from plasmid vectors is crucial for gene therapy and gene function studies.
• Transcriptional regulatory elements, including promoter/enhancers, introns, and polyadenylation signals, are key to achieving potent gene expression.

Purpose of the Study:

• To investigate the optimal combination of transcriptional regulatory elements for maximizing transgene expression.
• To compare the efficacy of various promoter/enhancer, intron, and polyadenylation signal sequences in vitro and in vivo.

Main Methods:

• Construction and evaluation of a series of plasmids differing in specific regulatory elements.
• Assessment of luciferase transgene expression in cell lines (HeLa, HepG2, ECV304) and mouse models (liver, skeletal muscle).
• Systematic comparison of promoter/enhancer, intron, and polyadenylation signal sequences for their impact on expression levels.

Main Results:

• Hybrid CA promoter/enhancer (CMV enhancer, chicken beta-actin promoter, beta-actin intron) and improved CMV promoter/enhancer (CMV enhancer, intron A) yielded the highest expression levels.
• Polyadenylation signal sequences significantly influenced transgene expression.
• Optimized plasmids pCASL3 and pCMVSL3 demonstrated superior performance.

Conclusions:

• The study provides a systematic reference for selecting effective regulatory elements in plasmid vector construction.
• Findings are applicable to advancing gene therapy, vaccine development, and gene transfer experiments.
• Optimized vector designs can significantly enhance transgene expression efficiency.