High-copy expression vector based on amplification-promoting sequences

Area of Science:

• Molecular Biology
• Cell Biology
• Biotechnology

Background:

• Efficient expression of heterologous proteins is crucial for research and biotechnology.
• Existing vector systems may have limitations in achieving high protein yields.
• Mouse L fibroblasts are a common cell line for protein expression studies.

Purpose of the Study:

• To develop a new vector system for enhanced heterologous protein expression in mouse L fibroblasts.
• To investigate the role of a specific DNA element in promoting high vector copy numbers.
• To assess the impact of high copy numbers on reporter gene expression levels.

Main Methods:

• Construction of a novel vector system incorporating a 370-bp amplification promoting element (muNTS1) from murine rDNA.
• Transfection of mouse L fibroblasts with the developed vector.
• Quantification of vector DNA copy number using established molecular biology techniques.
• Measurement of secreted alkaline phosphatase (SEAP) reporter gene expression levels.
• Analysis of the structural organization of integrated plasmid DNA in host cell chromosomes.

Main Results:

• The muNTS1 element mediated a 40- to 800-fold amplification of vector DNA in transfected L cells.
• High vector DNA copy numbers correlated with significantly increased expression of the SEAP reporter gene.
• Analysis revealed that multicopy plasmid DNA integrated into chromosomal DNA as reiterated head-to-tail concatamers.

Conclusions:

• The novel vector system enables efficient heterologous protein expression in mouse L fibroblasts.
• The muNTS1 element is effective in achieving high vector DNA persistence and copy numbers.
• This versatile system overcomes limitations in enzyme-deficient cell lines for protein production.