A versatile system for USER cloning-based assembly of expression vectors for mammalian cell engineering

Area of Science:

• Molecular Biology
• Biotechnology
• Mammalian Cell Engineering

Background:

• Developing versatile mammalian expression vectors is crucial for protein production, cell biology, and engineering cell factories.
• Existing methods for constructing complex expression vectors can be time-consuming and lack flexibility.

Purpose of the Study:

• To develop a versatile mammalian vector system for efficient protein production, cell biology analyses, and cell factory engineering.
• To enable rapid construction of mammalian expression vectors with multiple DNA fragments and high flexibility.

Main Methods:

• Utilized ligation-free uracil-excision based USER cloning for vector construction.
• Developed a toolbox of DNA building blocks with Flexible Assembly Sequence Tags (FASTs) for modular assembly.
• Assembled up to seven DNA fragments in a single cloning step with high efficiency (>90%).

Main Results:

• Successfully constructed a versatile mammalian vector system with basic vectors and a comprehensive toolbox.
• Demonstrated rapid swapping of gene, promoter, or selection markers using USER cloning with FASTs.
• Validated vector functionality via transient expression of fluorescent proteins in CHO, U-2-OS, and HEK293 cell lines.

Conclusions:

• The developed mammalian vector system offers a flexible and efficient platform for gene expression studies.
• This system facilitates high-throughput genome-scale studies and customizable gene expression vector assembly.
• The platform is extendable, supporting broad applications in mammalian cell research and biotechnology.