Epstein-Barr virus shuttle vector for stable episomal replication of cDNA expression libraries in human cells

Area of Science:

• Molecular Biology
• Cell Biology
• Genetics

Background:

• Efficiently introducing and expressing cDNA libraries in human cells is crucial for gene discovery.
• Existing methods may have limitations in efficiency and recovery of rare clones.

Purpose of the Study:

• To develop and validate an Epstein-Barr virus-based subcloning vector (EBO-pcD) for efficient cDNA library transfection and expression in human cells.
• To enable stable, extrachromosomal maintenance of plasmids and high-efficiency recovery of expressed genes.

Main Methods:

• Utilized an Epstein-Barr virus-based subcloning vector (EBO-pcD) containing a hygromycin B resistance marker, oriP, and EBNA gene.
• Transformed human lymphoblastoid cells with EBO-pcD expression libraries.
• Isolated and recovered intact cDNA clones from transformed cells via propagation in Escherichia coli.

Main Results:

• Achieved high-efficiency (10-15%) stable transformation of human lymphoblastoid cells with EBO-pcD plasmids.
• Demonstrated stable, extrachromosomal episomal maintenance of plasmids (2-8 copies/cell).
• Successfully recovered intact cDNA clones, including rare ones, from transformed cells.

Conclusions:

• EBO-pcD vectors facilitate efficient transfection, stable expression, and recovery of cDNA libraries in human cells.
• This system is particularly valuable for cloning genes where traditional screening methods are not applicable.
• Enables direct selection and recovery of rare gene expression events in human cell lines.