An efficient method for high-fidelity BAC/PAC retrofitting with a selectable marker for mammalian cell transfection

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Large-scale genomic sequencing yields vast gene data, necessitating functional studies for biological understanding.
• Bacterial artificial chromosomes (BACs) and P1-derived artificial chromosomes (PACs) are crucial for functional genomics due to their large insert capacity and stability.
• Transfecting mammalian cells with BAC/PAC clones requires a mammalian selectable marker, often absent in standard vectors.

Purpose of the Study:

• To develop a highly efficient and reliable method for retrofitting BACs and PACs with mammalian selectable markers.
• To overcome limitations of existing procedures regarding efficiency and fidelity in marker insertion.

Main Methods:

• A novel BAC/PAC retrofitting vector was designed.
• The vector utilizes in vivo cre/loxP site-specific recombination for targeted insertion into BAC/PAC vectors.
• Transformation into competent bacterial strains carrying BACs or PACs facilitates the retrofitting process.

Main Results:

• The procedure enables the retrofitting of dozens of BACs/PACs within a single day.
• No detectable deletions or unwanted recombination were observed in the modified BAC/PAC vectors.
• The method demonstrates high efficiency and fidelity in inserting mammalian selectable markers.

Conclusions:

• This simple and efficient retrofitting procedure significantly advances functional genomic studies using large insert clones.
• The cre/loxP-based system provides a robust solution for preparing BACs and PACs for mammalian cell transfection.
• The technique facilitates high-throughput functional analysis of genes within their native genomic context.