DNA modification and functional delivery into human cells using Escherichia coli DH10B

Area of Science:

• Genomics
• Molecular Biology
• Cell Biology

Background:

• Large human DNA clones in Bacterial Artificial Chromosome (BAC) libraries are valuable for functional genomics.
• Existing methods for modifying and transferring these large DNA fragments into mammalian cells are limited.

Purpose of the Study:

• To develop a novel Escherichia coli-based vector system for efficient modification, propagation, and delivery of large human BAC clones into mammalian cells.
• To enhance the utility of BAC libraries for gene expression and functional genomic studies.

Main Methods:

• Utilized the GET recombination system for homologous recombination in E. coli DH10B to insert an EGFPneo cassette into a ~200 kb human BAC clone.
• Engineered E. coli DH10B by deleting the asd gene for auxotrophy and introducing the Yersinia pseudotuberculosis invasin gene to facilitate mammalian cell invasion.
• Optimized conditions for DNA transfer and cell survival, including incubation time and multiplicity of infection.

Main Results:

• Successfully converted BAC clones into suitable vectors for mammalian cells using the E. coli-based system.
• Demonstrated targeted deletion of the asd gene, creating auxotrophic E. coli strains.
• Showcased the ability of engineered E. coli to invade HeLa cells and deliver DNA, evidenced by GFP expression and neomycin resistance.

Conclusions:

• The developed E. coli-based vector system, combining homologous recombination and invasion technologies, significantly improves the delivery of large genomic BAC clones into mammalian cells.
• This approach facilitates gene expression and functional genomic studies using BAC libraries from human and other genomes.