An infectious transfer and expression system for genomic DNA loci in humanand mouse cells

The recent completion of the human genome sequence(1) allows genomics resea rch to focus on understanding gene complexity, expression, and regulation. However, the routine-use genomic DNA expression systems required to investi gate these phenomena are not well developed. Bacterial artificial chromosom es (BACs) and P1-based artificial chromosomes (PACs) have proved excellent tools for the human genome sequencing projects. We describe a system to rap idly and efficiently deliver and express BAC and PAC library clones in huma n and mouse cells by converting them into infectious amplicon vectors. We s how packaging and intact delivery of genomic inserts of > 100 kilobases wit h efficiencies of up to 100%. To demonstrate that genomic loci transferred in this way are functional, the complete human hypoxanthine phosphoribosylt ransferase (HPRT) locus contained within a 115-kilobase BAC insert was show n to be expressed when delivered by infection into both a human HPRT-defici ent fibroblast cell line and a mouse primary hepatocyte culture derived fro m Hprt(-/-) mice. Efficient gene delivery to primary cells is especially im portant, as these cells cannot be expanded using antibiotic selection. This work is the first demonstration of infectious delivery and expression of g enomic DNA sequences of > 100 kilobases, a technique that may prove useful for analyzing gene expression from the human genome.