An infectious transfer and expression system for genomic DNA loci in human and mouse cells.
The recent completion of the human genome sequence allows genomics research to focus on understanding gene complexity, expression, and regulation. However, the routine-use genomic DNA expression systems required to investigate these phenomena are not well developed. Bacterial artificial chromosomes (BACs) and P1-based artificial chromosomes (PACs) have proved excellent tools for the human genome sequencing projects. We describe a system to rapidly and efficiently deliver and express BAC and PAC library clones in human and mouse cells by converting them into infectious amplicon vectors. We show packaging and intact delivery of genomic inserts of >100 kilobases with efficiencies of up to 100%. To demonstrate that genomic loci transferred in this way are functional, the complete human hypoxanthine phosphoribosyltransferase (HPRT) locus contained within a 115-kilobase BAC insert was shown to be expressed when delivered by infection into both a human HPRT-deficient fibroblast cell line and a mouse primary hepatocyte culture derived from Hprt-/- mice. Efficient gene delivery to primary cells is especially important, as these cells cannot be expanded using antibiotic selection. This work is the first demonstration of infectious delivery and expression of genomic DNA sequences of >100 kilobases, a technique that may prove useful for analyzing gene expression from the human genome.
Wade-Martins
R
R
Molecular Neuro-Oncology Laboratories, Neurosurgery Service, Massachusetts General Hospital-East & Harvard Medical School, Building 149, 13th Street, Charlestown, MA 02129, USA.
Smith
E R
ER
Tyminski
E
E
Chiocca
E A
EA
Saeki
Y
Y
D000818:Animals; D002478:Cells, Cultured; D022202:Chromosomes, Artificial, Bacterial; D027042:Chromosomes, Artificial, P1 Bacteriophage; D002999:Clone Cells; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D016678:Genome; D015894:Genome, Human; D023281:Genomics; D018259:Herpesvirus 1, Human / Q000235:genetics; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D014162:Transfection
D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase