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

Abstract

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.

Constitutive and Inducible Innate Responses in Cells Infected by HSV-1- Derived Amplicon Vectors

“…Alternatively, or in addition, it would be certainly possible to engineer the amplicon genome in order to express functions potentially able to inhibit cellular-induced gene silencing without toxicity for the transduced cells, or to include tissue-specific regulatory DNA elements, thereby forcing the amplicon genome to generate a transcriptionally active chromatin configuration, thus allowing stable expression. Previous observations using tissue-specific promoters or enhancers actually support the great interest of this approach [27-30]. …”

“…As it carries no virus genes, this plasmid requires the presence of a helper virus genome to express the proteins necessary for its amplification and packaging into HSV-1 particles. Over the last decade, technological improvements have enabled the production of relatively large amounts of amplicon stocks that are, not or only, minimally contaminated with helper virus particles [23-26], and to use these vectors to deliver long genomic sequences to target cells [27-30]. These major breakthroughs have made it possible to study important aspects of the biological features of amplicons in the absence of helper HSV-1.…”

Infectious delivery of the 132 kb CDKN2A/CDKN2B genomic DNA region results in correctly spliced gene expression and growth suppression in glioma cells

“…The iBAC takes advantage of the packaging capacity of herpes simplex virus type 1 (approximately 150-160 kb) 10,11 and of recent advances in packaging of foreign DNA into the virus in a 'gutless' manner without helper virus contamination. 12 Genomic sequences delivered into cells by the iBAC persist as extrachromosomal elements.…”

“…12 Genomic sequences delivered into cells by the iBAC persist as extrachromosomal elements. 10,11 As such, they can be easily recovered and reanalyzed and/or re-engineered in bacteria, thus allowing for potentially exquisite experimental manipulations. As proof-of-principle of the iBAC technology, we have previously shown that the genomic locus for human hypoxanthine phosphoribosyltransferase (HPRT) contained within a 115 kb BAC insert could be efficiently expressed in primary cells.…”

“…As proof-of-principle of the iBAC technology, we have previously shown that the genomic locus for human hypoxanthine phosphoribosyltransferase (HPRT) contained within a 115 kb BAC insert could be efficiently expressed in primary cells. 10 We have further shown that transfer and expression of the human low-density lipoprotein receptor (LDLR) gene contained within a 135 kb BAC insert resulted in the expected and correct regulation of the LDLR gene promoter by sterols in primary human fibroblasts taken from familial hypercholesterolemia (FH) patients. 11 This result thus showed that iBAC-delivered genes retained physiological regulation of gene expression, even in their extrachromosomal environment.…”