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

Nature Biotechnology volume 19, pages 1067–1070 (2001)Cite this article

Abstract

The recent completion of the human genome sequence1 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.

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Figure 1: The high-capacity HSV-1 amplicon system delivers intact genomic DNA inserts of >100 kilobases.
Figure 2: The 115-kilobase HPRT genomic DNA locus present in pHSV-HPRT (pHSV-143) is intact within the virion and functional following infectious delivery.
Figure 3: Analysis of three clonal cell lines carrying episomal pHSV-HPRT following infection and selection for two months of continuous culture.
Figure 4: Infection and transfection of primary mouse hepatocyte cultures by pHSV-HPRT.

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Acknowledgements

This work was supported by NIH Grant (CA692460) and a Wellcome Trust grant to E.A.C. R.W-M. is a Wellcome Trust International Prize Travelling Research Fellow and E.R.S. is a Pete Gilmore/American Brain Tumour Association Fellow. We would like to thank Dr. John Mullen for his help in establishing hepatocyte cultures, and Drs. Cornel Fraefel, Xandra Breakefield, Michael James, Peter Cook, and Hiroshi Kimura for helpful discussion and advice.

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  1. Molecular Neuro-Oncology Laboratories, Neurosurgery Service, Massachusetts General Hospital-East & Harvard Medical School, Building 149, 13th Street, Charlestown, 02129, MA

    Richard Wade-Martins, Edward R. Smith, Edyta Tyminski, E. Antonio Chiocca & Yoshinaga Saeki

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  1. Richard Wade-Martins
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Correspondence to E. Antonio Chiocca.

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Wade-Martins, R., Smith, E., Tyminski, E. et al. An infectious transfer and expression system for genomic DNA loci in human and mouse cells. Nat Biotechnol 19, 1067–1070 (2001). https://doi.org/10.1038/nbt1101-1067

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