title: A herpes simplex virus vector system for expression of complex cellular cDNA libraries.
abstract: Viral vector-based gene expression libraries from normal or diseased tissues offer opportunities to interrogate cellular functions that influence or participate directly in specific biological processes. Here we report the creation and characterization of a herpes simplex virus (HSV)-based expression library consisting of cDNAs derived from PC12 pheochromocytoma cells. A replication-defective HSV vector backbone was engineered to contain both a bacterial artificial chromosome (BAC) and the Invitrogen in vitro Gateway recombination system, creating DBAC-GW. A cDNA library was produced and transferred into the DBAC-GW genome by in vitro recombination and selection in bacteria to produce DBAC-L. DBAC-L contained at least 15,000 unique cDNAs, as shown by DNA array analysis of PCR-amplified cDNA inserts, representing a wide range of cancer- and neuron-related cellular functions. Transfection of the recombinant DBAC-L DNA into complementing animal cells produced more than 1 million DBAC-L virus particles representing the library genes. By microarray analysis of vector-infected cells, we observed that individual members of this vector population expressed unique PC12 cDNA-derived mRNA, demonstrating the power of this system to transfer and express a variety of gene activities. We discuss the potential utility of this and similarly derived expression libraries for genome-wide approaches to identify cellular functions that participate in complex host-pathogen interactions or processes related to disease and to cell growth and development.
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, 428 Bridgeside Point 2, 450 Technology Drive, Pittsburgh, PA 15219, USA.
authors.forename: Darren
authors.initials: D
authors.lastname: Wolfe
authors.forename: April M
authors.initials: AM
authors.lastname: Craft
authors.forename: Justus B
authors.initials: JB
authors.lastname: Cohen
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Glorioso
mesh_terms: D000818:Animals; D015870:Gene Expression; D015723:Gene Library; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D016716:PC12 Cells; D051381:Rats; D018139:Simplexvirus / Q000235:genetics*
references.citation: Mol Cell Biochem. 2008 Dec;319(1-2):181-7
references.pmid: 18709335
references.citation: Proc Natl Acad Sci U S A. 2009 Jun 2;106(22):9039-44
references.pmid: 19458262
references.citation: Proc Natl Acad Sci U S A. 2006 Apr 4;103(14):5508-13
references.pmid: 16554374
references.citation: Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11319-20
references.pmid: 8876133
references.citation: Nat Methods. 2006 Feb;3(2):117-22
references.pmid: 16432521
references.citation: Mol Ther. 2003 Oct;8(4):530-42
references.pmid: 14529825
references.citation: J Virol. 2006 Jun;80(11):5435-50
references.pmid: 16699024
references.citation: Exp Neurol. 1999 Sep;159(1):34-46
references.pmid: 10486173
references.citation: J Virol. 1994 Sep;68(9):5523-31
references.pmid: 8057434
references.citation: Gene Ther. 1998 Nov;5(11):1517-30
references.pmid: 9930305
references.citation: Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9146-50
references.pmid: 7568090
references.citation: Nucleic Acids Res. 2002 Dec 15;30(24):e142
references.pmid: 12490733
references.citation: J Virol. 1993 Dec;67(12):7501-12
references.pmid: 8230470
references.citation: Nat Methods. 2007 Sep;4(9):733-9
references.pmid: 17676048
references.citation: Gene Ther. 1998 Dec;5(12):1593-603
references.pmid: 10023438
references.citation: J Virol. 2004 Oct;78(20):11352-9
references.pmid: 15452256
references.citation: Exp Neurol. 1997 Mar;144(1):103-21
references.pmid: 9126159
references.citation: J Virol. 2000 Nov;74(21):10132-41
references.pmid: 11024142
references.citation: J Virol. 1999 Jan;73(1):519-32
references.pmid: 9847358
references.citation: J Virol. 1985 Nov;56(2):558-70
references.pmid: 2997476
references.citation: Stem Cells. 2008 Dec;26(12):3119-29
references.pmid: 18787207
references.citation: J Virol. 1989 Jan;63(1):18-27
references.pmid: 2535723
references.citation: Eur J Neurosci. 2008 Oct;28(7):1241-54
references.pmid: 18973552