title: A multipurpose vector system for the screening of libraries in bacteria, insect and mammalian cells and expression in vivo.
abstract: We have constructed a novel tetra-promoter vector (pBVboostFG) system that enables screening of gene/cDNA libraries for functional genomic studies. The vector enables an all-in-one strategy for gene expression in mammalian, bacterial and insect cells and is also suitable for direct use in vivo. Virus preparation is based on an improved mini Tn7 transpositional system allowing easy and fast production of recombinant baculoviruses with high diversity and negligible background. Cloning of the desired DNA fragments or libraries is based on the recombination system of bacteriophage lambda. As an example of the utility of the vector, genes or cDNAs of 18 different proteins were cloned into pBVboostFG and expressed in different hosts. As a proof-of-principle of using the vector for library screening, a chromophoric Thr65-Tyr-Gly67-stretch of enhanced green fluorescent protein was destroyed and subsequently restored by novel PCR strategy and library screening. The pBVboostFG enables screening of genome-wide libraries, thus making it an efficient new platform technology for functional genomics.
authors.affiliation: Department of Molecular Medicine and Biotechnology, A.I. Virtanen Institute, University of Kuopio PO Box 1627 FIN-70211, Kuopio, Finland.
authors.forename: Olli H
authors.initials: OH
authors.lastname: Laitinen
authors.forename: Kari J
authors.initials: KJ
authors.lastname: Airenne
authors.forename: Vesa P
authors.initials: VP
authors.lastname: Hytönen
authors.forename: Erik
authors.initials: E
authors.lastname: Peltomaa
authors.forename: Anssi J
authors.initials: AJ
authors.lastname: Mähönen
authors.forename: Thomas
authors.initials: T
authors.lastname: Wirth
authors.forename: Miia M
authors.initials: MM
authors.lastname: Lind
authors.forename: Kari A
authors.initials: KA
authors.lastname: Mäkelä
authors.forename: Pyry I
authors.initials: PI
authors.lastname: Toivanen
authors.forename: Diana
authors.initials: D
authors.lastname: Schenkwein
authors.forename: Tommi
authors.initials: T
authors.lastname: Heikura
authors.forename: Henri R
authors.initials: HR
authors.lastname: Nordlund
authors.forename: Markku S
authors.initials: MS
authors.lastname: Kulomaa
authors.forename: Seppo
authors.initials: S
authors.lastname: Ylä-Herttuala
mesh_terms: D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D016367:Baculoviridae / Q000235:genetics*; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004926:Escherichia coli / Q000235:genetics*; D005260:Female; D015870:Gene Expression; D015723:Gene Library; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051381:Rats; D017208:Rats, Wistar; D018411:Spodoptera / Q000166:cytology / Q000235:genetics*
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
references.citation: Nucleic Acids Res. 1998 Apr 1;26(7):1718-23
references.pmid: 9512544
references.citation: Am J Pharmacogenomics. 2001;1(1):29-35
references.pmid: 12173311
references.citation: Curr Opin Chem Biol. 2004 Feb;8(1):98-102
references.pmid: 15036163
references.citation: Mol Ther. 2003 Nov;8(5):853-62
references.pmid: 14599820
references.citation: Appl Environ Microbiol. 1983 Mar;45(3):1085-93
references.pmid: 16346229
references.citation: J Virol. 2003 Sep;77(18):9799-808
references.pmid: 12941888
references.citation: Biochem J. 2002 May 1;363(Pt 3):609-17
references.pmid: 11964162
references.citation: Trends Biotechnol. 2003 Sep;21(9):383-8
references.pmid: 12948670
references.citation: J Biol Chem. 2005 Apr 8;280(14):13250-5
references.pmid: 15695809
references.citation: Curr Opin Chem Biol. 2004 Feb;8(1):20-5
references.pmid: 15036152
references.citation: Nucleic Acids Res. 2001 Feb 15;29(4):E22
references.pmid: 11160942
references.citation: Gene Ther. 2001 Jun;8(11):846-54
references.pmid: 11423932
references.citation: J Biol Chem. 2001 Mar 16;276(11):8219-24
references.pmid: 11076945
references.citation: Gene Ther. 2002 Dec;9(24):1693-9
references.pmid: 12457283
references.citation: Adv Biochem Eng Biotechnol. 2002;77:21-42
references.pmid: 12227735
references.citation: Genome Res. 2000 Nov;10(11):1788-95
references.pmid: 11076863
references.citation: Biotechniques. 1990 Apr;8(4):404-7
references.pmid: 2340178
references.citation: Nat Biotechnol. 2001 May;19(5):451-5
references.pmid: 11329015
references.citation: Biochem J. 2004 Dec 1;384(Pt 2):385-90
references.pmid: 15324300
references.citation: Gene. 1996;173(1 Spec No):33-8
references.pmid: 8707053
references.citation: Nat Rev Mol Cell Biol. 2001 Nov;2(11):806-14
references.pmid: 11715047
references.citation: FEBS Lett. 2003 Dec 18;555(3):449-54
references.pmid: 14675754
references.citation: J Biol Chem. 2004 Aug 27;279(35):36715-9
references.pmid: 15131113
references.citation: Nucleic Acids Res. 1987 Apr 24;15(8):3595-606
references.pmid: 3575102
references.citation: Gene. 1992 Jul 1;116(1):115-6
references.pmid: 1628838
references.citation: Mol Ther. 2004 Feb;9(2):282-91
references.pmid: 14759812
references.citation: Gene Ther. 2000 Sep;7(17):1499-504
references.pmid: 11001370
references.citation: J Immunol Methods. 1997 Apr 25;203(2):131-9
references.pmid: 9149806
references.citation: Circ Res. 2003 May 30;92(10):1098-106
references.pmid: 12714562
references.citation: Nucleic Acids Res. 2003 Sep 1;31(17):e101
references.pmid: 12930975
references.citation: J Neuropathol Exp Neurol. 2000 Jul;59(7):561-74
references.pmid: 10901227
references.citation: Methods Enzymol. 1990;184:51-67
references.pmid: 2388586
references.citation: Trends Biotechnol. 2004 Jul;22(7):346-53
references.pmid: 15245907
references.citation: Trends Biotechnol. 2002 Apr;20(4):173-80
references.pmid: 11906750
references.citation: Brief Bioinform. 2001 Dec;2(4):363-74
references.pmid: 11808748
references.citation: Adv Biochem Eng Biotechnol. 2002;77:103-12
references.pmid: 12227733
references.citation: Protein Expr Purif. 2000 Nov;20(2):252-64
references.pmid: 11049749
references.citation: Nat Biotechnol. 2002 Nov;20(11):1154-7
references.pmid: 12355097
references.citation: FASEB J. 1999 Jan;13(1):9-22
references.pmid: 9872925
references.citation: J Gen Virol. 2003 May;84(Pt 5):1173-8
references.pmid: 12692282
references.citation: Nat Biotechnol. 2001 Sep;19(9):851-5
references.pmid: 11533644
references.citation: Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7303-7
references.pmid: 1871136