title: Expression-independent gene trap vectors for random and targeted mutagenesis in embryonic stem cells.
abstract: Promoterless gene trap vectors have been widely used for high-efficiency gene targeting and random mutagenesis in embryonic stem (ES) cells. Unfortunately, such vectors are only effective for genes expressed in ES cells and this has prompted the development of expression-independent vectors. These polyadenylation (poly A) trap vectors employ a splice donor to capture an endogenous gene's polyadenylation sequence and provide transcript stability. However, the spectrum of mutations generated by these vectors appears largely restricted to the last intron of target loci due to nonsense-mediated mRNA decay (NMD) making them unsuitable for gene targeting applications. Here, we present novel poly A trap vectors that overcome the effect of NMD and also employ RNA instability sequences to improve splicing efficiency. The set of random insertions generated with these vectors show a significantly reduced insertional bias and the vectors can be targeted directly to a 5' intron. We also show that this relative positional independence is linked to the human beta-actin promoter and is most likely a result of its transcriptional activity in ES cells. Taken together our data indicate that these vectors are an effective tool for insertional mutagenesis that can be used for either gene trapping or gene targeting.
authors.affiliation: MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, UK.
authors.forename: Anestis
authors.initials: A
authors.lastname: Tsakiridis
authors.forename: Elena
authors.initials: E
authors.lastname: Tzouanacou
authors.forename: Afifah
authors.initials: A
authors.lastname: Rahman
authors.forename: Douglas
authors.initials: D
authors.lastname: Colby
authors.forename: Richard
authors.initials: R
authors.lastname: Axton
authors.forename: Ian
authors.initials: I
authors.lastname: Chambers
authors.forename: Valerie
authors.initials: V
authors.lastname: Wilson
authors.forename: Lesley
authors.initials: L
authors.lastname: Forrester
authors.forename: Joshua M
authors.initials: JM
authors.lastname: Brickman
mesh_terms: D000199:Actins / Q000235:genetics; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000378:metabolism*; D015870:Gene Expression; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D016296:Mutagenesis; D011401:Promoter Regions, Genetic
chemical_list: D000199:Actins
references.citation: EMBO J. 1994 Dec 1;13(23):5656-67
references.pmid: 7988563
references.citation: Proc Natl Acad Sci U S A. 2000 Feb 15;97(4):1536-41
references.pmid: 10677496
references.citation: Genome Res. 2007 Jul;17(7):1051-60
references.pmid: 17540781
references.citation: Genesis. 2007 Jun;45(6):353-60
references.pmid: 17492751
references.citation: Nat Rev Mol Cell Biol. 2004 Feb;5(2):89-99
references.pmid: 15040442
references.citation: Nat Genet. 2004 Jun;36(6):543-4
references.pmid: 15167922
references.citation: J Biol Chem. 1995 Dec 1;270(48):28995-9003
references.pmid: 7499432
references.citation: Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):10091-5
references.pmid: 8234261
references.citation: Cell Stem Cell. 2008 Oct 9;3(4):402-15
references.pmid: 18940732
references.citation: Trends Biochem Sci. 1998 Jun;23(6):198-9
references.pmid: 9644970
references.citation: Methods Mol Biol. 2008;435:127-38
references.pmid: 18370072
references.citation: Development. 2006 Sep;133(18):3709-22
references.pmid: 16936074
references.citation: Nucleic Acids Res. 2005 Mar 30;33(6):e54
references.pmid: 15800205
references.citation: PLoS One. 2007 Jul 18;2(7):e617
references.pmid: 17637833
references.citation: Nature. 1998 Apr 9;392(6676):608-11
references.pmid: 9560157
references.citation: Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):14109-14
references.pmid: 14610273
references.citation: Genome Res. 2008 Oct;18(10):1670-9
references.pmid: 18799693
references.citation: RNA. 2005 Feb;11(2):147-56
references.pmid: 15613535
references.citation: Nucleic Acids Res. 1999 Dec 15;27(24):e35
references.pmid: 10572187
references.citation: Science. 1989 Apr 28;244(4903):463-5
references.pmid: 2497519
references.citation: Nat Biotechnol. 2002 Jan;20(1):87-90
references.pmid: 11753368
references.citation: Oncogene. 1997 Sep;15(11):1255-62
references.pmid: 9315092
references.citation: BMC Genet. 2008 Dec 05;9:83
references.pmid: 19061508
references.citation: Blood. 2004 Aug 1;104(3):711-8
references.pmid: 15090446
references.citation: Hum Gene Ther. 2004 Jul;15(7):648-58
references.pmid: 15242525
references.citation: Cell. 1998 Oct 30;95(3):379-91
references.pmid: 9814708
references.citation: Dev Dyn. 1998 Jun;212(2):326-33
references.pmid: 9626507
references.citation: Brief Funct Genomic Proteomic. 2007 Sep;6(3):180-5
references.pmid: 17967808
references.citation: Proc Natl Acad Sci U S A. 2003 Aug 19;100(17):9918-22
references.pmid: 12904583
references.citation: Genome Res. 2005 Mar;15(3):428-35
references.pmid: 15741512
references.citation: Nucleic Acids Res. 2004 Jun 21;32(11):3304-15
references.pmid: 15210863
references.citation: Genes Dev. 1991 Sep;5(9):1513-23
references.pmid: 1653172
references.citation: Neural Netw. 2008 Oct;21(8):1056-69
references.pmid: 18603407
references.citation: Mol Cell Biol. 1997 Aug;17(8):4611-21
references.pmid: 9234718
references.citation: Proc Natl Acad Sci U S A. 2005 May 17;102(20):7221-6
references.pmid: 15870191
references.citation: Proc Natl Acad Sci U S A. 2005 Sep 13;102(37):13001-2
references.pmid: 16150712
references.citation: Development. 2006 May;133(10):2011-22
references.pmid: 16651543
references.citation: Genesis. 2008 Jul;46(7):347-56
references.pmid: 18615730
references.citation: J Biol Chem. 1996 Aug 30;271(35):21316-22
references.pmid: 8702910
references.citation: Proc Natl Acad Sci U S A. 1995 Jul 3;92(14):6592-6
references.pmid: 7604039
references.citation: Nature. 2007 Dec 20;450(7173):1230-4
references.pmid: 18097409
references.citation: Proc Natl Acad Sci U S A. 2005 Sep 13;102(37):13188-93
references.pmid: 16129827
references.citation: Proc Natl Acad Sci U S A. 1990 May;87(9):3435-9
references.pmid: 2159150
references.citation: Nature. 2004 Jun 3;429(6991):575-8
references.pmid: 15175755
references.citation: Nat Genet. 2004 Mar;36(3):304-12
references.pmid: 14981515
references.citation: Nucleic Acids Res. 2005 Feb 01;33(2):e20
references.pmid: 15687378
references.citation: Nucleic Acids Res. 2007;35(13):4542-51
references.pmid: 17586820
references.citation: J Biochem. 1993 Mar;113(3):343-9
references.pmid: 8387481
references.citation: Proc Natl Acad Sci U S A. 2004 Mar 23;101(12):4170-4
references.pmid: 15010531
references.citation: Nucleic Acids Res. 2003 Sep 15;31(18):5317-23
references.pmid: 12954767
references.citation: Nucleic Acids Res. 2006;34(20):e139
references.pmid: 17062627
references.citation: Cell. 2003 May 30;113(5):643-55
references.pmid: 12787505
references.citation: Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8392-6
references.pmid: 7690960