title: Tagging genes with cassette-exchange sites.
abstract: In an effort to make transgenesis more flexible and reproducible, we developed a system based on novel 5' and 3' 'gene trap' vectors containing heterospecific Flp recognition target sites and the corresponding 'exchange' vectors allowing the insertion of any DNA sequence of interest into the trapped locus. Flp-recombinase-mediated cassette exchange was demonstrated to be highly efficient in our system, even in the absence of locus-specific selection. The feasibility of constructing a library of ES cell clones using our gene trap vectors was tested and a thousand insertion sites were characterized, following electroporation in ES cells, by RACE-PCR and sequencing. We validated the system in vivo for two trapped loci in transgenic mice and demonstrated that the reporter transgenes inserted into the trapped loci have an expression pattern identical to the endogenous genes. We believe that this system will facilitate in vivo studies of gene function and large-scale generation of mouse models of human diseases, caused by not only loss but also gain of function alleles.
authors.affiliation: Telethon Institute of Genetics and Medicine, Via P. Castellino 111, 80131 Naples, Italy.
authors.forename: Gilda
authors.initials: G
authors.lastname: Cobellis
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Nicolaus
authors.forename: Mariangela
authors.initials: M
authors.lastname: Iovino
authors.forename: Antonio
authors.initials: A
authors.lastname: Romito
authors.forename: Emanuele
authors.initials: E
authors.lastname: Marra
authors.forename: Manlio
authors.initials: M
authors.lastname: Barbarisi
authors.forename: Marco
authors.initials: M
authors.lastname: Sardiello
authors.forename: Francesco P
authors.initials: FP
authors.lastname: Di Giorgio
authors.forename: Nicola
authors.initials: N
authors.lastname: Iovino
authors.forename: Massimo
authors.initials: M
authors.lastname: Zollo
authors.forename: Andrea
authors.initials: A
authors.lastname: Ballabio
authors.forename: Riccardo
authors.initials: R
authors.lastname: Cortese
mesh_terms: D000818:Animals; D002460:Cell Line; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D051379:Mice; D008822:Mice, Transgenic; D011995:Recombination, Genetic
chemical_list: D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase
references.citation: Cold Spring Harb Symp Quant Biol. 1986;51 Pt 1:151-7
references.pmid: 3472711
references.citation: Cell Mol Biol (Noisy-le-grand). 1999 Jul;45(5):737-50
references.pmid: 10512203
references.citation: Proc Natl Acad Sci U S A. 2000 Feb 1;97(3):1002-7
references.pmid: 10655474
references.citation: Genesis. 2000 Apr;26(4):245-52
references.pmid: 10748462
references.citation: Biotechniques. 2000 Nov;29(5):1024-8, 1030, 1032
references.pmid: 11084865
references.citation: Dev Biol. 2001 May 15;233(2):319-28
references.pmid: 11336498
references.citation: Nucleic Acids Res. 2001 May 15;29(10):E47
references.pmid: 11353092
references.citation: Genomics. 2002 Mar;79(3):344-8
references.pmid: 11863364
references.citation: Nucleic Acids Res. 2003 Jan 1;31(1):278-81
references.pmid: 12520002
references.citation: Nat Genet. 2003 Mar;33 Suppl:276-84
references.pmid: 12610537
references.citation: Proc Natl Acad Sci U S A. 2003 Aug 19;100(17):9918-22
references.pmid: 12904583
references.citation: Nature. 2003 Oct 30;425(6961):917-25
references.pmid: 14586460
references.citation: Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):14109-14
references.pmid: 14610273
references.citation: J Mol Biol. 1990 Oct 5;215(3):403-10
references.pmid: 2231712
references.citation: Genes Dev. 1992 Jun;6(6):903-18
references.pmid: 1592261
references.citation: Biochemistry. 1997 Feb 18;36(7):1740-7
references.pmid: 9048557
references.citation: Nucleic Acids Res. 1997 Apr 15;25(8):1531-6
references.pmid: 9092658
references.citation: Nat Genet. 1997 Aug;16(4):338-44
references.pmid: 9241269
references.citation: Biochemistry. 1998 May 5;37(18):6229-34
references.pmid: 9572836
references.citation: Exp Cell Res. 1998 Oct 10;244(1):125-36
references.pmid: 9770356
references.citation: Nucleic Acids Res. 1999 Dec 15;27(24):e35
references.pmid: 10572187