title: Site-specific integration of targeted DNA into animal cell genomes.
abstract: Novel genetically engineered retroviral vectors and targeting plasmids are described that enable the site-specific targeting of exogenous DNA into the genomes of cultured animal cells. The protocol involves the transduction of competent cells by a chimeric retroviral vector containing a transcription unit composed of two linked cassettes: an upstream marker gene under the control of the viral 5' LTR; and a downstream reporter trap containing a strong promoter 5' to a 48bp yeast FRT element. When cells containing such integrated units are co-transfected with a plasmid encoding yeast FLP recombinase and a promoterless targeting plasmid containing a reporter cDNA tract 3' to an homologous FRT element, the targeting plasmid recombines at the chromosomally preconfigured FRT site, and a new hemizygous function is introduced into the downstream cassette. These reagents provide a new portable system for site-specific targeting of chemically modified genes into uniform and unique sites in genomically integrated transcription units.
authors.affiliation: Department of Pharmacology, School of Medicine, University of California, San Diego 92093-0636, USA.
authors.forename: K S
authors.initials: KS
authors.lastname: Koch
authors.forename: T
authors.initials: T
authors.lastname: Aoki
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.forename: A E
authors.initials: AE
authors.lastname: Atkinson
authors.forename: A S
authors.initials: AS
authors.lastname: Gleiberman
authors.forename: O K
authors.initials: OK
authors.lastname: Glebov
authors.forename: H L
authors.initials: HL
authors.lastname: Leffert
mesh_terms: D000818:Animals; D001665:Binding Sites; D015139:Blotting, Southern; D002460:Cell Line; D004247:DNA / Q000235:genetics*; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D004274:DNA, Recombinant / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D016678:Genome; D051379:Mice; D010957:Plasmids; D051381:Rats; D015203:Reproducibility of Results; D012190:Retroviridae / Q000235:genetics; D014162:Transfection / Q000379:methods; D016662:Virus Integration
chemical_list: D004274:DNA, Recombinant; D004247:DNA; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase