title: Chromosomal manipulation by site-specific recombinases and fluorescent protein-based vectors.
abstract: Feasibility of chromosomal manipulation in mammalian cells was first reported 15 years ago. Although this technique is useful for precise understanding of gene regulation in the chromosomal context, a limited number of laboratories have used it in actual practice because of associated technical difficulties. To overcome the practical hurdles, we developed a Cre-mediated chromosomal recombination system using fluorescent proteins and various site-specific recombinases. These techniques enabled quick construction of targeting vectors, easy identification of chromosome-rearranged cells, and rearrangement leaving minimum artificial elements at junctions. Applying this system to a human cell line, we successfully recapitulated two types of pathogenic chromosomal translocations in human diseases: MYC/IgH and BCR/ABL1. By inducing recombination between two loxP sites targeted into the same chromosome, we could mark cells harboring deletion or duplication of the inter-loxP segments with different colors of fluorescence. In addition, we demonstrated that the intrachromosomal recombination frequency is inversely proportional to the distance between two recombination sites, implicating a future application of this frequency as a proximity sensor. Our method of chromosomal manipulation can be employed for particular cell types in which gene targeting is possible (e.g. embryonic stem cells). Experimental use of this system would open up new horizons in genome biology, including the establishment of cellular and animal models of diseases caused by translocations and copy-number variations.
authors.affiliation: Evolutionary Medicine, Shiga Medical Center Research Institute, Moriyama, Japan.
authors.forename: Munehiro
authors.initials: M
authors.lastname: Uemura
authors.forename: Youko
authors.initials: Y
authors.lastname: Niwa
authors.forename: Naoki
authors.initials: N
authors.lastname: Kakazu
authors.forename: Noritaka
authors.initials: N
authors.lastname: Adachi
authors.forename: Kazuo
authors.initials: K
authors.lastname: Kinoshita
mesh_terms: D000818:Animals; D002460:Cell Line; D002875:Chromosomes / Q000648:ultrastructure*; D004254:DNA Nucleotidyltransferases / Q000235:genetics*; D004330:Drosophila; D005456:Fluorescent Dyes / Q000378:metabolism*; D018628:Gene Dosage; D018390:Gene Targeting; D005821:Genetic Techniques; D005822:Genetic Vectors; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D019426:Integrases / Q000235:genetics; D011995:Recombination, Genetic; D014178:Translocation, Genetic
chemical_list: D005456:Fluorescent Dyes; C045073:Cre recombinase; D004254:DNA Nucleotidyltransferases; D019426:Integrases; C077570:Site-specific recombinase