title: A reliable lacZ expression reporter cassette for multipurpose, knockout-first alleles.
abstract: Alteration of the mouse genome through homologous recombination in embryonic stem (ES) cells is the most accurate and versatile way to dissect gene function in a vertebrate model. Most often, a selectable marker is used to create a knockout allele by replacing an essential part of the gene. However, knockout strategies are limited because the mutation is present constitutively. Conditional approaches based on the Cre-loxP site-specific recombination (SSR) system address this limitation; however, it requires that all parts of the targeted gene remain in ES cells. Here we report success with a "knockout-first" strategy that ablates gene function by insertion of RNA processing signals without deletion of any of the target gene. Incorporation of site-specific recombination target sites creates a multipurpose allele for both knockout and conditional applications.
authors.affiliation: Biotec, Technische Universität Dresden, c/o Max Planck Institute of Molecular cell Biology and Genetics, Dresden, Germany.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Testa
authors.forename: Julia
authors.initials: J
authors.lastname: Schaft
authors.forename: Frank
authors.initials: F
authors.lastname: van der Hoeven
authors.forename: Stefan
authors.initials: S
authors.lastname: Glaser
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Anastassiadis
authors.forename: Youming
authors.initials: Y
authors.lastname: Zhang
authors.forename: Thomas
authors.initials: T
authors.lastname: Hermann
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Stremmel
authors.forename: A Francis
authors.initials: AF
authors.lastname: Stewart
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D050612:Fatty Acid Transport Proteins; D005260:Female; D020868:Gene Silencing; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D019426:Integrases / Q000235:genetics* / Q000378:metabolism; D007763:Lac Operon / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics; D026901:Membrane Transport Proteins; D051379:Mice; D018345:Mice, Knockout; D009687:Nuclear Proteins / Q000235:genetics; D012313:RNA / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D013879:Thioredoxins / Q000235:genetics; D014162:Transfection
chemical_list: C489900:Aff1 protein, mouse; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D050612:Fatty Acid Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009687:Nuclear Proteins; C493572:Slc27a4 protein, mouse; C516112:Txn2 protein, mouse; D013879:Thioredoxins; D012313:RNA; C045073:Cre recombinase; D019426:Integrases