title: A promoter-hijack strategy for conditional shutdown of multiply spliced essential cell cycle genes.
abstract: We describe a method for the isolation of conditional knockouts of essential multiply spliced genes in which the entire body of the gene downstream of the ATG start codon is left untouched but can be switched off rapidly and completely by adding tetracycline to the culture medium. The approach centers on a "promoter-hijack" strategy in which the gene's promoter is replaced with a minimal promoter responsive to the tetracycline-repressible transactivator (tTA). Elsewhere in the genome, a cloned fragment of the gene's promoter is used to drive expression of a tTA. Thus, the gene is essentially regulated by its own promoter but through the intermediary tTA. Using this strategy, we generated a conditional knockout of chromokinesin KIF4A, an important mitotic effector protein whose mRNA is multiply spliced and whose cDNA is highly toxic when overexpressed in cells. We used chicken DT40 cells, but the same strategy should be applicable to ES cells and, eventually, to mice.
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, Swann Building, King's Buildings, Mayfield Road, Edinburgh EH9 3JR, United Kingdom.
authors.forename: Kumiko
authors.initials: K
authors.lastname: Samejima
authors.forename: Hiromi
authors.initials: H
authors.lastname: Ogawa
authors.forename: Carol A
authors.initials: CA
authors.lastname: Cooke
authors.forename: Damien F
authors.initials: DF
authors.lastname: Hudson
authors.forename: Damien
authors.initials: D
authors.lastname: Hudson
authors.forename: Fiona
authors.initials: F
authors.lastname: Macisaac
authors.forename: Susana A
authors.initials: SA
authors.lastname: Ribeiro
authors.forename: Paola
authors.initials: P
authors.lastname: Vagnarelli
authors.forename: Stefano
authors.initials: S
authors.lastname: Cardinale
authors.forename: Alastair
authors.initials: A
authors.lastname: Kerr
authors.forename: Fan
authors.initials: F
authors.lastname: Lai
authors.forename: Sandrine
authors.initials: S
authors.lastname: Ruchaud
authors.forename: Zuojun
authors.initials: Z
authors.lastname: Yue
authors.forename: William C
authors.initials: WC
authors.lastname: Earnshaw
mesh_terms: D000818:Animals; D002460:Cell Line; D002645:Chickens; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D020043:Genes, Essential / Q000235:genetics; D018816:Genes, cdc; D016678:Genome / Q000235:genetics; D016547:Kinesins / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012326:RNA Splicing / Q000235:genetics*; D019076:Transgenes / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C092349:chromokinesin; D016547:Kinesins