A promoter-hijack strategy for conditional shutdown of multiply spliced essential cell cycle genes.
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.
Samejima
Kumiko
K
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.
Ogawa
Hiromi
H
Cooke
Carol A
CA
Hudson
Damien F
DF
Hudson
Damien
D
Macisaac
Fiona
F
Ribeiro
Susana A
SA
Vagnarelli
Paola
P
Cardinale
Stefano
S
Kerr
Alastair
A
Lai
Fan
F
Ruchaud
Sandrine
S
Yue
Zuojun
Z
Earnshaw
William C
WC
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
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C092349:chromokinesin; D016547:Kinesins