A promoter-hijack strategy for conditional shutdown of multiply spliced essential cell cycle genes

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

Communicated by Don W. Cleveland, University of California at San Diego, La Jolla, CA, December 22, 2007 (received for review November 12, 2007)

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.

Footnotes

^§To whom correspondence should be addressed. E-mail: bill.earnshaw{at}ed.ac.uk
Author contributions: K.S. and W.C.E. designed research; K.S., H.O., and F.L. performed research; C.A.C., D.H., F.M., S.A.R., P.V., S.C., S.R., and Z.Y. contributed new reagents/analytic tools; K.S. and A.K. analyzed data; and K.S. and W.C.E. wrote the paper.
↵*Present address: Johns Hopkins University School of Medicine, 725 North Wolfe Street Baltimore, MD 21205.
↵ ^†Present address: Chromosome and Chromatin Research, Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne 3052, Australia.
↵ ^‡Present address: Centre for Genomic Regulation, PRBB, Barcelona C/Dr. Aiguader 88, 08003, Spain.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0712083105/DC1.