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

K. Samejima, H. Ogawa, C.A. Cooke, D. Hudson, F. MacIsaac, S.A. Ribeiro, P. Vagnarelli, S. Cardinale, A. Kerr, F. Lai, Bill Earnshaw

Research output: Contribution to journalArticlepeer-review

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.
Original languageEnglish
Pages (from-to)2457-2462
Number of pages1
JournalProceedings of the National Academy of Sciences
Volume105
Issue number7
Publication statusPublished - 2008

Keywords

  • DT40
  • kif4
  • knockout
  • method
  • mitosis

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