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.

Promoter hijack reveals pericentrin functions in mitosis and the DNA damage response

“…34 Given the complexities of gene expression from the pericentrin (Pcnt) locus, we employed a promoter-hijack strategy to disrupt Pcnt and to ablate pericentrin expression. 35 As shown in Figure 1A, this involved the replacement of 5.5 kb upstream of the Pcnt coding sequence, which we presumed to contain the principal Pcnt promoter sequence, with a Tet operator array, using gene targeting and subsequent Cre recombinase deletion of the selectable cassette. Successful targeting of this sequence and cassette excision were verified by Southern blot analysis (Fig.…”

“…1B) and two targeted, recombined clones were used for further analysis (Pcnt Δ/Δ #1 and #2). As previously published work had suggested that pericentrin might be essential in cell lines, we transfected Pcnt heterozygote cells with a plasmid that encoded the tet transactivator (tTA) under the control of the Kif4A promoter, pKif4A-tTA2, 35 prior to targeting of the remaining Pcnt allele. While Pcnt Δ/Δ cells showed 2% of wild-type Pcnt expression, the Kif4A promoter-driven tTA led to various levels of Pcnt expression in Pcnt Δ/Δ clones, as determined by quantitative real-time using antibodies specific for the centrosome-associated kinase, Aurora A and the centriole components, centrin2, Cep135, glutamylated tubulin, Ninein and Cep76 (Fig.…”

“…Given the size of the Pcnt locus and the multiple spliced transcripts it encodes, the promoter-hijack strategy described by Samejima and coworkers seemed ideal for our purpose. 35 This approach allowed us to obtain clones with greatly attenuated Pcnt expression and, importantly, by varying tTA levels, we generated rescue clones that showed both wild-type and increased Pcnt expression.…”

“…Five μl of a 1:50 dilution was used as a template for each quantitative described previously. 35 The 5' homology arm was PCR-amplified from DT40 genomic DNA using KOD polymerase (Novagen/ Merck) with the following primers:…”

Characterisation of a stably integrated expression system for exogenous protein expression in DT40

“…The Discussion could include mention of the promoter analyses performed in Yang et al (2006) 1 , Samejima et al (2008) 2 , Vazquez et al (2003) 3 .…”