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Published December 8, 1998 | Published
Journal Article Open

Interference with gene regulation in living sea urchin embryos: Transcription factor Knock Out (TKO), a genetically controlled vector for blockade of specific transcription factors

Abstract

"TKO" is an expression vector that knocks out the activity of a transcription factor in vivo under genetic control. We describe a successful test of this concept that used a sea urchin transcription factor of known function, P3A2, as the target. The TKO cassette employs modular cis-regulatory elements to express an encoded single-chain antibody that prevents the P3A2 protein from binding DNA in vivo. In normal development, one of the functions of the P3A2 transcription factor is to repress directly the expression of the CyIIIa cytoskeletal actin gene outside the aboral ectoderm of the embryo. Ectopic expression in oral ectoderm occurs if P3A2 sites are deleted from CyIIIa expression constructs, and we show here that introduction of an αP3A2⋅TKO expression cassette causes exactly the same ectopic oral expression of a coinjected wild-type CyIIIa construct. Furthermore, the αP3A2⋅TKO cassette derepresses the endogenous CyIIIa gene in the oral ectoderm and in the endoderm. αP3A2⋅TKO thus abrogates the function of the endogenous SpP3A2 transcription factor with respect to spatial repression of the CyIIIa gene. Widespread expression of αP3A2⋅TKO in the endoderm has the additional lethal effect of disrupting morphogenesis of the archenteron, revealing a previously unsuspected function of SpP3A2 in endoderm development. In principle, TKO technology could be utilized for spatially and temporally controlled blockade of any transcription factor in any biological system amenable to gene transfer.

Additional Information

© 1998 by The National Academy of Sciences. Contributed by Eric H. Davidson, October 1, 1998. We express great appreciation to Drs. James Coffman and Carl Parker for critical reviews of the manuscript. We thank Drs. R. Andrew Cameron and Carmen Kirchhamer for their invaluable insight and discussions, and Armenia Gregorian, Vanessa Moy, and Gayle Zborowski for their excellent technical assistance. We are grateful to Drs. Robert Maxson of University of Southern California and Lynne Angerer of the University of Rochester for providing promoter clones. Research was supported by National Institutes of Health Grant HD-05753 and by the Stowers Institute for Medical Research. L.D.B. was supported by a California Institute of Technology Gosney Fellowship and by National Institutes of Health Grant HD-07257. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ''advertisement'' in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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August 19, 2023
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