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

"TKO" is an expression vector that knocks out the activity of a transcripti on factor in vivo under genetic control. We describe a successful test of t his concept that used a sea urchin transcription factor of known function, P3A2, as the target. The TKO cassette employs modular cis-regulatory elemen ts to express an encoded single-chain antibody that prevents the P3A2 prote in 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 C yIIIa expression constructs, and we show here that introduction of an alpha P3A2.TKO expression cassette causes exactly the same ectopic oral expressi on of a coinjected wild-type CyIIIa construct. Furthermore, the alpha P3A2. TKO cassette derepresses the endogenous CyIIIa gene in the oral ectoderm an d in the endoderm. alpha P3A2.TKO thus abrogates the function of the endoge nous SpP3A2 transcription factor with respect to spatial repression of the CyIIIa gene. Widespread expression of alpha P3A2.TKO in the endoderm has th e additional lethal effect of disrupting morphogenesis of the archenteron, revealing a previously unsuspected function of SpP3A2 in endoderm developme nt. In principle, TKO technology could be utilized for spatially and tempor ally controlled blockade of any transcription factor in any biological syst em amenable to gene transfer.