Interference with gene regulation in living sea urchin embryos: Transcription factor Knock Out (TKO), a genetically controlled vector for blockade of specific transcription factors
Interference with gene regulation in living sea urchin embryos: Transcription factor Knock Out (TKO), a genetically controlled vector for blockade of specific transcription factors
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DOI：
10.1073/pnas.95.25.14827
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发表时间：
1998-12-08
影响因子：
11.1
通讯作者：
Davidson, EH
中科院分区：
文献类型：
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作者：
Bogarad, LD;Arnone, MI;Davidson, EH
“TKO”是一种在基因控制下敲除体内转录因子活性的表达载体。我们描述了使用已知功能的海胆转录因子P3A2作为靶标对这一概念进行的成功测试。TKO盒使用模块化的顺式调节元件来表达编码的单链抗体，该抗体可以防止P3A2蛋白在体内与DNA结合。在正常发育中，P3A2转录因子的功能之一是直接抑制胚胎败育外胚层外CyIIIa细胞骨架肌动蛋白基因的表达。如果从CyIIIa表达结构中删除P3A2位点，那么口腔外胚层中就会发生异位表达，在这里，我们证明了引入αP3A2.TKO表达盒导致了与共同注射的野生型CyIIIa构建物完全相同的异位口头表达。此外，αP3A2.TKO盒下调了口腔外胚层和内胚层中内源CyIIIa基因的表达。因此，Alpha P3A2.TKO取消了内源性SpP3A2转录因子关于CyIIIa基因空间抑制的功能。αP3A2.TKO在内胚层的广泛表达还具有破坏原肠形态发生的额外致死作用，揭示了SpP3A2在内胚层发育中先前未被怀疑的功能。原则上，TKO技术可以用来在空间和时间上控制对任何生物系统中的任何转录因子的阻断，这些生物系统符合基因转移的要求。
"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 alpha P3A2.TKO expression cassette causes exactly the same ectopic oral expression of a coinjected wild-type CyIIIa construct. Furthermore, the alpha P3A2.TKO cassette derepresses the endogenous CyIIIa gene in the oral ectoderm and in the endoderm. alpha P3A2.TKO thus abrogates the function of the endogenous SpP3A2 transcription factor with respect to spatial repression of the CyIIIa gene. Widespread expression of alpha P3A2.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.