ALTERING GENE EXPRESSION WITH DESIGNED ZINC FINGERS

描述 这个项目的目标是开发一种新的方法， 植物中的基因表达。多指锌指蛋白将成为 从锌指库构建，并选择结合独特的 要沉默的基因中所含的DNA序列。锌指结构域 将与转录阻遏物结构域融合，并在 转基因植物以沉默靶基因的表达。的in 体外试验将用于确定最有效的转录 植物中的阻遏结构域，并确定最佳位置， 锌指粘合剂基因沉默将通过以下方法在体内证实： 用阻遏物构建体瞬时转染烟草原生质体。 转基因植物将产生表达锌指阻遏物 设计用于沉默内源基因的构建体。最后，锌指 阻遏蛋白将被设计成沉默病毒的表达， 复制所需的蛋白质。这些结构将用于 产生具有增加的病毒抗性的转基因植物， 感染该技术为基因沉默提供了一种有效的方法 在植物中表达以增加其抗病性，增强其 营养价值，或改变其他农业上重要的特性。的 在这个项目中开发的概念也可以扩展到改变基因 在其他真核生物中表达。

DESCRIPTION The objective of this project is to develop a novel method of silencing the gene expression in plants. Polydactyl zinc finger proteins will be constructed from a library of zinc fingers and selected to bind a unique DNA sequence contained in the gene to be silenced. The zinc finger domain will be fused to a transcriptional repressor domain and expressed in transgenic plants to silence the expression of the target gene. An in vitro assay will be used to identify the most effective transcriptional repression domains in plants and to determine the optimum position for the zinc finger to bind. Gene silencing will be demonstrated in vivo by transiently transfecting tobacco protoplasts with the repressor construct. Transgenic plants will be generated the express a zinc finger repressor construct designed to silence an endogenous gene. Finally, zinc finger repressor proteins will be designed to silence the expression of viral proteins necessary for replication. These constructs will be used to generate transgenic plants that have increased resistance in viral infection. This technique will provide a useful method for silencing gene expression in plants to increase their disease resistance, enhance their nutritional value, or alter other agriculturally important properties. The concepts developed in this project can also be extended to alter gene expression in other eukaryotes.