Dimeric Cys2His2 Zinc Finger Proteins for Gene Targeting

用于基因打靶的二聚 Cys2His2 锌指蛋白

• 批准号: 6845071
• 负责人: SCOT A WOLFE
• 金额: $ 29.34万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6845071
• 关键词: Drosophilidae; Saccharomyces cerevisiae; X ray crystallography; alleles; athymic mouse; biotechnology; chromatin immunoprecipitation; fungal proteins; gene expression; genetic regulation; genetically modified animals; microarray technology; nucleic acid sequence; peptide chemical synthesis; protein engineering; tissue /cell culture; transcription factor

DESCRIPTION (provided by applicant): Cys2His2 zinc finger proteins can be engineered to recognize a particular DNA sequence, such as a site in the promoter of a particular gene of interest. These proteins, when fused to activation or repression domains, can function as artificial transcription factors. They can be used as tools for the study of gene function in model organisms and potentially as gene therapy reagents for the treatment of disease. However, fundamental questions remain regarding the specificity of these proteins in vivo. This proposal describes the development of dimeric zinc finger proteins that can regulate a single endogenous gene. Dimeric zinc finger proteins should be superior to monomeric zinc finger proteins with regards to sequence specificity and in vivo utility, and they offer a unique system for dissecting the importance of specificity and affinity in in vivo function: Their specificity can be changed by adding or subtracting fingers and their affinity can be varied by increasing or decreasing the strength of the dimerization domain. In Aim #1, we will develop dimeric zinc finger proteins that can regulate a single gene in S. cerevisiae. The in vivo specificity of a series of different dimeric proteins will be characterized, and in conjunction with the analysis of their in vitro specificity and affinity, we will deconvolute the requirements for regulating a single gene in budding yeast. In Aim #2, we will investigate the same question in D. melanogaster. This organism provides unique tools, such as polytene chromosomes, for analyzing the role of specificity and affinity in gene regulation. By defining the increases in specificity and affinity required when moving from yeast to flies to regulate a single endogenous gene, these results should allow us to extrapolate to even more complex organisms such as mice. In Aim #3, we will develop dimeric zinc finger proteins capable of discriminating between two alleles based on single base pair differences. This study should define the limits of single base pair discrimination for zinc finger proteins, and in the future could provide a method for targeting cancer cells by tailoring a therapy specifically to their genotype. Our long-term goal is to develop tools for the regulation of a single gene or allele in a complex genome by understanding the relationship between specificity and affinity and in vivo function.

描述（由申请人提供）：CYS2HIS2锌指蛋白可以设计以识别特定的DNA序列，例如特定感兴趣基因的启动子中的位点。这些蛋白质与激活或抑制域融合时，可以用作人工转录因子。它们可以用作研究模型生物中基因功能的工具，并可能用作疾病治疗的基因治疗试剂。但是，关于这些蛋白质在体内的特异性仍然存在的基本问题。 该建议描述了可以调节单个内源基因的二聚体锌指蛋白的发展。关于序列特异性和体内效用，二聚体锌指蛋白应优于单体锌指蛋白，并且它们提供了一种独特的系统来阐明特异性和亲和力在体内功能中的特异性和亲和力的重要性：它们的特异性可以通过添加或减去亲和力来通过增加或降低性能来改变或降低效果。 在AIM＃1中，我们将开发二聚锌指蛋白，该蛋白可以调节酿酒酵母中的单个基因。将表征一系列不同二聚体蛋白的体内特异性，并结合其体外特异性和亲和力的分析，我们将反应调节在萌芽酵母中调节单个基因的要求。在AIM＃2中，我们将在D. Melanogaster中调查相同的问题。该生物提供了独特的工具，例如多丹染色体，用于分析特异性和亲和力在基因调节中的作用。通过定义从酵母到果蝇转移以调节单个内源基因时所需的特异性和亲和力的增加，这些结果应该使我们能够推断出更复杂的生物（如小鼠）。在AIM＃3中，我们将开发二聚体锌指蛋白，能够根据单基对差异区分两个等位基因。这项研究应定义单基对锌蛋白的单基对歧视的限制，将来可以通过针对其基因型定制一种治疗方法来提供一种靶向癌细胞的方法。 我们的长期目标是通过了解特异性与亲和力与体内功能之间的关系来开发复杂基因组中单个基因或等位基因的工具。