Inducible transgenes expressing EJC enriched mRNAs: a post-genomic innovation

描述(申请人提供)：本修订提案最初是针对PA-07-336，“开发用于研究的动物模型和相关生物材料”而提交的。该研究计划需要建立和初步鉴定具有新型可诱导转基因表达人组织因子(AsTF)的新型可诱导转基因，asTF是凝血的主要触发因素，其异常表达和活性导致许多心血管系统衰弱疾病、各种形式的肿瘤和增殖性糖尿病视网膜病变的病因，因为TF的表达将局限于单核/巨噬细胞系的细胞，这是循环中公认的生物活性因子的来源。创新的转基因设计确保了产生的asTF mRNA将在外显子-外显子连接复合体(EJC)-多聚体蛋白形成中得到丰富，这些多聚体蛋白沉积在新合成的mRNA分子上，对于有效的mRNA核输出、细胞质内靶向、翻译以及最关键的翻译后修饰和分泌蛋白的输出是必不可少的。如果该项目成功，将极有可能极大地推动转基因动物研究领域的发展，因为它将1)建立和验证一种概念性的新的转基因设计方法，通过利用EJC丰富的表达盒来优化蛋白质表达；2)增强现有的在单核/巨噬细胞中可逆的体内基因表达的转基因技术，单核/巨噬细胞是生物学和疾病中许多重要(病理)生理过程的中心细胞类型；3)建立一种技术先进的体内方法来研究循环转录因子，其在健康和疾病中的作用(S)将引起美国国立卫生研究院系统内许多研究所的兴趣，包括国立卫生研究院、美国国立卫生研究院、NIDDK、美国国家信息学研究所和NEI。与公共卫生相关：本申请建议开发质量上新颖的可诱导转基因，表达丰富的外显子-外显子连接复合体(EJC)-多聚体蛋白形成的mRNA，其沉积在新合成的mRNA分子上对于有效的转录后mRNA处理至关重要。这需要建立表达选择性剪接形式的循环人组织因子(AsTF)的小鼠转基因模型，asTF是凝血的主要触发因素，其表达和活性的升高被认为是许多心血管系统衰弱疾病以及各种形式的肿瘤的病因之一。如果成功，该项目将包括转基因技术的质的进步，因为它将建立一种主要的新方法来研究人类转铁蛋白，其在健康和疾病方面的功能属于几个NIH研究所的范畴，最著名的是NHLBI、NIDDK和NCI。

DESCRIPTION (provided by applicant): This revised proposal was originally submitted in response to PA-07-336, "Development of Animal Models and Related Biological Materials for Research." The research plan entails generation and initial characterization of murine transgenic models featuring novel inducible transgenes expressing the soluble, alternatively spliced form of human Tissue Factor (asTF), the principal trigger of coagulation whose aberrant expression and activity contributes to etiology of many debilitating disorders of the cardiovascular system, various forms of neoplasia, and proliferative diabetic retinopathy asTF expression will be restricted to the cells of monocyte/macrophage lineage, a well established source of bioactive TF in circulation. Innovative transgene design ensures that the produced asTF mRNA will be enriched in exon-exon junction complexes (EJC)-multimeric protein formations whose deposition on newly synthesized mRNA molecules was recently shown to be essential for effective mRNA nuclear export, intra-cytoplasmic targeting, translation and, most critically, post-translational modifications and export of secreted proteins. If successful, the project is very likely to substantially advance the field of transgenic animal research, as it will 1) establish and validate a conceptually novel approach to transgene design, via utilization of EJC enriched expression cassettes for optimal protein expression; 2) enhance the existing transgenic technologies of reversible in vivo gene expression in monocytes/macrophages, a cell type central to many vital (patho)physiologic processes in biology and disease, and 3) establish a technologically advanced in vivo approach to study circulating TF, whose function(s) in health and disease are of interest to many institutes within the NIH system, including NHLBI, NCI, NIDDK, NIA, and NEI. PUBLIC HEALTH RELEVANCE: This application proposes development of qualitatively novel inducible transgenes expressing mRNA enriched in exon-exon junction complexes (EJC)-multimeric protein formations whose deposition on newly synthesized mRNA molecules is essential for effective post-transcriptional mRNA processing. It entails generation of murine transgenic models expressing the alternatively spliced form of circulating human Tissue Factor (asTF), the principal trigger of coagulation whose elevated expression and activity is known to contribute to etiology of many debilitating disorders of the cardiovascular system, as well as various forms of neoplasia. If successful, the project will comprise a qualitative advance in transgenic technology as it will establish a principally novel methodology to study human TF, whose functions in health and disease fall within the categorical interests of several NIH institutes, most notably NHLBI, NIDDK, and NCI.