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

描述（由申请人提供）：本修订提案最初是为了响应PA-07-336，“动物模型和相关生物材料的研究开发”而提交的。该研究计划需要建立小鼠转基因模型并对其进行初步表征，该模型具有表达可溶性、可选择性剪接形式的人组织因子（asTF）的新型可诱导转基因，asTF是凝血的主要触发因子，其异常表达和活性导致许多心血管系统衰弱性疾病、各种形式的肿瘤、而增殖性糖尿病视网膜病变asTF的表达将局限于单核细胞/巨噬细胞谱系，这是循环中生物活性TF的良好来源。创新的转基因设计确保产生的asTF mRNA将富含外显子-外显子连接复合物(EJC)-多聚体蛋白结构，其沉积在新合成的mRNA分子上，最近被证明对于有效的mRNA核输出、胞质内靶向、翻译以及最重要的翻译后修饰和分泌蛋白的输出至关重要。如果成功，该项目很可能大大推进转基因动物研究领域，因为它将1)建立和验证一种概念上新颖的转基因设计方法，通过利用EJC富集表达盒进行最佳蛋白质表达；2)加强现有的单核/巨噬细胞体内可逆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TF，其在健康和疾病中的功能是NIH系统内许多研究所感兴趣的，包括NHLBI， NCI， NIDDK， NIA和NEI。公共卫生相关性：该应用程序提出了开发质量新颖的可诱导转基因，表达富含外显子-外显子连接复合物（EJC）的mRNA -多聚体蛋白结构，其沉积在新合成的mRNA分子上对于有效的转录后mRNA加工至关重要。它需要产生表达循环人组织因子（asTF）的可选剪接形式的小鼠转基因模型，asTF是凝血的主要触发因子，其表达和活性的升高已知有助于许多心血管系统衰弱性疾病的病因学，以及各种形式的肿瘤。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TF，其在健康和疾病中的功能属于几个NIH研究所的分类兴趣，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.