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“研究用动物模型和相关生物材料的开发”而提交的。“该研究计划需要产生和初步表征小鼠转基因模型，其特征在于表达可溶性、可变剪接形式的人组织因子（asTF）的新型诱导型转基因，这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性疾病的病因学，各种形式的肿瘤，和增殖性糖尿病视网膜病变，因为TF表达将限于单核细胞/巨噬细胞谱系的细胞，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所产生的asTF mRNA将富含外显子-外显子连接复合物（EJC）-多聚体蛋白形成物，其在新合成的mRNA分子上的沉积最近被证明对于有效的mRNA核输出、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。如果成功的话，该项目很可能会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，通过利用富含EJC的表达盒进行最佳蛋白质表达; 2）增强单核细胞/巨噬细胞中可逆体内基因表达的现有转基因技术，一种对生物学和疾病中的许多重要（病理）生理过程至关重要的细胞类型，和3）建立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系统内的许多研究所感兴趣的，包括NHLBI、NCI、NIDDK、NIA，的NEI。公共卫生相关性：本申请提出开发表达富含外显子-外显子连接复合物（EJC）-多聚体蛋白形成物的mRNA的定性新型诱导型转基因，所述多聚体蛋白形成物沉积在新合成的mRNA分子上对于有效的转录后mRNA加工是必需的。它需要产生表达可变剪接形式的循环人组织因子（asTF）的鼠转基因模型，所述组织因子是凝血的主要触发物，已知其表达和活性升高有助于心血管系统的许多衰弱性疾病以及各种形式的瘤形成的病因学。如果成功，该项目将包括转基因技术的质的进步，因为它将建立一个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研究所的绝对利益，最值得注意的是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.