Analysis of GFP Transgenes in Odontoblast in Differentiation

成牙本质细胞分化过程中 GFP 转基因的分析

• 批准号: 7456466
• 负责人: Mina Mina
• 金额: $ 34.47万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7456466
• 关键词: Apoptosis; Biological Assay; Biological Markers; Cell Separation; Cells; Congenital Disorders; Cultured Cells; Dental Papilla; Dental Pulp; Dentin; Dentin Formation; Dentinogenesis; Disease; Experimental Models; Fibroblasts; Gene Expression Profile; Genes; Goals; Green Fluorescent Proteins; In Vitro; Injury; Insulin-Like Growth Factor I; Microarray Analysis; Mitotic; Molecular; Molecular Analysis; Mus; Natural regeneration; Odontoblasts; Pattern; Population; RNA; Reporter; Research Personnel; Signal Pathway; Site; Somatomedins; Sorting - Cell Movement; Staging; Stem Cell Development; Stem cells; Testing; Tooth structure; Transgenes; Transgenic Animals; Transgenic Mice; Transplantation; Tubular formation; base; bone; in vivo; insight; novel; programs; promoter; repaired; research study; transgene expression

DESCRIPTION (provided by applicant): Dentinogenesis is regulated by a single layer of highly differentiated post-mitotic odontoblasts originating from the dental papilla. Despite significant progress in the discovery of key molecules and signaling pathways regulating tooth initiation and patterning, the mechanisms leading to dentinogenesis are relatively unknown. This has been due, in part, to difficulties in obtaining homogenous populations of progenitor cells for molecular analysis and the lack of suitable molecular markers for identifying the intermediate stages of odontoblast differentiation. The overall goals of this proposal are to use promoter-GFP reporter transgenic mice as a novel experimental model to unravel the underlying molecular mechanisms regulating the linear stepwise progression of pulp progenitor cells into odontoblasts. This proposal will use the activation of Col1a1-GFP transgenes to identify and isolate cells at early stages of differentiation by FACS sorting for further cellular and molecular analyses including an examination of their differentiation potential and gene expression profile. 3 specific aims are proposed. Aim #1 will test the hypothesis that stage-specific activation of Col1a1-GFP transgenes can serve as markers to distinguish and identify sub-populations of cells from dental pulp for further lineage analysis in vitro. Aim #2 will test the hypothesis that insulin-like growth factor (IGF-I) stimulates odontoblast differentiation. In Aim 2, Col1a1-GFP transgenes will be used to examine the underlying mechanisms by which IGF-I stimulates odontoblast differentiation. In this aim the effects of the altered levels of IGF-I on proliferation, apoptosis, and differentiation of pulp cells derived from transgenic mice with bone/odontoblast targeted over-expression of IGF-I (pOBCo!3.6-IGF), and transgenic mice with IGF-I haploinsufficiency (Igf1, HET) will also be examined. Aim #3 will test the hypothesis that Col1a1-GFP transgenes can be used as a marker to assess the dentin forming potential of adherent pulp fibroblasts in an in vivo transplantation assay at an ectopic site. It can be envisioned that results obtained from these experiments, will provide fundamental insights in the development of stem cell-based treatments for the repair and regeneration of dentin and eventually whole tooth that has been compromised by congenital disorders, diseases, and injuries.

描述（由申请人提供）：牙齿发生受源自牙齿乳头的高度分化后的牙胶细胞的单层调节。尽管在调节牙齿启动和模式的关键分子的发现和信号通路方面取得了重大进展，但导致牙齿生成的机制相对尚不清楚。这部分是由于难以获得祖细胞的同质种群进行分子分析，并且缺乏适当的分子标记来识别odontoblast分化的中间阶段。该提案的总体目标是将启动子GFP报告基因转基因小鼠用作一种新型的实验模型，以揭示调节纸浆祖细胞线性逐步发展为牙糖细胞的基本分子机制。该建议将使用COL1A1-GFP转基因的激活来识别和分离通过FACS分类的早期分化阶段和分离细胞，以进一步进行细胞和分子分析，包括检查其分化潜力和基因表达谱。提出了3个具体目标。 AIM＃1将检验以下假设：Col1a1-GFP转基因的特异性激活可以用作区分和鉴定细胞与牙髓的亚群的标记，以在体外进一步谱系分析。 AIM＃2将检验以下假设：胰岛素样生长因子（IGF-I）刺激牙糖细胞分化。在AIM 2中，COL1A1-GFP转基因将用于检查IGF-I刺激Odontoblast分化的潜在机制。在此目标中，IGF-I水平改变的影响对源自骨/牙骨质细胞的转基因小鼠的增殖，凋亡和分化，其靶向IGF-I（POBCO！3.6-IGF）的过表达，以及具有IGF-I单倍型（IGF-I单倍型）的影响。 AIM＃3将检验以下假设：COL1A1-GFP转基因可以用作评估牙本质在异位部位的体内移植测定中粘附纸浆成纤维细胞的潜力的标记。可以预见的是，从这些实验中获得的结果将提供基于干细胞处理的基本见解，以修复和再生牙本质的修复和再生，并最终受到先天性疾病，疾病和伤害的损害。