FLP-MEDIATED CELL LINEAGE ANALYSIS IN THE MOUSE EMBRYO

FLP 介导的小鼠胚胎细胞谱系分析

• 批准号: 2203174
• 负责人: Susan M. Dymecki
• 金额: $ 12.36万
• 依托单位: CARNEGIE INSTITUTION OF WASHINGTON, D.C.
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-15 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2203174
• 关键词: biomarker; cell differentiation; cell migration; cell population study; cell type; cytogenetics; embryo /fetus tissue /cell culture; genetic mapping; genetically modified animals; histogenesis; laboratory mouse; mammalian embryology; molecular cloning; neural crest; nucleic acid sequence; polymerase chain reaction; recombinase

The overall objectives of this project are to develop a binary recombination-based system to fate map and manipulate somatic lineages in the mouse embryo, and to utilize this new tool to reveal mechanisms underlying establishment of neural crest cell lineages. A central issue in mammalian development -understanding how lineage and environment interact to determine phenotype - has been limited by the inability to follow the fate of specific cells in situ and to observe the distribution of their progeny in the embryo throughout gestation. Such precise lineage mapping is prerequisite to understanding developmental determinants of cell proliferation, differentiation, and migration. Knowledge of these mechanisms is fundamental to understanding the origins of congenital malformations. We have engineered an in situ lineage marking system in transgenic mice that should mark specific populations of cells in a heritable, cell autonomous, non-diluting fashion during embryonic development. Additionally, this system can be used in conjunction with homologous recombination to perform lineage/tissue-specific in vivo mutagenesis. We have exploited an excisional recombination system found in yeast for these purposes: the recombinase FLP catalyzes recombination between direct repeats of FLP recombination targets (FRTs) excising the intervening DNA. Since initiating this project in 1993 we have: (1) constructed a modular set of FLP and universal target (FRT-disrupted lacZ) vectors; (2) demonstrated efficient FLP-activation of the target transgene in embryonic stem (ES) cells; (3) generated separate FLP and target (FRT-disrupted lacZ) transgenic mouse lines; and (4) demonstrated FLP-mediated recombination in embryos from a FLPxtarget cross. We are now in the unique position to fully characterize this system in vivo as a tool for marking cell lineages and for directed modifications of the mouse genome. Because neural crest cells migrate extensively and differentiate to form a variety of cell types they are optimal to study how lineage and environment interact to determine cell fate. Derangements of neural crest cell development are implicated in numerous congenital malformations including neural tube, limb, cranial, enteric ganglion and cardiac defects, deafness, and thymic agenesis. A major unanswered question is when and how does this population of cells generate such phenotypic diversity. Toward the proposed aims, we have generated transgenic mice expressing FLP in the dorsal CNS as a means to activate lacZ in neural crest progenitors. We will: (1) utilize this marking system to map the murine neural crest; (2) identify similarities and differences between the murine map and that of the chick, the latter providing much of our current knowledge; (3) move from descriptions of cell fate to analysis of underlying mechanisms through lineage studies in mutant embryos. Mechanisms of pathogenesis associated with Splotch (Pax-3), lethal spotting (Is), and W (c-kit) phenotypes will give insight into the pathogenesis of parallel human syndromes (e.g. Waardenburg Syndrome and Hirschsprung's disease).

该项目的总体目标是开发二进制 基于重组的系统，以命运图和操纵躯体谱系 在鼠标胚胎中，并利用这种新工具来揭示机制 基础神经rest细胞谱系的建立。一个中心问题 在哺乳动物的发展中 - 理解谱系和环境如何 相互作用以确定表型 - 受到无法限制的 遵循特定细胞原位的命运，并观察分布 整个妊娠的胚胎中的后代。如此精确的血统 映射是理解发展决定因素的先决条件 细胞增殖，分化和迁移。了解这些 机制对于理解先天性的起源至关重要 畸形。 我们已经在转基因小鼠中设计了原位谱系标记系统 这应该标记一个可遗传的细胞中的特定细胞种群 在胚胎开发过程中，自主，不浸润的方式。 此外，该系统可以与同源 重组以进行谱系/组织特异性体内诱变。我们 已经利用了在酵母中发现的解放重组系统 这些目的：重组酶FLP催化重组 直接重复FLP重组靶标（FRT）切除 中间的DNA。自1993年启动该项目以来，我们有：（1） 构建了一组模块化的FLP和通用目标（FRT破坏了 lacz）向量； （2）证明了目标有效的FLP激活 胚胎干细胞中的转基因； （3）生成单独的FLP和 靶（FRT干扰LACZ）转基因小鼠系； （4）证明 FLP介导的重组来自Flpxtarget十字架的胚胎。我们现在 以独特的位置将这个系统在体内充分表征为一个 用于标记细胞谱系和定向修改的工具 小鼠基因组。 因为神经rest细胞广泛迁移并分化为形成 他们最适合研究谱系和谱系的多种细胞类型 环境相互作用以确定细胞命运。神经 细胞发育与许多先天性畸形有关 包括神经管，肢体，颅，肠神经节和心脏 缺陷，耳聋和胸腺发育不全。一个主要的未解决问题是 这些细胞群体何时以及如何产生这种表型 多样性。针对拟议的目的，我们已经产生了转基因小鼠 在背CNS中表达FLP作为激活神经LACZ的一种手段 波峰祖先。我们将：（1）利用此标记系统映射 鼠神经rest; （2）确定相似性和差异 鼠地图和小鸡的地图，后者提供了我们的大部分 当前知识； （3）从细胞命运的描述转移到分析 通过突变胚胎中的谱系研究的基本机制。 与斑点（PAX-3）相关的发病机理的机制，致死 发现（IS）和W（c-kit）表型将洞悉 平行人类综合征的发病机理（例如Waardenburg综合征和 Hirschsprung病）。