Homologous Recombination in a Secretory Mouse Cell Line

分泌型小鼠细胞系中的同源重组

• 批准号: 6572294
• 负责人: AMY B HARKINS
• 金额: $ 18.3万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-12-15 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6572294
• 关键词: animal tissue; biotechnology; calcium flux; cell line; electrical measurement; electrophysiology; exocytosis; gene targeting; membrane activity; molecular genetics; neural transmission; neurogenetics; neuroregulation; pheochromocytoma; protein structure function; recombinant DNA; synaptic vesicles; synaptotagmin; technology /technique development; transfection; voltage /patch clamp

DESCRIPTION (Provided By Applicant): Disruptions of the molecular events that regulate exocytosis/synaptic transmission give rise to a variety of neuropathologies. Model systems that allow exploration of the underlying molecular machinery that control these processes are problematic. This exploratory/developmental grant (R21) application seeks to develop a mouse cell line that can be genetically modified to study vesicle release at the single-cell level, and to provide pilot data for subsequent grant applications. In the nervous system, synaptic transmission is initiated by the entry of Ca2+ into the presynaptic terminal, which activates the fusion of transmitter-filled vesicles with the presynaptic membrane. Synaptotagmin I (syt I), an integral vesicular protein, has been postulated to act as the Ca2+ sensor for exocytosis. One genetic approach to study the function of syt I as a Ca2+ sensor has been the generation of knockout mice. The syt I homozygous knockout mice are viable until 48 hours after birth, and the heterozygous animals are phenotypically indistinguishable from wild-type. These problems, not uncommon in knockout animals, make it difficult to study the functional role of syt I. 1. The first aim is to introduce a targeting vector to disrupt the syt I gene by homologous recombination in a mouse cell line of non-embryonic origin. We have identified a mouse pheochromocytoma cell line that exhibits Ca2+-dependent exocytosis of membrane-bound vesicles. We have designed specific targeting vectors to the syt I gene of the cells. We will attempt to introduce the specific targetig vectors into the cells, to screen cells with positive and negative selection, and to obtain and test the genomic DNA from the screened cells for targeted interruption of the syt I gene. We believe that this unique cell line will serve as an excellent model system to probe the function of syt I with biophysical techniques. 2. The second aim is to characterize Ca 2+-regulated exocytosis in the knockout cell line. We will use capacitance and amperometric measurements, in conjunction with [Ca2+]i measurements, to measure exocytosis from patch-clamped single cells. Cells will be stimulated to elicit Ca2+-dependent secretion. From the analysis of Ca2+-influx and vesicle release, we will determine the Ca2+ dependency of secretion for the knockout cell line and compare it to secretion in wild-type cells. Our short-term goal is to establish a syt I homozygous knockout cell line that can be used to determine whether syt I is the Ca2+ sensor for rapid Ca2+-dependent secretion in these secretory cells. Our long-term goal is to knock out each of the syt isoforms in the mouse cell line, using different selection vectors, and then add each one back to the cell by stable transfection to study the role that different syt isoforms play in secretion.

描述（由申请人提供）： 调节胞吐作用/突触的分子事件的破坏 传播引起多种神经病理学。模型系统 允许探索控制这些的潜在分子机制 流程有问题。这项探索性/发展补助金 (R21) 申请旨在开发一种可以进行基因改造的小鼠细胞系 研究单细胞水平的囊泡释放，并提供试点数据 用于后续拨款申请。在神经系统中，突触传递 由 Ca2+ 进入突触前末梢启动， 激活充满递质的囊泡与突触前的融合 膜。突触结合蛋白 I (syt I) 是一种完整的囊泡蛋白，已被 假定充当胞吐作用的 Ca2+ 传感器。一种遗传方法 研究 syt I 作为 Ca2+ 传感器的功能已经产生 基因敲除小鼠。 syt I 纯合基因敲除小鼠可存活 48 小时 出生后，杂合动物在表型上无法区分 来自野生型。这些问题在基因敲除动物中并不少见，使得 很难研究 syt I 的功能作用。 1. 第一个目标是引入打靶载体来破坏syt I基因 通过在非胚胎来源的小鼠细胞系中同源重组。我们 已鉴定出具有 Ca2+ 依赖性的小鼠嗜铬细胞瘤细胞系 膜结合囊泡的胞吐作用。我们设计了具体的目标 载体到细胞的 syt I 基因。我们将尝试介绍 特定的靶向载体进入细胞，筛选阳性和阴性细胞 阴性选择，并从筛选的基因组DNA中获取并测试 用于有针对性地中断 syt I 基因的细胞。我们相信，这种独特的 细胞系将作为一个优秀的模型系统来探测syt的功能 我用生物物理技术。 2. 第二个目标是表征敲除中 Ca 2+ 调节的胞吐作用 细胞系。我们将使用电容和电流测量， 结合 [Ca2+]i 测量，测量膜片钳的胞吐作用 单细胞。细胞将受到刺激，引发 Ca2+ 依赖性分泌。从 通过分析 Ca2+ 流入和囊泡释放，我们将确定 Ca2+ 敲除细胞系的分泌依赖性并将其与分泌进行比较 在野生型细胞中。 我们的短期目标是建立一个 syt I 纯合敲除细胞系 可用于快速判断syt I是否为Ca2+传感器 这些分泌细胞中的 Ca2+ 依赖性分泌。我们的长期目标是 使用不同的方法敲除小鼠细胞系中的每种 syt 同工型 选择向量，然后通过 stable 将每个向量添加回单元格 转染以研究不同 syt 亚型在分泌中所起的作用。