Homologous Recombination in a Secretory Mouse Cell Line

描述(由申请人提供)： 调控胞吐/突触的分子事件的破坏 传播会引起各种神经病变。模型系统 允许探索控制这些的潜在分子机制 流程是有问题的。这项探索性/发展性赠款(R21) 应用寻求开发一种可转基因的小鼠细胞系 在单细胞水平上研究囊泡释放，并提供中试数据 以备日后拨款申请之用。在神经系统中，突触传递 是由Ca~(2+)进入突触前终末启动的 激活充满递质的囊泡与突触前的融合 薄膜。突触素I(Synaptopagmin I，Syt I)是一种完整的囊泡蛋白。 被认为是胞吐作用的钙传感器。一种遗传方法 研究了syt I作为钙离子传感器的功能已经产生 基因敲除老鼠。Syt i纯合子基因敲除小鼠存活到48小时。 出生后，杂合子动物在表型上是无法区分的 来自野生型。这些问题，在基因敲除动物中并不少见，使 很难研究syt I的功能作用。 1.第一个目的是引入靶向载体来干扰syt I基因 通过在非胚胎起源的小鼠细胞系中进行同源重组。我们 已经鉴定出一种小鼠嗜铬细胞瘤细胞系，该细胞系表现出钙依赖 膜结合囊泡的胞吐作用。我们已经设计了具体的目标 载体到细胞的syt I基因。我们将尝试介绍 将特定的靶标载体导入细胞，筛选阳性和阳性的细胞 负选择，并从筛选的菌株中获得基因组DNA并进行检测 靶向阻断syt I基因的细胞。我们相信这一独一无二的 细胞系将成为探索syt功能的良好模型系统。 我熟悉生物物理技术。 2.第二个目的是研究基因敲除过程中钙离子调控的胞吐作用 细胞系。我们将使用电容和安培测量，在 结合[Ca~(2+)]i测量膜片钳的胞吐作用 单细胞。细胞将被刺激以诱导钙依赖的分泌。从… 分析Ca~(2+)内流和囊泡释放，测定Ca~(2+) 基因敲除细胞株分泌的依赖性及其与分泌物的比较 在野生型细胞中。 我们的短期目标是建立一种syt I纯合子基因敲除细胞系， 可用于确定syt i是否是快速检测的钙离子传感器 这些分泌细胞的分泌依赖于钙离子。我们的长期目标是 用不同的方法敲除小鼠细胞系中的每一种syt亚型 选择向量，然后将每个选择向量按稳定的方式添加回细胞 目的：研究不同syt亚型在细胞分泌中的作用。

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.