Homologous Recombination in a Secretory Mouse Cell Line

描述（申请人提供）： 调节胞吐/突触的分子事件的中断 传播引起多种神经病理学。建模系统， 允许探索控制这些的潜在分子机制， 过程有问题。探索/发展补助金（R21） 一项申请试图开发一种可以遗传修饰的小鼠细胞系， 在单细胞水平上研究囊泡的释放，并提供试验数据 供以后的补助金申请使用。在神经系统中，突触传递 是由Ca 2+进入突触前末梢启动的， 激活充满发射器的囊泡与突触前 膜的突触结合蛋白I（Synaptotagmin I，syt I）是一种完整的囊泡蛋白， 假定作为胞吐作用的Ca 2+传感器。一种遗传学方法， 研究syt I作为钙离子传感器的功能， 敲除小鼠syt I纯合敲除小鼠可存活至48小时 在出生后，杂合子动物在表型上无法区分 从野生型。这些问题在基因敲除动物中并不少见， 难以研究syt I的功能作用。 1.第一个目的是引入靶向载体来破坏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 已经鉴定了一种小鼠嗜铬细胞瘤细胞系，其表现出钙依赖性 膜结合囊泡的胞吐作用。我们设计了特定的目标 载体连接到细胞的syt I基因。我们将尝试引入 将特异性靶向载体导入细胞中，以筛选具有阳性和 阴性选择，并从筛选的菌株中获得基因组DNA并进行检测， 细胞进行靶向中断syt I基因。我们认为，这一独特的 细胞系将作为一个很好的模型系统来探讨syt的功能 我用生物物理技术。 2.第二个目的是描述敲除中Ca 2+调节的胞吐作用。 细胞系我们将使用电容和安培测量， 结合[Ca 2 +]i测量，以测量膜片钳的胞吐作用。 单细胞将刺激细胞以引发Ca 2+依赖性分泌。从 Ca 2+内流和囊泡释放的分析，我们将确定Ca 2 + 敲除细胞系的分泌依赖性，并将其与分泌 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， 可用于确定syt I是否为快速测定Ca 2+的Ca 2+传感器。 这些分泌细胞中的Ca 2+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亚型 选择向量，然后将每个向量通过稳定 转染以研究不同SYT同种型在分泌中的作用。

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.