Using Transgenics to Identify Functional Gap Junctions

使用转基因技术来识别功能性间隙连接

• 批准号: 6902332
• 负责人: ROBERT J WYMAN
• 金额: $ 8.18万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6902332
• 关键词: Drosophilidae; arthropod genetics; beta galactosidase; beta lactamase; biotechnology; cell cell interaction; deoxyglucose; developmental neurobiology; egg /ovum; enzyme substrate; gap junctions; gene expression; glycine; interneurons; membrane transport proteins; method development; molecular genetics; salivary glands; small molecule; synaptogenesis; tissue mosaicism; transfection /expression vector; visual photoreceptor

DESCRIPTION (provided by applicant): Gap junctions (GJs) occur in all animals from very early stages of development. Yet, despite their ubiquity, gap junctions are the cell structures about which the least is known; their role in cell biology and development is still barely explored. GJs are difficult to detect. In vivo, microinjection is required which limits the technique to large and/or unusually accessible cells. We propose to develop a molecular biological method for the in-vivo detection of both enduring and transient GJs without the need for intracellular injection. Transgenic animals will be made with tissue specific expression of B-galactosidase. The animal will be injected with a B-gal substrate (C12FDG) which is taken up by cells and hydrolysed to a small fluorescent reporter molecule. B-gal is too large to pass through gap junctions, but the reporter molecule can. Cells expressing B-gal can be detected with antibodies; any cell not expressing 6-gal, but exhibiting the reporter color must have received its color via GJs. Another similar method will be tested which makes use of tissue specific expression of transporters to load the presynaptic cells with a small tracer which can pass through GJs and be detected in post-junctional cells. Controls include the demonstration that GJ mutants block the transmission. As a first use, we wish to apply the method, to the following problem. During the last 30 years, many authors have demonstrated temporary gap junctions (GJs) occurring in nervous system development just prior to the period when chemical synapses are formed. GJ communication has been hypothesized to play a direct causal role in the establishment of functional chemical synapses. However, it has not been possible to block the GJs and thus prove a causal link. In the adult eye, there are no retina-to-lamina gap junctions. Yet, we showed that mutations in two gap junction genes disrupt chemical synaptic transmission there. In mutants, transmission can be restored by GJ transgenes, but only if they are expressed during the period of eye development; expression of the genes in the adult does not rescue. We hypothesize that lamina gap junctions during development are essential for the later development of chemical synaptic transmission. We wish to use the method described herein to directly demonstrate such transient GJs

描述（由申请人提供）：从发展的早期阶段，所有动物的间隙连接（GJ）都出现在所有动物中。然而，尽管存在无处不在，但间隙连接是最少已知的细胞结构。它们在细胞生物学和发育中的作用几乎没有得到探索。 GJ很难检测到。在体内，需要显微注射，这将技术限制在大型和/或异常访问的单元格上。我们建议开发一种分子生物学方法，用于在体内检测持久和瞬时GJ，而无需细胞内注射。 转基因动物将用B-半乳糖苷酶的组织特异性表达制成。将向动物注入B-GAL底物（C12FDG），该基材被细胞吸收并水解为小荧光报告基因分子。 b-gal太大而无法通过间隙连接，但是记者分子可以。可以用抗体检测表达B-GAL的细胞。任何不表达6-GAL的细胞，但展示报告基因的颜色都必须通过GJ收到其颜色。将测试另一种类似的方法，该方法利用转运蛋白的组织特异性表达，用小型示踪剂加载突触前细胞，该示踪剂可以通过GJ并在后官能细胞中检测到。控件包括GJ突变体阻止传输的演示。 作为首次使用，我们希望将该方法应用于以下问题。在过去的30年中，许多作者表现出在形成化学突触之前的神经系统发育中发生的临时间隙连接（GJ）。假设GJ通信在功能化学突触的建立中起着直接的因果作用。但是，不可能阻止GJ，因此证明了因果关系。 在成人眼中，没有视网膜到薄片的间隙连接。然而，我们表明两个间隙连接基因中的突变破坏了那里的化学突触传递。在突变体中，GJ转基因可以恢复传播，但前提是它们在眼睛发育期间表达；基因在成年人中的表达不会营救。我们假设开发过程中的椎板间隙连接对于后来的化学突触传播至关重要。我们希望使用此处描述的方法直接演示这种瞬态GJ