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）：间隙连接（GJ）存在于所有动物的发育早期阶段。然而，尽管间隙连接无处不在，但我们对间隙连接的了解却最少。它们在细胞生物学和发育中的作用仍然很少被探索。 GJ 很难检测到。在体内，需要显微注射，这限制了该技术只能用于大型和/或异常容易接近的细胞。我们建议开发一种分子生物学方法，用于体内检测持久性和瞬时性 GJ，而无需细胞内注射。 将制备具有组织特异性表达 B-半乳糖苷酶的转基因动物。动物将被注射 B-gal 底物 (C12FDG)，该底物被细胞吸收并水解成小的荧光报告分子。 B-gal 太大而无法通过间隙连接，但报告分子可以。表达B-gal的细胞可以用抗体检测；任何不表达 6-gal，但显示报告颜色的细胞都必须通过 GJ 接收其颜色。另一种类似的方法将进行测试，该方法利用转运蛋白的组织特异性表达来向突触前细胞加载小示踪剂，该示踪剂可以穿过 GJ 并在连接后细胞中被检测到。对照包括证明 GJ 突变体阻断传播。 作为第一次使用，我们希望将该方法应用于以下问题。在过去的 30 年里，许多作者已经证明，在化学突触形成之前的神经系统发育过程中会出现临时间隙连接 (GJ)。假设 GJ 通讯在功能性化学突触的建立中发挥直接因果作用。然而，还不可能阻止 GJ 从而证明因果关系。 在成人眼中，没有视网膜与椎板间隙连接。然而，我们发现两个间隙连接基因的突变会破坏那里的化学突触传递。在突变体中，传输可以通过 GJ 转基因恢复，但前提是它们在眼睛发育期间表达；该基因在成人中的表达并不能挽救。我们假设发育过程中的层间隙连接对于化学突触传递的后期发育至关重要。我们希望使用本文描述的方法来直接证明这种瞬态 GJ