产生康涅素表达稳定细胞池
Abraham Tettey-Matey1, Chiara Di Pietro1, Viola Donati1,2
1Institute of Biochemistry and Cell Biology, Italian National Research Council, Rome, Italy.
Methods in molecular biology (Clifton, N.J.)
|April 5, 2024
概括
这项研究介绍了一种基于lentivirus的协议,用于创建稳定的细胞池以研究基因功能. 这种方法使得在体外和体内测试中实现一致的转基因表达,包括成像.
科学领域:
- 分子生物学分子生物学
- 细胞生物学 细胞生物学
- 遗传学 遗传学是一种遗传学.
背景情况:
- 稳定的细胞池提供可复制的转基因传播,而不是短暂的表达.
- 诱导性转基因表达对于在各种测试中研究基因功能至关重要.
- 莲氏病毒 (LV) 促进了转基因的传递,基因组的整合和在多种细胞类型中的表达.
研究的目的:
- 开发一种lentivirus载体,用于生成可诱导多克西环素的稳定细胞池.
- 建立一种用于研究感兴趣基因 (GOI) 功能,特别是像Cx32.2.这样的连接蛋白的协议.
- 为了实现功能测定,包括用GCaMP6s进行Ca2+成像.
主要方法:
- 开发一种可诱导多克西环素的双基斯特龙型晶状病毒转移载体.
- 通过短暂的转染和生成稳定的细胞池来产生LV粒子.
- 活细胞和固定细胞中转基因表达的共聚焦显微镜的逐步免疫标记协议.
主要成果:
- 使用开发的LV向量成功生成稳定细胞池.
- 通过免疫标记证明Cx32蛋白表达和局部化.
- 验证功能测试的协议,如Ca2+成像.
结论:
- 开发的lentivirus载体和协议为创建稳定的细胞池提供了强大的工具.
- 这种方法可以使用各种分子,免疫和光学测试来研究基因功能.
- 该协议可适应在体外和体内研究中表达任何GOI.
相关概念视频
01:37Gap Junctions
Multicellular organisms employ a variety of ways for cells to communicate with each other. Gap junctions are specialized proteins that form pores between neighboring cells in animals, connecting the cytoplasm between the two, and allowing for the exchange of molecules and ions. They are found in a wide range of invertebrate and vertebrate species, mediate numerous functions including cell differentiation and development, and are associated with numerous human diseases, including cardiac and...
01:27Gap Junctions
The cytoplasm of adjacent animal cells can exchange small molecules, ions, and secondary messengers via the communication channels which form the gap junctions. These junctions comprise a few hundred to thousands of molecular channels, each made of two halves, called the connexon hemichannel. A connexon is a hexamer of six transmembrane connexin proteins, which assemble radially, thus forming a pore or channel in the center. One connexon hemichannel docks with a corresponding connexon on the...