组织特异性和内源性蛋白质标签与分裂的光蛋白
Gloria D Ligunas1,2, German Paniagua1, Jesselynn LaBelle1,2
1Department of Molecular Cell Biology, University of California, Merced, CA USA.
bioRxiv : the preprint server for biology
|March 11, 2024
概括
研究人员开发了一种新的分裂光蛋白系统,用于精确的,组织特异的斑马鱼蛋白质标签. 这种方法克服了当前技术的局限性,使得可以详细研究整个生物体中的蛋白质动态.
科学领域:
- 分子生物学分子生物学
- 遗传学 是一个遗传学.
- 发展生物学 发展生物学
背景情况:
- 基因编码的光蛋白对于研究动态生物过程至关重要.
- 目前用于光蛋白融合表达的现有方法存在局限性,特别是在整个生物体中,包括过度表达文物和内源标记的技术困难.
研究的目的:
- 为了克服当前蛋白质标签技术的局限性.
- 建立一种使用分裂光蛋白系统在斑马鱼中标记组织特异性和内源性蛋白质的方法.
主要方法:
- 使用了分割mNeonGreen2 (分割-mNG2) 系统,包括mNG21-10和mNG211片段.
- 通过标准转基因通过组织特异性促进剂来表达mNG21-10.
- 使用CRISPR/Cas9基因编辑插入mNG211到感兴趣的内源基因中.
- 共同表达了两个片段,以实现自我组装成一个功能光复合体.
主要成果:
- 在各种斑马鱼组织中成功实现了细胞骨架基因 (tubb4b和krt8) 的差异标记.
- 通过将mNG21-10元件固定在特定细胞区内,证明了操纵蛋白质功能的能力.
- 验证了分裂-mNG2系统用于内源蛋白标记和组织特定研究.
结论:
- 分裂mNG2系统为克服当前蛋白质标签策略的局限性提供了一个强大的工具.
- 这种方法使得斑马鱼的精确,组织特异和内生蛋白质标记成为可能.
- 该系统对分子和发育生物学中的各种研究领域具有广泛的应用性.
更多相关视频
11:06Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells
Published on: June 30, 2018
8.5K
11:04Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks
Published on: December 24, 2016
9.9K
相关概念视频
01:24Tagging and Fusion Proteins
6.6K
Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and...
6.6K
01:19Protein Dynamics in Living Cells
2.1K
Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
2.1K
03:31Labeling DNA Probes
8.2K
DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
8.2K