| Abstract [eng] |
Single-protein tracking can provide crucial information about cell biology. Electron microscopy can visualize molecules with higher resolution, than widely used fluorescent microscopy. To visualize specific proteins, EM tags are needed to introduce into the cells. CRISPR-Cas9 technology, derived from S.pyogenes, revolutionized the gene engineering approaches. It allowed for the precise and easy knock-in of the donor DNA sequences. EM tags for single proteins should localize molecules with high precision and give a low signal-to-noise ratio on the EM images. One of the most suitable tags for single-protein visualization is metallothionein. The application of this tag was checked in procaryotic and eukaryotic mammal cells. For a better understanding of disease and drug development, it is important to localize molecules in human cells. We successfully cloned EM reporters for visualization of mitochondria, endoplasmic reticulum, and inner and outer membranes of the human cells, and quality controlled them with Sangar sequencing. |
