IN VIVO LABELING · Labelling of fusion proteins in living mice with synthetic probes
6РП — Действия „Мария Кюри“
- Период
- 2007-01-01 → 2008-12-31
- Финансиране от ЕС
- 173 264 €
- Участници
- 1
- Схема
- EIF
Линиите свързват координатора с партньорите.
Накратко на български
Концентрацията на калций в конкретни части от живи клетки, например в ядрата на мускулни клетки на мишки, се измерва чрез специални синтетични маркери. Това помага за по-точното проследяване на процесите по предаване на сигнали в организма.
Кратко обяснение, генерирано от езиков модел по текста на CORDIS. Оригиналът е по-долу.
Резултати накратко
Final Activity Report Summary - IN VIVO LABELING (Labelling of fusion proteins in living mice with synthetic probes)
The role of calcium in signal transduction relies on the precise spatial and temporal control of its concentration. The existing means to detect fluctuations in Ca2+ concentrations with adequate temporal and spatial resolution are limited. We introduced in this project a method to measure Ca2+ concentrations in defined locations in living cells that is based on linking the Ca2+-sensitive dye Indo-1 to SNAP-tag fusion proteins. Fluorescence spectroscopy of SNAP-Indo-1 conjugates in vitro showed that the conjugates retained the Ca2+-sensing ability of Indo-1. In a proof-of-principle experiment, local Ca2+ sensing was demonstrated in cultured primary muscle cells of mice expressing a nucleus-localised SNAP-tag fusion. Ca2+ concentrations inside nuclei of resting cells were measured by SEER (shifted excitation and emission ratio-ing) of confocal microscopic images of fluorescence. After permeabilising, the plasma membrane, changes in the bathing solution induced corresponding changes in nuclear [Ca2+] that were readily detected and used for a preliminary calibration of the technique. This work thus demonstrates the synthesis and application of SNAP-tag-based Ca2+ indicators that combine the spatial specificity of genetically encoded calcium indicators with the advantageous spectroscopic properties of synthetic indicators.
Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз
Цел на проекта
Characterizing the movement, interactions and chemical microenvironment of a protein inside the living cell is the key towards a detailed understanding of its function. While in the past most functional studies of proteins have been performed in vitro or with cell culture experiments, it has become increasingly evident that protein function also needs to be studied on an organismal level. To achieve this objective, there is a generally acknowledged need for new and innovative approaches to study individual proteins in living animals. In this project I propose to develop a method for the covalent and specific labelling of fusion proteins in transgenic mice with synthetic probes that allows performing in vivo imaging studies of selected proteins in the living animal.The approach is based on expression of the protein of interest as fusion with mutants of O6-alkylguanine-DNA alkyltransferase and its labelling with synthetic benzylguanine derivatives, a technology that has been developed in the host laboratory for this project and has already been applied to specifically label proteins in cell cultures. Transgenic mice will be generated using the lentivirus technique. In summary, the proposed research aims at the development of new and innovative approaches to study central biological problems and should allow establishing the covalent labelling of fusion proteins in living animals as a key technology in life sciences.
Оригинален текст от CORDIS (на английски).
Участници
- ECOLE POLYTECHNIQUE FEDERALE DE LAUSANNE · LAUSANNEКоординаторШвейцария
Връзки
Данни: CORDIS, © Европейски съюз