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Assessing cell lines with inducible depletion of cohesin and condensins components through analysis of metaphase chromosome morphologyстатья
Статья опубликована в журнале из списка RSCI Web of Science
Информация о цитировании статьи получена из
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Статья опубликована в журнале из перечня ВАК
Статья опубликована в журнале из списка Web of Science и/или Scopus
Дата последнего поиска статьи во внешних источниках: 23 января 2026 г.
- Авторы: Yunusova A.M., Smirnov A.V., Pristyazhnuk I.E., Shnaider T.A., Maltseva E.K., Afonnikova S.D., Gusev O.A., Battulin N.R.
- Журнал: Вавиловский журнал генетики и селекции
- Том: 28
- Номер: 2
- Год издания: 2024
- Издательство: ИЦиГ СО РАН
- Местоположение издательства: Новосибирск
- Первая страница: 138
- Последняя страница: 147
- DOI: 10.18699/vjgb-24-16
- Аннотация: One of the most productive strategies for finding the functions of proteins is to study the consequences ofloss of protein function. For this purpose, cells or organisms with a knockout of the gene encoding the protein of interest are obtained. However, many proteins perform important functions and cells or organisms could suddenly losefitness when the function of a protein is lost. For such proteins, the most productive strategy is to use inducible proteindegradation systems. A system of auxin-dependent protein degradation is often implemented. To use this system,it is sufficient to introduce a transgene encoding a plant-derived auxin-dependent ubiquitin ligase into mammaliancells and insert a sequence encoding a degron domain into the gene of interest. A crucial aspect of developmentof cell lines engineered for inducible protein depletion is the selection of cell clones with efficient auxin-dependentdegradation of the protein of interest. To select clones induced by depletion of the architectural chromatin proteinsRAD21 (a component of the cohesin complex) and SMC2 (a component of the condensin complex), we propose touse the morphology of metaphase chromosomes as a convenient functional test. In this work, we obtained a series of clones of human HAP1 cells carrying the necessary genetic constructs for inducible depletion of RAD21 andSMC2. The degradation efficiency of the protein of interest was assessed by flow cytometry, Western blotting andmetaphase chromosome morphology test. Based on our tests, we showed that the clones we established with theSMC2 degron effectively and completely lose protein function when induced by auxin. However, none of the HAP1clones we created with the RAD21 degron showed complete loss of RAD21 function upon induction of degradationby auxin. In addition, some clones showed evidence of loss of RAD21 function even in the absence of induction. Thechromosome morphology test turned out to be a convenient and informative method for clone selection. The resultsof this test are in good agreement with flow cytometry analysis and Western blotting data.
- Добавил в систему: Баттулин Нариман Рашитович