TY - JOUR

T1 - Assessing cell lines with inducible depletion of cohesin and condensins components through analysis of metaphase chromosome morphology

AU - Yunusova, A. M.

AU - Smirnov, A. V.

AU - Pristyazhnuk, I. E.

AU - Shnaider, T. A.

AU - Maltseva, E. K.

AU - Afonnikova, S. D.

AU - Gusev, O. A.

AU - Battulin, N. R.

N1 - We thank Masato Kanemaki for sharing the HCT-116 RAD21-mAC and SMC2-mAC cell lines. This work was supported by Russian Science Foundation grant No. 23-74-00055. Cell culture was performed at the Collective Center of ICG SB RAS\u201CCollection of Pluripotent Human and Mammalian Cell Cultures for Biological and Biomedical Research\u201D, project number FWNR-2022=0019 (https://ckp.icgen.ru/cells/; http://www.biores.cytogen.ru/brc_cells/collections/ICG_SB_RAS_CELL). Cryoarchiving and primary analysis of control cell lines HCT-116 RAD21-mAC and SMC2-mAC was supported by the Ministry of Science and Higher Education of the Russian Federation, grant 075-15-2021-1344.

PY - 2024/4

Y1 - 2024/4

N2 - One of the most productive strategies for finding the functions of proteins is to study the consequences of loss 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 lose fitness when the function of a protein is lost. For such proteins, the most productive strategy is to use inducible protein degradation 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 mammalian cells and insert a sequence encoding a degron domain into the gene of interest. A crucial aspect of development of cell lines engineered for inducible protein depletion is the selection of cell clones with efficient auxin-dependent degradation of the protein of interest. To select clones induced by depletion of the architectural chromatin proteins RAD21 (a component of the cohesin complex) and SMC2 (a component of the condensin complex), we propose to use 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 and SMC2. The degradation efficiency of the protein of interest was assessed by flow cytometry, Western blotting and metaphase chromosome morphology test. Based on our tests, we showed that the clones we established with the SMC2 degron effectively and completely lose protein function when induced by auxin. However, none of the HAP1 clones we created with the RAD21 degron showed complete loss of RAD21 function upon induction of degradation by auxin. In addition, some clones showed evidence of loss of RAD21 function even in the absence of induction. The chromosome morphology test turned out to be a convenient and informative method for clone selection. The results of this test are in good agreement with flow cytometry analysis and Western blotting data.

AB - One of the most productive strategies for finding the functions of proteins is to study the consequences of loss 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 lose fitness when the function of a protein is lost. For such proteins, the most productive strategy is to use inducible protein degradation 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 mammalian cells and insert a sequence encoding a degron domain into the gene of interest. A crucial aspect of development of cell lines engineered for inducible protein depletion is the selection of cell clones with efficient auxin-dependent degradation of the protein of interest. To select clones induced by depletion of the architectural chromatin proteins RAD21 (a component of the cohesin complex) and SMC2 (a component of the condensin complex), we propose to use 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 and SMC2. The degradation efficiency of the protein of interest was assessed by flow cytometry, Western blotting and metaphase chromosome morphology test. Based on our tests, we showed that the clones we established with the SMC2 degron effectively and completely lose protein function when induced by auxin. However, none of the HAP1 clones we created with the RAD21 degron showed complete loss of RAD21 function upon induction of degradation by auxin. In addition, some clones showed evidence of loss of RAD21 function even in the absence of induction. The chromosome morphology test turned out to be a convenient and informative method for clone selection. The results of this test are in good agreement with flow cytometry analysis and Western blotting data.

KW - SMC proteins

KW - chromosome condensation

KW - degron

UR - https://www.scopus.com/record/display.uri?eid=2-s2.0-85190507251&origin=inward&txGid=95fbf8cfb28130ccb8792cdbfcfdc842

UR - https://www.elibrary.ru/item.asp?id=65616483

UR - https://www.mendeley.com/catalogue/29112c51-b386-30a4-9190-6563b6da8d32/

U2 - 10.18699/vjgb-24-16

DO - 10.18699/vjgb-24-16

M3 - Article

C2 - 38686135

VL - 28

SP - 138

EP - 147

JO - Вавиловский журнал генетики и селекции

JF - Вавиловский журнал генетики и селекции

SN - 2500-0462

IS - 2

M1 - 2

ER -