303 V. V. Holiar, N. Ya. Gotsulyak, A. I. Khoruzhenko © 2019 V. V. Holiar et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Bio- polymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 576.322, 577.22 Generation and characterization of the MCF-7 cell line with a knockout of a p85-S6K1 isoform of the ribosomal protein S6 kinase 1 V. V. Holiar, N. Ya. Gotsulyak, A. I. Khoruzhenko, I. V. Zaiets Institute of Molecular Biology and Genetics, NAS of Ukraine 150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03143 zaiets.i@gmail.com Aim. To generate the p85-S6K1 knockout MCF-7 breast cancer cell line and to evaluate the effect of p85-S6K1 on cell growth, migration and survival under stress conditions. Methods. CRISPR/Cas9 genome editing, Western blotting, immunofuorescence staining, MTT assay, in vitro scratch assay. Results. We generated two clones of the p85-S6K1 knockout MCF-7 cell line and tested their survival upon hydrogen peroxide treatment as well as the proliferation and migration rates. The generated cell clones display an impaired ability to survive under oxidative stress, exhibit inhibition of cell growth, cell motility and downregulation of rpS6 phosphorylation on Ser235/236/240/244 under cell starvation compared to the control cells. Conclusions. The p85-S6K1 isoform could be involved in modulation of cancer cell behaviour promoting cell growth, migration and survival. The obtained clones can be further used to study the participation of different S6K1 isoforms in the control of cell function. Keywords: mTOR/S6K1 signaling, CRISPR/Cas9, p85-S6K1 isoform. Introduction S6 ribosomal protein kinase 1 (S6K1) is an extensively studied serine/threonine protein kinase which functions as a component of the mTOR/S6K1-dependent signaling pathway. It is involved in the regulation of protein, lipid and nucleotide synthesis as well as cell proli- feration, survival, motility and other cellular processes (reviewed in [1, 2]). Overactivation of this kinase leads to the development of a number of disease states, including obesity, diabetes and cancer (reviewed in [2–4]). For example, S6K1 is frequently overexpressed in breast, lung, ovary, endometrial and thyroid cancers [5–9]. Its expression correlates with poor prognosis in patients with breast, kidney and hepatocellular carcinomas which makes S6K1 a potential therapeutic target for cancer treatment (reviewed in [5]). Furthermore, dys- regulation of mTOR/S6K1 signaling is of spe- cial signifcance in breast cancer. This signal- ISSN 1993-6842 (on-line); ISSN 0233-7657 (print) Biopolymers and Cell. 2019. Vol. 35. N 4. P 303–312 doi: http://dx.doi.org/10.7124/bc.000A0B