Generation of HEK-293 stable cell lines with disrupted expression of ribosomal protein S6 kinase (S6K1) isoforms using the CRISPR/Cas9 genome editing system

• Main Authors: Zaiets, I.V., Sivchenko, A.S., Khoruzhenko, A.I., Filonenko, V.V.
• Format: Article
• Language: English
• Published: Інститут молекулярної біології і генетики НАН України 2017
• Series: Вiopolymers and Cell
• Subjects: Molecular and Cell Biotechnologies
• Online Access: http://dspace.nbuv.gov.ua/handle/123456789/152987

Aim. To generate HEK-293 cells with disrupted expression of S6K1 isoforms: p85, p70 and p60. Methods. CRISPR/Cas9 gene editing, Western blotting, immunofluorescent analysis, RT-PCR analysis, MTT assay, scratch assay. Results. Several clones of HEK-293 cells with a complete loss of p85/p70/p60-S6K1 protein expression were generated. The effects of p85/p70/p60-S6K1 knockout on Akt/mTORC1/S6K1 signaling and cell proliferation and migration were assessed. Conclusions. The generated cell lines can be used to study a role played by S6K1 in cell physiology and to gain more detailed insight into cellular functions of the S6K1 isoforms. The HEK-293 cells exhibit down-regulation of Akt phosphorylation on Ser473 and subsequent attenuation of cell growth rate, as well as inhibition of cell motility.