For lentiviral packaging, HEK293T cells were seeded in a 10 cm dish 1 day before transfection. The indicated viral plasmids Lentidcas9-vp64-blast (Addgene #61425) or FgH1tUTG (Addgene #70183) were co-transfected with lentivirus packaging plasmids pMD2.G (Addgene #12259) and psPAX2 (Addgene #12260) in a 4:2:3 ratio using PEI MAX (molecular weight 40,000) (Polysciences, Inc., USA). The next day after transfection, media were changed to Opti-MEM® I Reduced Serum Media (Thermo Fisher Scientific). At 48 h after transfection, virus-containing media were collected by Amicon Ultra-15 Centrifugal Filter Units 100 kDa (Millipore), passed through a 0.45 µm polyethersulfone filter (Millipore), aliquoted and stored at −80 °C before infection or titration. mESCs were seeded 1 day before transduction. The next day, the medium was changed to fresh and protamine sulfate 50 μg/ml (Sigma) and Lentidcas9-vp64-blast or FgH1tUTG were added. Infected green fluorescent protein-positive cells mESCs were sorted in BD FACSAria™ III. FgH1tUTG-transduced cells were infected with Lentidcas9-vp64-blast and selected by 5 µg/ml of blasticidin (Sigma) for 1 week. Oligonucleotides were designed using the online tools http://sam.genome-engineering.org/database and https://www.nature.com/articles/nbt.2647: F-TCCCGAGGCGGGGCGGGGCTTAGTT; R-AAACAACTAAGCCCCGCCCCGCCTC; and cloned into FgH1tUTG vector.
Suvorova I.I., Knyazeva A.R., Petukhov A.V., Aksenov N.D, & Pospelov V.A. (2019). Resveratrol enhances pluripotency of mouse embryonic stem cells by activating AMPK/Ulk1 pathway. Cell Death Discovery, 5, 61.
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