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For the generation of cell lines carrying randomly integrated transgenes using lentiviral transduction, DNA constructs were first packaged into lentiviral particles. For this, 1 × 106 Hek293T cells were seeded into one well of a 6-well plate and transfected the next day with the lentiviral packaging vectors: 1.2 μg pLP1, 0.6 μg pLP2, and 0.4 μg VSVG (Thermo Fisher Scientific), together with 2 μg of the desired construct using Lipofectamine 2000 (Thermo Fisher Scientific). Hek293T supernatant containing the viral particles was harvested after 48 h. 0.2 × 106 mESCs were seeded per 12-well and transduced the next day with 500 μl of viral supernatant and 8 ng/μl polybrene (Sigma). Antibiotic selection was started 2 days after transduction and kept for at least 3 passages.

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