Lenticrispr v2 egfp

Dbt cells were previously transduced either with a constitutive MYCN expression construct (Dbt/MYCN) or with MYCN and an inducible P3F expression construct (Dbt/MYCN/iP3F) (5 (link),6 (link)). The pCMV6-Entry vector expressing FGF8b cDNA or the empty vector EV (Origene, Rockville, MD) was stably transfected using lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA). CRISPR/Cas9 targeting of FGF8 was performed with single-guide RNAs cloned into the lentiCRISPR v2-eGFP (GenScript, Piscataway NJ) and compared with the control non-targeting vector lentiCRISPR v2-eGFP (CR-control). Dbt/MYCN/iP3F cells were transduced with a green fluorescent protein (GFP) expression construct as previously described (6 (link)). Lentiviral particles were packaged using HEK293T cells with the human immunodeficiency virus-based pPACK-H1 Packaging Plasmid Mix (System Biosciences, Mountain View, CA).