To generate the Cas9 Expressing Immortalized Chicken Embryonic Fibroblast (DF1) Cell Line, Cas9-2A-PuroR was cloned into pT2KCAGGS using Gibson Assembly (NEB). The DF1 cells were then transfected, and selected for using puromycin. The constitutive nuclear expression of Cas9 was validated by immunostaining using an antibody against Cas9, followed by DAPI-counterstaining. This DF1 cell line can be used to greatly streamline the process of experimental designs for future CRISPR/Cas9 knockout experiments in chick embryos.Organism: ChickenSource Organ: EmbryoBioSafety Level: IIQuantity: 10⁶ cells/1.0 mlGrowth Properties: AdherentMorphology: FibroblastMarkers: PuromycinPropagation: The base medium for this cell line is Prigrow III medium available at abm, Cat. No. TM003. To make the complete growth medium, add the following components to the base medium: fetal bovine serum (TM999)* to a final concentration of 10% and Penicillin/Streptomycin Solution (G255) to a final concentration of 1%.Change media every 2-3 days.Carbon dioxide (CO2): 5%, Temperature: +37.0°C.* Do not use heat-inactivated FBS for cell culture unless specified otherwise.Freeze Thaw:Quality Control: 1) ImmunostainingShipping Conditions: Dry IceStorage Conditions: -180°C