Cas9 Expressing Immortalized Chicken Embryonic Fibroblast (DF1) Cell Line

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.