Figure 1: Methods for establishing hepatocyte cell lines using conventional approaches (a), HGD-based gene delivery (b) and EP-based gene delivery (c). In (a), the liver is first perfused with collagenase to isolate single hepatocytes, to which in vitro gene delivery using EP, liposomes, or virus is applied. In (b), HGD is performed with transposon vectors, and 2 days later, perfusion with collagenase is performed to isolate single hepatocytes. In (c), transposon vectors are directly introduced into the parenchyma of the livers of anesthetized mice, and then, the injected portion is immediately subjected to in vivo EP using tweezer-type electrodes and a square-pulse generator. The treated mice are kept for 7 days prior to collagenase perfusion. These resulting collagenase-dissociated hepatocytes cells are then cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% fetal bovine serum (FBS), 1 μg/mL of insulin, and 4 μg/mL dexamethasone on a collagen-coated dish (d). One day after hepatocyte isolation, puromycin (0.3 μg/mL) is added to the medium to eliminate untransfected hepatocytes and then kept for 7-10 days for generation of viable colonies.