Porcine hepatocyte isolation and reversible immortalization mediated by retroviral transfer and site-specific recombination

Figure 1 Flow diagram of the preparation of isolated hepatocytes with a modified four-step retrograde perfusion technique.

Figure 2 Isolation technique and microscopic appearance of primary and immortalized porcine hepatocytes. A: Perfusion was conducted by inserting a suitable pipette into vessels exposed on a cut surface of the sample; B: Non-immortalized primary porcine hepatocytes grow slowly with low plating efficiency and a short life span; C: Immortalized porcine hepatocytes grow rapidly as islands and had an extended life span. Magnifications: B and C 100 ×.

Figure 3 Schematic drawings of the integrating component of retroviral vector SSR#69 before and after Cre-recombination. SSR#69 contains the hygromycin B resistance gene (Hyg R) as a positive selectable marker and the herpes simplex virus thymidine kinase gene (HSV-TK) as a negative selectable marker. The SV40T, Hyg R and HSV-TK genes are flanked by loxP sites.

Figure 4 Gene expression of liver-specific functions in immortalized and reverted cells. Lines 1 to 7, from left to right, Marker, Albumin’ (immortalized hepatocytes), Albumin (reverted hepatocytes), GAPDH’ (immortalized hepatocytes), GAPDH (reverted hepatocytes), SV40T’ (immortalized hepatocytes), SV40T (reverted hepatocytes), respectively.

Figure 5 Scheme of reversible immortalization. The primary porcine hepatocytes were immortalized by transfer of an oncogene (SV40T). After expansion of the immortalized cells, Cre/loxP recombination was performed to remove the oncogene (SV40T) and the cells reverted to their pre-immortalized state. (A-C) Double immunofluorescence of immortalized hepatocytes stained with DAPI (blue), which binds, together with monoclonal antibody anti-SV40T revealed with texas red-antibody conjugate (red). Blue and red fluorescence merged as purple. SV40T expression is revealed by intense staining of the cell nucleus.