(a)
(b)
(c)
Figure 3: Establishing hepatocyte cell lines by in vivo gene transfer of PB transposons in murine liver. (a) Schematic illustrating the vectors used. pT-LT, pT-E6/E7, pT-Liv#11, pT-EGFP, pT-tdTomato, and pT-ALB/pac are transposon vectors. pTrans is a vector conferring expression of PB transposase. Upon in vivo gene delivery, pT-EGFP, pT-E6/E7, and pT-Liv#11 (or pT-LT and pT-ALB/pac) are cotransfected with pTrans to obtain the 567 cell line (carrying EGFP, pac, E6, E7, and hTERT genes) or LT line (carrying EGFP, pac, and SV40T genes). Arrows under each vector show the orientation of transcription in each expression unit. Abbreviation: PB: PB ITRs; CAG: chicken β-actin-based promoter; p(A): poly(A) signal; E6: HPV18-derived E6 protein gene; E7: HPV18-derived E7 protein gene; SV40T: SV40 T antigen gene; EGFP: enhanced green fluorescent protein gene; hTERT: hemagglutinin-tagged human telomerase reverse transcriptase gene; CMVp: cytomegalovirus promoter; ALBp: human albumin promoter; pac: puromycin acetyltransferase gene; Transposase: PB transposase; pA: poly(A) sites. (b) Cell colonies 10 days after puromycin selection. Both LT (A-D) and 567 (E-H) lines are viable, showing bright EGFP-derived fluorescence. However, there are no viable cells in the control group (data not shown). From these colonies, we obtained clonal lines called LT1-1 and LT1-2, LT2-1 and LT2-2, 5671-1 and 5671-2, and 5672-1 and 5672-2. . (c) RT-PCR analysis of hepatocyte marker gene expression in the isolated clones. The primer sets used for albumin, HNF4α, and C/EBPα were 5-ctcaggtgtcaaccccaa-3 and 5-tccacacaaggcagtctc-3, 5-tgccaacctcaattcatcca-3 and 5-gctcgaggctccgtagtgtt-3, and 5-aagaagtcggtggacaagaacag-3 and 5-gttgcgttgtttggctttatctc-3, respectively. These primer sets yielded 103 bp (for albumin), 94 bp (for HNF4α), and 100 bp (for C/EBPα) PCR products. Notably, almost all of the clones tested still exhibited expression of albumin. F9 cells: murine embryonal carcinoma cells used as negative control; intact liver: adult murine liver used as positive control. M: 100 bp ladder size marker.