Improvement of Hydrodynamics-Based Gene Transfer of Nonviral DNA Targeted to Murine Hepatocytes
Figure 1
(a) The plasmid vectors, pCEIL and phRL-SV40, used for HGD are targeted to murine liver. (b) A liver dissected one day after the HGD of pCEIL DNA dissolved in PBS(-). The whole liver is photographed under light (upper panel) and under light and UV (lower panel). There is notable EGFP-derived green fluorescence in the region surrounding the large aorta. Particularly, the area in the right median lobe (enclosed by the circle) is extensively transfected. We therefore focused our study on this region. CAG: cytomegalovirus enhancer + chicken β-actin promoter; CAP site: transcription start site; EGFP: enhanced green fluorescent protein cDNA; IRES: internal ribosomal entry site; Pa: poly(A) sites; SVp: SV40 early promoter. (c) The measurement of luc activity in the liver one day after transfection with pCEIL/PBS(-) (PBS); pCEIL/PBS(-)/DDMC (DDMC); pCEIL/PBS(-)/SugarFect (Sugar); pCEIL/PBS(-)/in vivo-jet PEI (PEI); pCEIL/PBS(-)/DMRIE-C (DMRIE); pCEIL/PBS(-)/FuGENE HD (FuGENE); pCEIL/PBS(-)/F/P MPs (F/P); or PBS(-) alone (Mock). In each group, phRL-SV40 is included as the gene transfer control. The data (in relative light units (RLU) per milligram of protein) are presented as the mean ± the standard error (). For comparison of pCEIL/PBS(-) (PBS) and experimental groups, Scheffe’s post hoc test was used with findings of marked with double asterisk and with an asterisk, respectively. (d) Fluorescence in the liver samples one day after transfection with PBS(-) alone (Mock); pCEIL/PBS(-) (DNA/PBS); or pCEIL/PBS(-)/in vivo-jet PEI (DNA/PEI). Unfixed liver samples of the right medial lobe (upper row panels) and immunohistochemical specimens (lower row panels) were examined with a fluorescence stereomicroscope to detect EGFP. Note the widespread bright cytoplasmic fluorescence in the liver sample after the introduction of pCEIL/PBS(-)/in vivo-jet PEI.
