Improvement of Hydrodynamics-Based Gene Transfer of Nonviral DNA Targeted to Murine Hepatocytes
Figure 2
(a) The schematic for Cre-loxP-based recombination, using pCRTEIL as the reporter transgene. Before recombination, the floxed HcRed1/CAT hybrid sequence is expressed under the control of the CAG promoter, whereas the EGFP and luc cDNAs are silent. Cre-mediated recombination results in the deletion of the floxed sequence and the subsequent generation of recombined pCRTEIL. This induces the expression of the EGFP and luc cDNAs. CAG: cytomegalovirus enhancer + chicken β-actin promoter; CAT: chloramphenicol acetyltransferase gene; EGFP: enhanced green fluorescent protein cDNA; HcRed1: far-red fluorescent variant protein derived from the sea anemone Heteractis crispa; IRES: internal ribosomal entry site; pA: poly(A) sites; TTRp: mouse transthyretin promoter. (b) In vivo Cre-mediated recombination in pCRTEIL.Unfixed liver samples were dissected one day after HGD with pCRTEIL and pTR/NCre; pCRTEIL alone; pTR/NCre alone; or PBS(-) alone (Mock) and then were immediately inspected for HcRed1-derived red fluorescence (red arrows) or EGFP-derived green fluorescence (green arrows). Mixed fluorescence (indicated by green and red arrows in the “merge” column) is present only in the liver samples transfected with pCRTEIL and pTR/NCre. (c) Luc activity of the liver samples described in A. The data (in RLU per milligram of protein) are presented as the mean ± the standard error (). For comparison of PBS(-) alone (Mock) and the other groups, Scheffe’s post hoc test was used with findings of marked with double asterisk.
