Evaluation of viral and mammalian promoters for driving transgene expression in mouse liver.
Fifteen luciferase plasmid constructs driven by various promoters including cytomegalovirus (CMV), Rous sarcoma virus (RSV), human serum albumin (SA), alpha-1 antitrypsin (AAT), cytochrome P450 CYP1A2, CYP2C9, CYP2C18, CYP2D6, CYP3A4, mouse CYP2b10, human amyloid precursor protein (APP), chicken beta actin (ACT), nuclear factor kappa B (NFkappaB), and heat shock protein 70 (HS) promoters were hydrodynamically introduced into mouse hepatocytes, and the level and persistence of luciferase gene expression were examined. Eight hours post-gene transfer, the CMV and AAT promoters showed the highest activity, followed by the CYP2D6, HS, and RSV promoters which were slightly less active. The human serum albumin promoter exhibited the lowest activity among the promoters examined. The time course of gene expression showed a two-phase decline in luciferase activity with a rapid phase within the first 5-7 days and a slower decline thereafter. Results from Southern and Northern blot analyses revealed a good correlation between the decline of luciferase activity and the decrease in mRNA level, suggesting promoter silencing as the possible mechanism for the observed transient luciferase gene expression. Inclusion of EBN1 and oriP sequences of Epstein-Barr virus into the plasmid extended the period of active transcription for about one week. These results provide important information concerning the role of promoters in regulating transgene expression and for the proper design of plasmids for gene expression and gene therapy.
Al-Dosari
Mohammed
M
Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, PA 15261, USA.
Zhang
Guisheng
G
Knapp
Joseph E
JE
Liu
Dexi
D
D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics*; D015152:Blotting, Northern; D015139:Blotting, Southern; D003587:Cytomegalovirus / Q000235:genetics*; D005786:Gene Expression Regulation / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D008099:Liver / Q000378:metabolism*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D013997:Time Factors; D019076:Transgenes / Q000235:genetics*
D008156:Luciferases