Transfection of liver in vivo by biolistic particle delivery: its use in the investigation of cytochrome P450 gene regulation.
We describe the delivery of reporter gene constructs to rat liver through the use of the Helios Gene Gun system. The effectiveness of this transfection method is illustrated by describing its use for determining in vivo the role of a DNA element that regulates cytochrome P450 2B1 (CYP2B1) gene expression in response to xenobiotics. DNA was delivered to the liver of an anesthetized animal via DNA-coated gold microcarriers. The highest level of reporter gene expression was obtained about six hours posttransfection; however, at this time endogenous CYP2B1 mRNA is transiently induced by the anesthetic treatment. The optimal time for investigating expression of a reporter gene under the control of CYP2B1 regulatory elements was 24 h after transfection, by which time the inductive effect of the anesthetic had ceased. Reporter gene expression subsequently declined rapidly to a low level by 48 h. In the transfected liver the heterologous SV40 promoter was about eight-fold stronger than the minimal CYP2B1 promoter. However, when attached to the phenobarbital response element both promoters give the same fold-induction of reporter activity in response to phenobarbital.
Muangmoonchai
Roongsiri
R
Department of Biochemistry and Molecular Biology, University College London, UK.
Wong
Siew Cheng
SC
Smirlis
Despina
D
Phillips
Ian R
IR
Shephardl
Elizabeth A
EA
D000818:Animals; D019470:Biolistics / Q000295:instrumentation / Q000379:methods*; D019362:Cytochrome P-450 CYP2B1 / Q000235:genetics* / Q000378:metabolism*; D004790:Enzyme Induction / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D010634:Phenobarbital / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection / Q000295:instrumentation / Q000379:methods*
D019362:Cytochrome P-450 CYP2B1; D010634:Phenobarbital