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Joseph E Knapp

Publications and source records attributed to Joseph E Knapp.

6 recordsLinked to original sources
PubMed2006

Direct assessment of promoter activity of human cytochrome p450 genes using optimized transfection in vitro and in vivo.

Although transcription regulation of human cytochrome P450 enzymes (CYP) is known to play an important role in drug metabolism and homeostasis, factors influencing the expression of various CYP genes in humans remain largely undefined. We used three cell lines and CD-1 mice to assess the activity of genomic promoter sequences of human CYP2D6, 1A2, 3A4, 2C9, 2C18, and 2E1 genes. CYP promoter sequences were amplified by PCR using human liver genomic DNA as the template and cloned into pGL3-Basic vectors that contain a luciferase reporter gene but lack promoter or enhancer sequences. Each plasmid construct was transfected into cells in vitro using polyethylenimine (PEI) as the transfection reagent and into mice using the recently developed hydrodynamics-based procedure. Relative promoter strength was determined by the level of luciferase expression in transfected cells. All six human CYP promoters are active in driving reporter gene expression in cultured hepatic HepG2 cells and non-hepatic cells such as human embryonic kidney fibroblasts (293 cells) and murine melanoma cells (BL-16) as well as cells in intact mouse liver, lung, heart, kidney and spleen. The order of strength among CYP promoters examined was found to be 2D6 > 1A2 > 3A4 > 2C9 > 2C18 > 2E1.

Animals↗
PubMed2005

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.

Animals↗
PubMed2005

Hydrodynamic delivery.

Hydrodynamic delivery has emerged as a near-perfect method for intracellular DNA delivery in vivo. For gene delivery to parenchymal cells, only essential DNA sequences need to be injected via a selected blood vessel, eliminating safety concerns associated with current viral and synthetic vectors. When injected into the bloodstream, DNA is capable of reaching cells in the different tissues accessible to the blood. Hydrodynamic delivery employs the force generated by the rapid injection of a large volume of solution into the incompressible blood in the circulation to overcome the physical barriers of endothelium and cell membranes that prevent large and membrane-impermeable compounds from entering parenchymal cells. In addition to the delivery of DNA, this method is useful for the efficient intracellular delivery of RNA, proteins, and other small compounds in vivo. This review discusses the development, current application, and clinical potential of hydrodynamic delivery.

Animals↗
PubMedDate not supplied

Activation of human CYP2C9 promoter and regulation by CAR and PXR in mouse liver.

The activity of various genomic segments at the 5'-flanking region of the human CYP2C9 gene in driving gene expression and their involvement in pregnane X receptor (PXR) and constitutive androstane receptor (CAR) mediated activation were evaluated in mouse hepatocytes. Using the genomic sequence of human CYP2C9 as a template, segments covering different regions of CYP2C9 5'-flanking sequences starting from the translation start site were amplified by PCR and inserted into a pGL-3 luciferase vector. Plasmid DNA containing the 0.2K, 1K, 2K, 3K, 5K, or 10K upstream sequences of the CYP2C9 gene were transfected into mouse liver by hydrodynamic delivery, and the activity of each fragment in driving reporter gene expression was assessed. With the exception of the 10K fragment, the level of luciferase activity in transfected mouse liver was similar among the constructs examined. Cotransfection of these reporter constructs with the pCMX-PXR or pCMX-CAR plasmids resulted in a slight increase in luciferase gene expression that could be significantly enhanced by chemical inducers. In mice cotransfected with pCMX-PXR, pregnenolone-16 alpha-carbonitrile (PCN) induced a 20-fold increase in the luciferase level compared to a 70-fold increase induced by rifampicin. Similarly, when animals were cotransfected with the pCMX-CAR plasmid, phenobarbital and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene enhanced luciferase gene expression by 10- and 57-fold, respectively. The element responsible for PXR- and CAR-mediated activation of luciferase gene expression by chemical inducers was found to reside in the -2000 to -1000 bp region of the 5'-flanking sequence of the CYP2C9 gene. These results prove that PXR and CAR are transcription factors regulating CYP2C9 gene expression.

5' Flanking Region↗
PubMedDate not supplied

Activities of the USP Analytical Microbiology Expert Committee during the 2000-2005 revision cycle.

This article is a comprehensive review of the published activities of the Analytical Microbiology Expert Committee (AMB) for the 2000-2005 revision cycle. The major thrust of the activities during this revision cycle were directed at international harmonization, and to provide guidance in the changing field of pharmaceutical microbiology. In addition to reviewing the changes accomplished, this article discusses the rationale for many of the changes and some background information regarding new initiatives underway. Where appropriate, changes in the USP that did not fall under the direct purview of the AMB Expert Committee (EC) but of interest to the microbiology community are also discussed.

Advisory Committees↗