65 2.1 Material and Methods 66 2.1.1 Plasmid and cloning 66 2.1.2 Cell culture and transfection 66 2.1.3 Assessment of cell viability 67 2.1.4 Assessment of Neurite process length and branching point. 68 2.1.5 Ras Pull-Down Assay 68 2.1.6 Generation of Human LRRK2 Transgenic Flies. 69 2.1.7 Western Blot Analysis. 70 2.1.8 Immunoprecipitation. 71 2.1.9 Optical Neutralization Technique. 71 2.1.10...

We developed a simple, rapid, high-throughput detection and genotyping method for noroviruses using real-time reverse transcription-PCR (RT-PCR) and high-resolution melting (HRM) analysis to create a difference plot. The capsid gene was amplified by real-time RT-PCR in the presence of ResoLight HRM dye, a saturating DNA dye. Following optimization of the HRM assay conditions, the major noroviru...

BACKGROUND Oral immunization with vaccines may be an effective strategy for prevention of Clostridium difficile infection (CDI). However, application of previously developed vaccines for preventing CDI has been limited due to various reasons. Here, we developed a recombinant Lactococcus lactis oral vaccine and evaluated its effect on a C. difficile-infected animal model established in golden ha...

Cationic liposome-mediated gene transfection is a promising method for gene therapy. In this study, the transfection efficiency and histological patterns were evaluated in rat lung after intravenous administration via femoral vein of naked plasmid DNA, naked plasmid DNA with pretreatment of DOTAP, and DOTAP-cholesterol-plasmid DNA complex. Plasmid DNA encoding bacterial LacZ gene was used. For ...

cyp3a4 probably has the broadest catalytic activity of any cytochrome p450. it is a crucial task to test new drug candidates in a reliable system for their ability to induce expression of this enzyme. firstly, a total of 300 bp core distal enhancer of cyp3a4 xrem region (-7972/-7673) were amplified from human genomic dna. the pcr product was then ligated into a human secretory alkaline phosphat...

Background: Colorectal cancer (CRC) is one of the most common causes of cancer-related death in the world. The expression of N-myc downstream-regulated gene 2 (NDRG2) is down-regulated in CRC. The aim of this study was to investigate the effect of NDRG2 overexpression on cell proliferation and invasive potential of SW48 cells.Methods: SW48 cells were transfected with a plasmid overexpressing ND...

Gene targeting by microRNAs is important in health and disease. We developed a functional assay for identifying microRNA targets and applied it to the K(+) channel K(ir)2.1 [KCNJ2 (potassium inwardly-rectifying channel, subfamily J, member 2)] which is dysregulated in cardiac and vascular disorders. The 3'UTR (untranslated region) was inserted downstream of the mCherry red fluorescent protein c...

BACKGROUND The wide variety of real-time amplification platforms currently available has determined that standardisation of DNA measurements is a fundamental aspect involved in the comparability of results. Statistical analysis of the data arising from three different real-time platforms was conducted in order to assess inter-platform repeatability. On three consecutive days two PCR reaction mi...

The integrase from the Streptomyces phage phiC31 carries out efficient recombination between the attP site in the phage genome and the attB site in the host bacterial chromosome. In this paper, we show that the enzyme also functions in human cells. A plasmid assay system was constructed that measured intramolecular integration of attP into attB. This assay was used to demonstrate that in the pr...