Dear microscopists, our next FILM CLUB is coming up, again with a very interesting presentation: Charanjit Singh (Eric Alton lab) will tell us about "QUANTITATIVE BIOLOGICAL IMAGING OF PLASMID DNA IN LIVE HUMAN AIRWAY EPITHELIAL CELLS FOLLOWING NON-VIRAL GENE TRANSFER" FRIDAY 25 MAY 2012, 1PM, SAFB-120, SOUTH KENSINGTON CAMPUS The UK CF Gene Therapy Consortium is interested in non-viral gene therapy for cystic fibrosis (CF). It is widely accepted that in addition to extracellular barriers responsible for inefficient uptake, there are key intracellular obstacles to the nuclear delivery of the therapeutic plasmid DNA (pDNA). Thus, we are investigating the intracellular fate of pDNA following transfection, using the clinically relevant cationic Genzyme Lipid (GL) 67A formulation, using transmission electron microscopy (TEM) combined with three-dimensional Spinning-Disk confocal microscopy (SDCM), to track, quantitate and provide high resolution 'snapshots' of pDNA at the single molecule level in transfected primary human airway epithelial cells (AECs) grown at the air-liquid interface (ALI). The pDNA was tagged with 1.4 nm gold nanoparticles (NG- pDNA) or fluorescent quantum dots (QDot-pDNA) for use in TEM or fluorescence studies, respectively. We were able to detect significantly (p<0.05) more electron-dense gold spots in gold-enhanced TEM sections from NG-pDNA-transfected cells compared with those transfected with unconjugated pDNA. Approximately 50% and 10% of the total internalised pDNA localised to nuclei within 1 hour transfection in SDCM (123 AECs, 8 independent experiments) and TEM (48 AECs, 6 independent experiments) studies respectively. In experiments to track the intracellular trafficking of the NG-pDNA, we found that the nuclear delivery of NG-pDNA increase with increasing transfection time from 15 to 60 min but there was no any change after 360 min transfection. Parallel confocal experiments following the intracellular fate of Qdot-pDNA over 120 min in living AECs confirm the TEM experiments. See you there, good imaging, Martin -- ------------------------------------------------------------------------ *Martin Spitaler, PhD* *FILM - Facility for Imaging by Light Microscopy* - Facility Manager - Sir Alexander Fleming Building, desk 401 Imperial College London / South Kensington Exhibition Road London SW7 2AZ UK Tel. +44-(0)20-759-42023 E-mail m.spitaler@imperial.ac.uk <mailto:m.spitaler@imperial.ac.uk> Website: http://imperial.ac.uk/imagingfacility