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Fig. 5. Construction and expression of Ad-shTeM, Ad-shChM-I and Ad-hTeM in adenoviral vectors. (A) The structure of the pALC3 cosmid vector with an expression cassette containing either a shTeM, shChM-I or hTeM insert. The 34-kb adenoviral genome is indicated as a closed ring. The 7-kb cosmid vector backbone is flanked by loxP sites and includes a cos site, a bacterial ori site and the ampicillin resistance gene (Amp). The expression cassette consisting of the cytomegalovirus immediate-early enhancer-chicken ß-actin hybrid (CAG) promoter, the preprotrypsin secretion signal (ss), the indicated FLAG inserts, an internal ribosome entry site (IRES) and enhanced green fluorescent protein (EGFP) cDNA followed by a polyadenylation signal (pA) was cloned into the SwaI site of the pALC3 vector, resulting in the pALC3-shChM-I, pALC3-shTeM or pALC3-hTeM cosmid. Recombinant adenoviruses were generated upon cotransfection of a Cre-expression plasmid with pALC3-shChM-I, pALC3-shTeM or pALC3-hTeM cosmids into 293 cells and are designated as Ad-shChM-I, Ad-shTeM or Ad-hTeM. Empty vector expressing EGFP alone was designated as Ad-EGFP. (B) Phase-contrast microscopic image (upper panel) indicating the cobblestone-like morphology of HUVECs 24 hours after adenoviral infection. The fluorescence microscopic image (lower panel) shows efficient adenoviral gene transduction in HUVECs. Scale bar: 40 µm. (C) Fluorescence microscope images of uninfected HUVECs (upper panel) or HUVECs transduced with Ad-EGFP. Dil-AcLDL uptake is observed as red spots regardless of adenoviral transduction. Cell nuclei were stained blue-violet with Hoechst dye 33342 (upper panel). Green fluorescence is observed in the HUVECs expressing EGFP (lower panel). Scale bar: 40 µm. (D) Cell lysates from uninfected HUVECs (wild) or the HUVECs infected with Ad-hTeM were directly harvested with 1x SDS sample buffer and then subjected to western blotting (left panel). Secreted proteins were concentrated by a Butyl-Toyopearl column from conditioned medium derived from wild type HUVECs (wild) or HUVECs transduced by Ad-EGFP, Ad-shChM-I or Ad-shTeM, and subjected to western blot analysis by anti-FLAG monoclonal antibodies.





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