cadmium-selenium quantum dots (qdot 545)  (Quantum Dot Inc)

Journal: Bioscience Reports

Article Title: Peptides mediating DNA transport on microtubules and their impact on non-viral gene transfer efficiency

doi: 10.1042/BSR20170995

Figure Lengend Snippet: X-rhodamine-labelled MTs (red) were polymerized in vitro and incubated with Qdot streptavidin (green) conjugate 545 labelled with biotinylated P79-98 peptide (bio-P79-98: VVMVGEKPITITQHSVETEG-Ttdsbiotin; left panel) or biotinylated P38-57 peptide (bio-P38-57: VNLHQCKRGIFCLVKQAKVTTtds-biotin; right panel) in HeLa cells cytosolic extracts supplemented with 10 mM ATP .

Article Snippet: To evidence that P79-98 can mediate the binding of a cargo to MTs, the biotinylated P79-98 peptide (P79-98-bio) was linked to Qdot 545 streptavidin (P79-98-Qdot) ( ).

Techniques: In Vitro, Incubation

Journal: Bioscience Reports

Article Title: Peptides mediating DNA transport on microtubules and their impact on non-viral gene transfer efficiency

doi: 10.1042/BSR20170995

Figure Lengend Snippet: ( a ) Scheme of assembly of biotinylated (bio) and Cy3-pDNA with biotinylated P79-98 peptide loaded streptavidin (STR). ( b ) P79-98 allows intracellular movement of pDNA on MTs. HeLa cells stably expressing eGFP-α-tubulin were transfected for 90 min with P79-98/Cy3-pDNA/His-lPEI complexes. Panels (1–4) show a representative time lapse acquisition (interval between each frame: 10 s) performed by videomicroscopy.

Article Snippet: To evidence that P79-98 can mediate the binding of a cargo to MTs, the biotinylated P79-98 peptide (P79-98-bio) was linked to Qdot 545 streptavidin (P79-98-Qdot) ( ).

Techniques: Stable Transfection, Expressing, Transfection

Journal: Bioscience Reports

Article Title: Peptides mediating DNA transport on microtubules and their impact on non-viral gene transfer efficiency

doi: 10.1042/BSR20170995

Figure Lengend Snippet: HeLa cells were transfected with 2.5-µg pCMV-eGFP (5130 bp) encoding Yellow Fish enhanced GFP gene under the control of the CMV promoter. pDNA was biotinylated (pDNA-bio) (DNA/biotin molar ratio of 3) and associated either with bio-P79-98 (P4) or bio-P38-57 (P1) via streptavidin as described in a. The equipped plasmid was then complexed with His–lPEI (DNA/polymer weight ratio of 1/6). The transfection was performed for 4 h at 37°C in the absence (grey bar) or the presence (black bar) of 33 µM nocodazole. Then the cells were washed and incubated for 48 h in fresh medium in the absence of nocodazole and any polyplexes. The fluorescence of cells was measured after 48 h transfection by flow cytometry. % stands for the percentage of transfected cells, MFI for the mean of the fluorescence intensity of the transfected cells and global transfection efficiency (TE) for the mathematical product of the MFI of cells and the percentage of fluorescent cells.

Article Snippet: To evidence that P79-98 can mediate the binding of a cargo to MTs, the biotinylated P79-98 peptide (P79-98-bio) was linked to Qdot 545 streptavidin (P79-98-Qdot) ( ).

Techniques: Transfection, Plasmid Preparation, Incubation, Fluorescence, Flow Cytometry

Journal: Bioscience Reports

Article Title: Peptides mediating DNA transport on microtubules and their impact on non-viral gene transfer efficiency

doi: 10.1042/BSR20170995

Figure Lengend Snippet: HeLa cells were incubated for 4 h in the absence and the presence of 2.5 µg pDNA. Fluorescein-labelled pDNA was biotinylated (pDNA-bio) (DNA/biotin molar ratio of 3) and associated either with bio-P79-98 (P4) or bio-P38-57 (P1) via streptavidin as described in a. The equipped fluorescent plasmid was then complexed with His–lPEI (DNA/polymer weight ratio of 1/6). The incubation was performed for 4 h at 37°C in the absence or the presence of 33 µM nocodazole. Then the cells were washed and the fluorescence of cells was measured by flow cytometry in the absence (white bar) and in the presence (black bar) of Trypan Blue.

Article Snippet: To evidence that P79-98 can mediate the binding of a cargo to MTs, the biotinylated P79-98 peptide (P79-98-bio) was linked to Qdot 545 streptavidin (P79-98-Qdot) ( ).

Techniques: Incubation, Plasmid Preparation, Fluorescence, Flow Cytometry

Journal: Bioscience Reports

Article Title: Peptides mediating DNA transport on microtubules and their impact on non-viral gene transfer efficiency

doi: 10.1042/BSR20170995

Figure Lengend Snippet: Hydrodynamic injection of 15 µg of biotinylated p3NF-CMV-luc-3NF conjugated with streptavidin (pDNA-STR) or biotinylated p3NF-CMV-luc-3NF conjugated bio-P79-98 linked to streptavidin (pDNA-P4) was performed in the tail vein of CD1-Swiss mice (six mice each). Mice were injected in less than 8 s via the tail vein with polyplexes in 2.5 ml of isotonic NaCl + 5% glucose. Three days post-injection, mice were anaesthetized by isofluran inhalation then killed by cervical dislocation. The liver was removed, crushed in passive lysis buffer (Promega, Charbonnières Les Bains, France) using the gentle MACS dissociator (Miltenyi Biotech, Paris, France). After centrifugation of homogenates, the luciferase activity was measured and expressed as RLU per mg of proteins. p3NF-CMVLuc-3NF (5556 bp) encoding the firefly luciferase cassette under the control of a CMV promoter and containing 3NF sequences recognized by NFκB was constructed from p3NF-Luc-3NF by replacing pTAL promoter by CMV promoter.

Article Snippet: To evidence that P79-98 can mediate the binding of a cargo to MTs, the biotinylated P79-98 peptide (P79-98-bio) was linked to Qdot 545 streptavidin (P79-98-Qdot) ( ).

Techniques: Injection, Lysis, Centrifugation, Luciferase, Activity Assay, Construct

Journal: Bioscience Reports

Article Title: Peptides mediating DNA transport on microtubules and their impact on non-viral gene transfer efficiency

doi: 10.1042/BSR20170995

Figure Lengend Snippet: HeLa cells were transfected with 2.5-µg pCMV-eGFP. The plasmid was biotinylated (pDNA-bio) (biotin/DNA molar ratio of 3) and associated via streptavidin (STR) either with bio-P79-98 (P4) or bio-P38-57 (P1) as described in a. The equipped plasmid was then complexed either with (white bar) KLN25/MM27 (DNA/lipid weight ratio of 1/2) or (black bar) Lipofectamine cationic liposomes. The fluorescence of cells was measured after 48-h transfection by flow cytometry and data were given as the percentage of transfected cells (% of positive cells), the mean of the fluorescence intensity (MFI) of the transfected cells and the global transfection efficiency (global TE) (i.e. the mathematical product of the MFI of cells and the percentage of fluorescent cells).

Article Snippet: To evidence that P79-98 can mediate the binding of a cargo to MTs, the biotinylated P79-98 peptide (P79-98-bio) was linked to Qdot 545 streptavidin (P79-98-Qdot) ( ).

Techniques: Transfection, Plasmid Preparation, Fluorescence, Flow Cytometry

Journal: Bioscience Reports

Article Title: Peptides mediating DNA transport on microtubules and their impact on non-viral gene transfer efficiency

doi: 10.1042/BSR20170995

Figure Lengend Snippet: pDNA (pCMV-eGFP) was substituted with various amounts of biotin residues. Polyplexes were formed with His–lPEI (black bar) at DNA/polymer weight ratio of 1/6. Lipoplexes were formed with KLN25/MM27 liposomes (white bar) at DNA/lipid weight ratio of 1/2. DNA complexes were mixed with fluorescein-labelled streptavidin and then the fluorescence intensity of polyplexes and lipoplexes was measured by flow cytometry.

Article Snippet: To evidence that P79-98 can mediate the binding of a cargo to MTs, the biotinylated P79-98 peptide (P79-98-bio) was linked to Qdot 545 streptavidin (P79-98-Qdot) ( ).

Techniques: Fluorescence, Flow Cytometry