Journal: Gene therapy

Article Title: Recombinant elastin based nanoparticles for targeted gene therapy

doi: 10.1038/gt.2017.54

Figure Lengend Snippet: A) Schematic of heterogeneous nanoparticle. B) Average ± SD of the normalized fold increase of cell uptake. A549 cells were starved for 24 hours then were treated in serum free media with the indicated treatments fluorescein-labeled 2 µM LDLR3-ELP and combination of fluorescein-labeled 2 µM LDLR3-ELP and ELP and KGF-ELP, and LDLR3-ELP for 48 hours. Lentiviral vectors were not added in this experiment. After 48 hours, flow cytometry was used to quantify the data. Before analysis, trypan blue was used in each sample suspension to capture only the internalized labeled fusion proteins and not the ones that are bound to the periphery of the cells. These experiments were repeated two more times with triplicates. ***indicates P<0.001when compared to control (2 µM LDLR3-ELP). Error bars represent ± SD.

Article Snippet: The PUC57 plasmids containing the genes (VPGVG) 2 VPGCG(VPGVG) 2 , LDLR3, and KGF were purchased from GenScript (Piscataway, NJ).

Techniques: Labeling, Flow Cytometry, Suspension, Control

Journal: Gene therapy

Article Title: Recombinant elastin based nanoparticles for targeted gene therapy

doi: 10.1038/gt.2017.54

Figure Lengend Snippet: A549 cells were starved for 24 hours and then treated with lentiviral vector bound to either LDLR-3-ELP homogeneous or KGF-ELP LDLR3-ELP heterogeneous nanoparticles in the presence or absence of recombinant KGF (rKGF). After 24 hours, the old media was discarded, and fresh 37°C serum free media was added to each well. 72 hours later, the transduction efficiency was quantified using flow cytometry. Treatments were normalized to control (1µM LDLR3-ELP + 1µM LDLR3-ELP +Virus). These experiments were repeated two more times with triplicates. ***indicates P<0.001 when compared to control (1 µM LDLR3-ELP + 1 µM LDLR3-ELP). Error bars represent ± SD.

Article Snippet: The PUC57 plasmids containing the genes (VPGVG) 2 VPGCG(VPGVG) 2 , LDLR3, and KGF were purchased from GenScript (Piscataway, NJ).

Techniques: Plasmid Preparation, Recombinant, Transduction, Flow Cytometry, Control, Virus

Journal: Gene therapy

Article Title: Recombinant elastin based nanoparticles for targeted gene therapy

doi: 10.1038/gt.2017.54

Figure Lengend Snippet: A) GFP expression in A549 cells. B) Average ± SD of the normalized GFP expression of supernatant. A549 cells were starved for 24 hours. A binding assay was performed and cells were treated with the supernatant. Cells were treated for 24 hours with the lentiviral vector containing 12 µM ELP, 12 µM KGF-ELP, and 2 µM LDLR3-ELP were added respectively After 24 hours, the old media was discarded, and fresh 37°C serum free media was added to each well. Then, 72 hours later, the transduction efficiency was quantified using flow cytometry. Treatments were normalized to control (virus). These experiments were repeated two more times with triplicates. ***indicates P<0.001 when compared to control (virus). Error bars represent ± SD.

Article Snippet: The PUC57 plasmids containing the genes (VPGVG) 2 VPGCG(VPGVG) 2 , LDLR3, and KGF were purchased from GenScript (Piscataway, NJ).

Techniques: Expressing, Binding Assay, Plasmid Preparation, Transduction, Flow Cytometry, Control, Virus

Journal: Gene therapy

Article Title: Recombinant elastin based nanoparticles for targeted gene therapy

doi: 10.1038/gt.2017.54

Figure Lengend Snippet: A549 were starved for 24 hours then were treated in serum free media with the indicated treatments. Cells were treated for 24 hours with the lentiviral vector containing nanoparticles that consisted of 1 µM of biotinylated LDLR3-ELP and 1 µM KGF-ELP. At the indicated time points, cells were put on ice for five minutes followed by three ice cold washed in PBS. The cells were lysed with radio immune protection assay (RIPA) buffer containing 2X halt protease and phosphatase inhibitors cocktail with 1X ethylene diamine triacetic acid (EDTA). The lysates were immunoprecipitated using streptavidin conjugated magnetic beads (dynabeads). Then a magnetic bar was used to separate the beads from the supernatant. Following a few washes, the immunoprecipitated complexes (dynabeads) and the supernatant were analyzed for the presence of virus using western blots. Anti VSV-G antibody was used to detect the lentiviral vector.

Article Snippet: The PUC57 plasmids containing the genes (VPGVG) 2 VPGCG(VPGVG) 2 , LDLR3, and KGF were purchased from GenScript (Piscataway, NJ).

Techniques: Plasmid Preparation, Immunoprecipitation, Magnetic Beads, Virus, Western Blot