Journal: Frontiers in Molecular Neuroscience

Article Title: Development of non-viral vehicles for targeted gene transfer into microglia via the integrin receptor CD11b

doi: 10.3389/fnmol.2014.00079

Figure Lengend Snippet: Antibodies and reagents used for immunostaining.

Article Snippet: In vitro experiments for specificity and internalization of the OX42 antibody as well as transfections were performed in mixed cultures and isolated microglia obtained from 1 to 3 day old Sprague-Dawley rat brains as outlined in Section “Primary Cell Culture.” A total of five brains of 9–10 weeks old male Sprague-Dawley rats (300–350 g ) were used to test specificity of CD11b for microglia and for in vivo transfections.

Techniques: Immunostaining, Plasmid Preparation

Journal: Frontiers in Molecular Neuroscience

Article Title: Development of non-viral vehicles for targeted gene transfer into microglia via the integrin receptor CD11b

doi: 10.3389/fnmol.2014.00079

Figure Lengend Snippet: Integrin receptor CD11b is specific for microglia in vitro and in vivo . (A) CD11b-positive cells (green) in mixed glia culture stained for the selective microglia marker Iba1 (red). Many cells that stained for the nuclear dye Hoechst were negative for CD11b and Iba1 (white arrow heads). (B) GFAP-positive cells (red) did not immunostain for CD11b (green) in vitro . (C) Immunohistochemistry of rat brains injected with OX42-Alexa 488 (green) demonstrated co-labeling with microglial marker Iba1 (red, white arrow heads). (D) GFAP-IR (red) and CD11b-IR (green) did not co-localize showing that astrocytes do not express CD11b or take up OX42 antibody.

Article Snippet: In vitro experiments for specificity and internalization of the OX42 antibody as well as transfections were performed in mixed cultures and isolated microglia obtained from 1 to 3 day old Sprague-Dawley rat brains as outlined in Section “Primary Cell Culture.” A total of five brains of 9–10 weeks old male Sprague-Dawley rats (300–350 g ) were used to test specificity of CD11b for microglia and for in vivo transfections.

Techniques: In Vitro, In Vivo, Staining, Marker, Immunohistochemistry, Injection, Labeling

Journal: Frontiers in Molecular Neuroscience

Article Title: Development of non-viral vehicles for targeted gene transfer into microglia via the integrin receptor CD11b

doi: 10.3389/fnmol.2014.00079

Figure Lengend Snippet: OX42 antibody is internalized into microglia and accumulates in perinuclear acidic vesicles. (A) Microglial cells incubated on ice with OX42-FITC (green) immunostained for CD11b in the membrane only. Microglia incubated with OX42-FITC at 37°C showed green fluorescent vesicles close to the cell nucleus (Hoechst-dye, blue) indicating the uptake of OX42 antibody into microglia. The negative control antibody X63 did not bind and internalize in microglia. (B) Quantification of FITC fluorescence demonstrated a rapid increase and accumulation of OX42 antibody in the perinuclear region of microglia. (C) The internalization of OX42 antibody was confirmed by quenching the extracellular fluorescence with trypan blue. Confocal images revealed that OX42 antibody accumulates in perinuclear acidic vesicles in microglial cells as observed by the co-localization of the acidic organelle marker Lysotracker Red. Values are plotted as Mean ± SEM. n ≥ 77 cells per time-point. **** P < 0.0001 vs. control; N.S., not significant; AUs, arbitrary units.

Article Snippet: In vitro experiments for specificity and internalization of the OX42 antibody as well as transfections were performed in mixed cultures and isolated microglia obtained from 1 to 3 day old Sprague-Dawley rat brains as outlined in Section “Primary Cell Culture.” A total of five brains of 9–10 weeks old male Sprague-Dawley rats (300–350 g ) were used to test specificity of CD11b for microglia and for in vivo transfections.

Techniques: Incubation, Membrane, Negative Control, Fluorescence, Marker, Control

Journal: Frontiers in Molecular Neuroscience

Article Title: Development of non-viral vehicles for targeted gene transfer into microglia via the integrin receptor CD11b

doi: 10.3389/fnmol.2014.00079

Figure Lengend Snippet: Gel retardation assay demonstrates the ability of the OX42-immunoporter in binding and retarding plasmid DNA. Non-linearized pDNA alone (lane 2) migrated in two main bands. PEI (lanes 3–5) and PEI–PEG (lanes 6–8) completely retarded plasmid DNA at N/P-ratios of ≥5. The OX42-immunoporter (lanes 9–12) bound and retarded DNA with increasing N/P-ratios forming an OX42-immunogene. PEI, polyethyleneimine; PEG, polyethylene glycol; IP, immunoporter; bps, base pairs; pDNA, plasmid DNA.

Article Snippet: In vitro experiments for specificity and internalization of the OX42 antibody as well as transfections were performed in mixed cultures and isolated microglia obtained from 1 to 3 day old Sprague-Dawley rat brains as outlined in Section “Primary Cell Culture.” A total of five brains of 9–10 weeks old male Sprague-Dawley rats (300–350 g ) were used to test specificity of CD11b for microglia and for in vivo transfections.

Techniques: Electrophoretic Mobility Shift Assay, Binding Assay, Plasmid Preparation

Journal: Frontiers in Molecular Neuroscience

Article Title: Development of non-viral vehicles for targeted gene transfer into microglia via the integrin receptor CD11b

doi: 10.3389/fnmol.2014.00079

Figure Lengend Snippet: The OX42-immunogene reduces off-target transfection of PEI–PEG in vitro . (A) Representative confocal images of transfected cells in mixed culture. Non-targeting PEI–PEG (upper panel) transfected few microglial cells (yellow arrow heads) with an EGFP reporter plasmid as judged by EGFP fluorescence (green). EGFP-transfected cells were either Iba1-immunoreactive (IR, red, microglia) or lacked GFAP expression (red). PEI–PEG also transfected GFAP-IR astrocytes (white arrow heads). The targeting OX42-immunogene (lower panel) transfected few GFAP-negative cells that potentially are microglial cells (yellow arrow heads), but it also delivered the reporter gene into few GFAP-IR astrocytes (white arrow heads). (B) Quantification of transfected cells per coverslip. PEI–PEG (214 ± 37 cells) transfected significantly more cells than the OX42-immunogene (5 ± 2 cells). Values are plotted as Mean ± SEM. *** P < 0.005. n = 6 coverslips for PEI–PEG and n = 4 coverslips for OX42-immunogene. EGFP, green fluorescent protein; GFAP, glial fibrillary acidic protein.

Article Snippet: In vitro experiments for specificity and internalization of the OX42 antibody as well as transfections were performed in mixed cultures and isolated microglia obtained from 1 to 3 day old Sprague-Dawley rat brains as outlined in Section “Primary Cell Culture.” A total of five brains of 9–10 weeks old male Sprague-Dawley rats (300–350 g ) were used to test specificity of CD11b for microglia and for in vivo transfections.

Techniques: Transfection, In Vitro, Plasmid Preparation, Fluorescence, Expressing

Journal: Frontiers in Molecular Neuroscience

Article Title: Development of non-viral vehicles for targeted gene transfer into microglia via the integrin receptor CD11b

doi: 10.3389/fnmol.2014.00079

Figure Lengend Snippet: The OX42-immunogene is taken up in microglia but does not cause gene expression in vitro . (A) PEI–PEG transfected few GFAP-negative (red) cells that potentially are microglia. Green fluorescence of high (yellow arrow heads) and low intensity (blue arrow heads) was visible for mixed cultures treated with either PEI–PEG or the OX42-immunogene. The green fluorescence of low intensity almost exclusively co-localized with GFAP-negative cells (blue arrow heads). (B) The green fluorescence of high intensity was EGFP-specific (green) and visible in the cytoplasm and cell nucleus. Specific fluorescence caused by EGFP-expression was confirmed with an anti-EGFP antibody (red). The green fluorescence of low intensity was unrelated to EGFP-expression (non-specific) as it did not co-localize with EGFP-IR. Non-specific fluorescence (green) was mainly localized in CD11b-IR microglia (red), appeared vesicle-like and accumulated in the perinuclear region. Note: when imaging non-specific fluorescence, laser power and gain were increased compared to imaging EGFP-specific fluorescence by confocal microscopy.

Article Snippet: In vitro experiments for specificity and internalization of the OX42 antibody as well as transfections were performed in mixed cultures and isolated microglia obtained from 1 to 3 day old Sprague-Dawley rat brains as outlined in Section “Primary Cell Culture.” A total of five brains of 9–10 weeks old male Sprague-Dawley rats (300–350 g ) were used to test specificity of CD11b for microglia and for in vivo transfections.

Techniques: Gene Expression, In Vitro, Transfection, Fluorescence, Expressing, Imaging, Confocal Microscopy

Journal: Frontiers in Molecular Neuroscience

Article Title: Development of non-viral vehicles for targeted gene transfer into microglia via the integrin receptor CD11b

doi: 10.3389/fnmol.2014.00079

Figure Lengend Snippet: Transfection experiments with a control vector that lacked the EGFP reporter gene caused an increase in non-specific fluorescence after treatment with PEI–PEG and the OX42-immunogene. Untreated control mixed cultures displayed very low levels of non-specific green background fluorescence.

Article Snippet: In vitro experiments for specificity and internalization of the OX42 antibody as well as transfections were performed in mixed cultures and isolated microglia obtained from 1 to 3 day old Sprague-Dawley rat brains as outlined in Section “Primary Cell Culture.” A total of five brains of 9–10 weeks old male Sprague-Dawley rats (300–350 g ) were used to test specificity of CD11b for microglia and for in vivo transfections.

Techniques: Transfection, Control, Plasmid Preparation, Fluorescence

Journal: Frontiers in Molecular Neuroscience

Article Title: Development of non-viral vehicles for targeted gene transfer into microglia via the integrin receptor CD11b

doi: 10.3389/fnmol.2014.00079

Figure Lengend Snippet: The OX42-immunogene does not cause gene expression in vivo . (A) Representative confocal images show the OX42-immunogene injected into the right lateral ventricle caused an increase in green fluorescence that co-localized in Iba1-IR microglia which exhibited an amoeboid shape (yellow arrow heads). (B) Confocal imaging of brain sections (dorsal striatum) revealed that the green fluorescence observed is non-specific, because non-specific fluorescence was also seen in the red filter in absence of red fluorophore-labeled cell markers. Further, an anti-EGFP antibody did not detect EGFP-expression.

Article Snippet: In vitro experiments for specificity and internalization of the OX42 antibody as well as transfections were performed in mixed cultures and isolated microglia obtained from 1 to 3 day old Sprague-Dawley rat brains as outlined in Section “Primary Cell Culture.” A total of five brains of 9–10 weeks old male Sprague-Dawley rats (300–350 g ) were used to test specificity of CD11b for microglia and for in vivo transfections.

Techniques: Gene Expression, In Vivo, Injection, Fluorescence, Imaging, Labeling, Expressing

Journal: Frontiers in Molecular Neuroscience

Article Title: Development of non-viral vehicles for targeted gene transfer into microglia via the integrin receptor CD11b

doi: 10.3389/fnmol.2014.00079

Figure Lengend Snippet: The OX42-immunogene forms large aggregates and triggers an immune response in microglia. (A) Comparison of the size-distribution profiles between complete cell culture medium (DMEM + 10% FBS) and the OX42-immunoporter obtained by DLS shows that the non-viral vehicle did not form aggregates. After adding pDNA to form the OX42-immunogene (N/P = 4), aggregates formed over a large range (≈50–1300 nm). (B) Adding FBS to polyplexes after maturation in HBS and maturation of polyplexes in FBS-containing medium caused a shift in size-distribution to lower aggregate sizes, but it had no effect on the width of size-distributions. Values are plotted as Mean ± SEM (two experiments). FBS, fetal bovine serum; OX42-IP, OX42-immunoporter; HBS, HEPES-buffered saline; D (+/–), DMEM with/without 10% FBS. (C) ROS production in microglial cells was measured by quantifying ROS-indicator fluorescence after 60 min and reported as percentage (%) of the internal standard PDBu. The aggregated OX42-immunogene triggered the respiratory burst (38.8 ± 3.3%) which was inhibited by the respiratory burst inhibitor diphenyliodonium (–7.02 ± 4.49%). The positive control zymosan caused the release of ROS at similar levels (45.8 ± 4.7%) to the OX42-immunogene. However, PEI–PEG (–0.97 ± 3.74%), OX42 antibody (2.57 ± 1.68%) and the OX42-immunoporter (9.83 ± 2.84%) did not trigger significant ROS production. Values are plotted as Mean ± SEM. ** P < 0.01 vs. control; **** P < 0.0001 vs. control; N.S., not significant. DPI, diphenyliodonium; PDBu, phorbol 12,13-dibutyrate.

Article Snippet: In vitro experiments for specificity and internalization of the OX42 antibody as well as transfections were performed in mixed cultures and isolated microglia obtained from 1 to 3 day old Sprague-Dawley rat brains as outlined in Section “Primary Cell Culture.” A total of five brains of 9–10 weeks old male Sprague-Dawley rats (300–350 g ) were used to test specificity of CD11b for microglia and for in vivo transfections.

Techniques: Comparison, Cell Culture, Saline, Fluorescence, Positive Control, Control

Journal: Frontiers in Molecular Neuroscience

Article Title: Development of non-viral vehicles for targeted gene transfer into microglia via the integrin receptor CD11b

doi: 10.3389/fnmol.2014.00079

Figure Lengend Snippet: The ability of PEI to facilitate intracellular escape in microglia is limited. (A) PEI–PEG (424 ± 42 vs. 74 ± 13) and the aggregated OX42-immunogene (71 ± 8 vs. 8 ± 3) transfected significantly more cells in the presence of chloroquine. (B) Chloroquine also increased specificity for microglia when transfected with PEI–PEG (20.3 ± 1.2% vs. 14.3 ± 1.7%) and the OX42-immunogene (32.3 ± 2.4% vs. 12.7 ± 8.0%). Values are plotted as Mean ± SEM. n = 6 coverslips (three independent experiments). * P < 0.05, **** P < 0.0001. Cq, chloroquine.

Article Snippet: In vitro experiments for specificity and internalization of the OX42 antibody as well as transfections were performed in mixed cultures and isolated microglia obtained from 1 to 3 day old Sprague-Dawley rat brains as outlined in Section “Primary Cell Culture.” A total of five brains of 9–10 weeks old male Sprague-Dawley rats (300–350 g ) were used to test specificity of CD11b for microglia and for in vivo transfections.

Techniques: Transfection