astrocyte specific promoter gfap sequence  (Addgene inc)

Journal: Scientific reports

Article Title: Nkx2.1 regulates the generation of telencephalic astrocytes during embryonic development.

doi: 10.1038/srep43093

Figure Lengend Snippet: Figure 9. Binding of Nkx2.1 to conserved binding sequences in the promoters of various glial regulatory genes. Amplification of the putative Nkx2.1 binding sequences located in a 206 bp PCR product within the GFAP promoter (a) and in a 391 bp PCR fragment from the Lhx6 promoter (b) after chromatin immunoprecipitation (ChIP) with the Nkx2.1 antibody. Input DNA was added as the positive control as it contains the cross-linked sonicated genomic DNA taken before ChIP with the Nkx2.1 antibody; a strong signal was observed for all the promoter regions. No amplification of the Nkx2.1 core sequence (tcaag) located in two, 410 bp and 352 bp, PCR products from the Ngn2 promoter was detected (c). No signal was detected in the negative control where no antibody was added for ChIP nor when no DNA was added while performing the PCR (a–c). A very faint signal was detected in some samples immunoprecipitated with non-specific control IgG. These results suggest that Nkx2.1 binds in vivo to the promoter region of GFAP at putative Nkx2.1 binding sequences. The relative intensities of all the bands were calculated by assigning an arbitrary value of 1 to the input band. The quantifications are indicated below the gels. The Figure represents one of three independently performed assays. As a control, human embryonic kidney 293 (HEK293) cells were co-transfected with two reporter-constructs, namely, the pDRIVE-mGFAP-LacZ and the pCAG-IRES-Tomato plasmids (d–f). Cell nuclei were counterstained in blue with Hoechst (d). In the control, only 4.26% of Tomato+cells were LacZ+, n = 94. To test the binding of Nkx2.1 to the GFAP promoter, HEK293 cells were co-transfected with two reporter constructs, namely, the pDRIVE-mGFAP-LacZ and the pCAG-Nkx2.1-IRES-Tomato plasmids (g–i). Cell nuclei were counterstained in blue with Hoechst (g). Activation of the LacZ reporter was seen upon addition of the Nkx2.1 expression vector, thus confirming that Nkx2.1 activates the GFAP promoter, most probably by binding the sequence identified as Nkx2.1 consensus. Here, 98% of Tomato+cells were LacZ+, n = 50. Bar = 50 μm.

Article Snippet: The mouse GFAP promoter sequence from the pDRIVE-mGFAP plasmid (#pdrive-mgfap, InvivoGen) used for transfection experiments is shown in Supp.

Techniques: Binding Assay, Amplification, Chromatin Immunoprecipitation, Positive Control, Sonication, Sequencing, Negative Control, Immunoprecipitation, Control, In Vivo, Transfection, Construct, Activation Assay, Expressing, Plasmid Preparation

Journal: Cellular & molecular biology letters

Article Title: Genetically encoded phosphatidylserine biosensor for in vitro, ex vivo and in vivo labelling.

doi: 10.1186/s11658-023-00472-7

Figure Lengend Snippet: Fig. 4 The expression of C2 fusion proteins in ex vivo organotypic hippocampal slices after AAV delivery. a OHSC were transduced with AAV carrying either C2-SNAP or C2m2-SNAP at 5 days in vitro (5DIV) and maintained for 9 days for protein expression. The secretion of recombinant proteins was inhibited by 10 µg/ml brefeldin A (BFA) for 5 h. Fixed control (CTRL) and inhibited (BFA) slices were labelled by immunohistochemistry and SNAP-tag substrate for fluorescent imaging. Created by Biorender.com. b Confocal images of OHSC transduced with either AAV-C2-SNAP or AAV-C2m2-SNAP. Cell nuclei were stained with DAPI, fused SNAP-tag was labelled with AlexaFluor647 (AF647). Astrocytes, microglia and neurons were labelled with GFAP, Iba1 antibodies, or expressed EGFP, respectively. Scale bar 20 µm. c, d Quantification of C2-SNAP and C2m2-SNAP fluorescence within astrocytes with or without BFA treatment. Data presented as means ± standard error of the mean (n = 10–15 images per replicate, two independent replicates). Means were compared by one-way ANOVA and post-hoc Tukey test. p values < 0.05 were considered as significant. RFU relative fluorescence units

Article Snippet: The resulting pET21aC2m2 plasmid was used to construct pET21a-C2m2-SNAP and pET21a-C2m2-mKate plasmids, following the procedure described above. pAAV plasmid for virus production The plasmids for virus production were designed to carry astrocyte specific promoter GFAP sequence from pAAV-GFAP-mKate plasmid (Addgene plasmid #99129, RRID:Addgene_99129).

Techniques: Expressing, Ex Vivo, Transduction, In Vitro, Recombinant, Control, Immunohistochemistry, Imaging, Staining, Fluorescence