pcag smfp ha vector  (Addgene inc)

Journal: Disease models & mechanisms

Article Title: Autism-linked NLGN3 is a key regulator of gonadotropin-releasing hormone deficiency.

doi: 10.1242/dmm.049996

Figure Lengend Snippet: Fig. 3. Two novel NLGN3 nonsense variants found in patients with GnRH deficiency (GD) and autism spectrum disorder (ASD). (A) Family pedigrees of the two probands (Case 1 and Case 2) identified in this study and presenting GD and ASD features. Sequence chromatograms of nucleotides 70367955-70367975 and nucleotides 70367752-70367772 of the NLGN3 coding sequence in Case 1, Case 2 and their unaffected mothers (the positions of the C>T and G>A are highlighted by the red boxes). (B) Schematic representation of NLGN3 protein (encoded by the NM_181303.1 transcript): signal peptide (light blue), extracellular (green), transmembrane (yellow) and intracellular (orange) domains. All missense variants (gray dots) reported in gnomAD database (v2.1.1) are plotted according to their amino acid (aa) position and Combined Annotation-Dependent Depletion (CADD) score. Pathogenic variants related to ASD are indicated in black: R471C (Jamain et al., 2003), P534S (Quartier et al., 2019) and R617W (Redin et al., 2014). Identified variants (R55* and W122*) are indicated in red and have a higher CADD score compared to others. Multi-species alignment of partial protein sequences of vertebrate NLGN3 ortholog proteins shows that the R55 and W122 residues are evolutionarily conserved in humans and other vertebrate species with a high conservation degree, calculated by GERP++.

Article Snippet: To introduce the c.163C>T and c.366G>A variants into human NLGN3 gene, the WT human HA-tagged NLGN3 expression vector (Addgene, 59318) was mutagenized using a QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies) and specific oligonucleotides for NLGN3 R55* (fw, 5′-GGCAGTGGTACTCAGGCACCCCTTAGC-3′; rev, 5′-GCTAAGGGGTGCCTGAGTACCACTGCC-3′) and W122* (fw, 5′-GTCATGCTGCCGGTCTGATTCACTGCCAACTTGGATATCG-3′; rev, 5′-TCAGACCGGCAGCATGACTTCGGGCACAGCTGTGTGGATG-3′).

Techniques: Sequencing

Journal: Disease models & mechanisms

Article Title: Autism-linked NLGN3 is a key regulator of gonadotropin-releasing hormone deficiency.

doi: 10.1242/dmm.049996

Figure Lengend Snippet: Fig. 4. NLGN3 is developmentally regulated in GnRH neurons. (A) Quantitative PCR analysis performed on GN11 and GT1-7 cells revealed higher Nlgn3 expression levels in GT1-7 cells (logFC=8.12, P<0.01). This result is in line with microarray experiments (logFC=4.15; P<0.00001). Data are presented as mean±s.d. of three biological replicates. Unpaired two-tailed Student’s t-test (**P<0.01). (B) Immunoperoxidase staining for NLGN3 on GN11 and GT1-7 revealed different levels of endogenous NLGN3 protein in these cells. Scale bar: 25 μm. (C,D) Coronal sections of E14.5 mouse heads were immunolabeled for NLGN3 together with GnRH (C) or PLXND1 (D) to detect GnRH neurons. Sections are shown at the level of the VNO (nose; C) or MPOA (forebrain; D). White dashed line boxes indicate areas shown at higher magnification on the right of the corresponding panel, with single channels also shown adjacent to the panel. Open arrowheads indicate examples of GnRH-positive cells that lack NLGN3; filled arrowheads indicate examples of GnRH-positive cells with NLGN3. Arrows indicate examples of NLGN3- positive cells in the nasal parenchyma. Sections were counterstained with DAPI. MPOA, medial preoptic area; OB, olfactory bulb; OE, olfactory epithelium; VNO, vomeronasal organ. Scale bars: 250 μm (right panels), 150 μm (middle panels) or 50 μm (left panels).

Article Snippet: To introduce the c.163C>T and c.366G>A variants into human NLGN3 gene, the WT human HA-tagged NLGN3 expression vector (Addgene, 59318) was mutagenized using a QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies) and specific oligonucleotides for NLGN3 R55* (fw, 5′-GGCAGTGGTACTCAGGCACCCCTTAGC-3′; rev, 5′-GCTAAGGGGTGCCTGAGTACCACTGCC-3′) and W122* (fw, 5′-GTCATGCTGCCGGTCTGATTCACTGCCAACTTGGATATCG-3′; rev, 5′-TCAGACCGGCAGCATGACTTCGGGCACAGCTGTGTGGATG-3′).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Microarray, Two Tailed Test, Immunoperoxidase Staining, Immunolabeling

Journal: Disease models & mechanisms

Article Title: Autism-linked NLGN3 is a key regulator of gonadotropin-releasing hormone deficiency.

doi: 10.1242/dmm.049996

Figure Lengend Snippet: Fig. 5. Mutant NLGN3 proteins induce endoplasmic reticulum retention and impair neuritogenesis in immortalized GnRH neurons. (A) Immunoblot analysis with anti-HA or anti-GAPDH antibodies on whole-cell lysates and conditioned media from COS7 cells overexpressing control vector (GFP), HA- tagged wild-type (WT) or mutant NLGN3. GAPDH is shown as a loading control for cell lysates. (B) Confocal images of GN11 cells transfected cells with mEmerald-ER-3 (green) and human WT or mutated HA-tagged NLGN3 vectors and stained for HA (red) after 24 h. HA-NLGN3 single-channel images are shown below each panel. (C) Confocal images of GN11 transfected with human WT or mutated NLGN3 HA-tagged encoding vector and stained for HA (green) and F-actin (red). Arrowheads point at neurites in NLGN3 WT-expressing GN11 cells. (D) Quantification of cell perimeter (P), cell area (A), aspect ratio (AR) and complexity index (CI) in GN11 cells transfected with indicated plasmids. Column graph quantifications show a significant increase in the number of neurites extending from NLGN3 WT-expressing cells compared to NLGN3 mutants and GFP-transfected control cells. Data are presented as mean±s.d. of three biological replicates. Significant differences were determined by one-way ANOVA followed by Tukey’s multiple comparison test (***P<0.001; ****P<0.0001). Nuclei were counterstained with DAPI. Scale bars: 25 μm.

Article Snippet: To introduce the c.163C>T and c.366G>A variants into human NLGN3 gene, the WT human HA-tagged NLGN3 expression vector (Addgene, 59318) was mutagenized using a QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies) and specific oligonucleotides for NLGN3 R55* (fw, 5′-GGCAGTGGTACTCAGGCACCCCTTAGC-3′; rev, 5′-GCTAAGGGGTGCCTGAGTACCACTGCC-3′) and W122* (fw, 5′-GTCATGCTGCCGGTCTGATTCACTGCCAACTTGGATATCG-3′; rev, 5′-TCAGACCGGCAGCATGACTTCGGGCACAGCTGTGTGGATG-3′).

Techniques: Mutagenesis, Western Blot, Control, Plasmid Preparation, Transfection, Staining, Expressing, Comparison

Journal: Biotechnology Progress

Article Title: Generation of a host cell line containing a MAR ‐rich landing pad for site‐specific integration and expression of transgenes

doi: 10.1002/btpr.3254

Figure Lengend Snippet: Schematic representation of the generation of LPs‐cMAR containing cells. Cells were transfected with the landing pad vectors harboring the 5′ chicken lysozyme MAR and then selected by G418 selection and/or by sorting double positive fluorescent cells. pLP_EGFP vector contains the chicken 5′ lysozyme MAR, pCMV, AttB‐WT site for BxB1 recombinase, EGFP reporter gene, and a ghb polyA tail. pLP_DsRed vector contains the chicken 5′ lysozyme MAR, pCMV, AttB‐GA (mutated) site for BxB1 recombinase, DsRed reporter gene, and a ghb polyA tail. In addition, both vectors contain the Neomicyn resistance cassette (pSV40‐NeoR‐SV40polyA) for G418 antibiotic selection

Article Snippet: The BxB1 expression vector pCAG–NLS–HA– BxB1 was purchased from Addgene (#51271).

Techniques: Transfection, Selection, Plasmid Preparation