Journal: iScience

Article Title: Combined metabolomic and transcriptomic profiling approaches reveal the cardiac response to high-fat diet

doi: 10.1016/j.isci.2022.104184

Figure Lengend Snippet:

Article Snippet: Plasmid: pCMV Flag ERR1 , Addgene , #10975.

Techniques: Marker, Extraction, Transfection, Lysis, Luciferase, Protease Inhibitor, RNA Sequencing, Recombinant, Plasmid Preparation, Software

Journal: Genetics in medicine : official journal of the American College of Medical Genetics

Article Title: Elucidating the clinical and molecular spectrum of SMARCC2-associated NDD in a cohort of 65 affected individuals.

doi: 10.1016/j.gim.2023.100950

Figure Lengend Snippet: Figure 1 SMARCC2 linear domain structure, distribution of pathogenic variants in the BAF complex and N-terminal variants causing protein loss. A. Linear protein model of SMARCC2 and its domains: an N-terminal module containing a MarR-like helix-turn-helix domain (eggshell) with DNA-binding ability,25 as well as a BRCT domain (orange) with an inserted non-functional chromodomain (red), which have been proposed to mediate protein-protein interactions.25 The SWIRM domain (magenta) mediates protein-protein interaction,34,35 the SANT domain (berry) is the chromatin binding domain of the protein which was proposed to recognize unmodified histone tails,36,37 the dimerization region (purple) and core assembly region (dark blue) are coiled-coil domains involved in the formation of the core BAF complex. The first is necessary for heterodimerization with SMARCC1 and the latter interacts with SMARCD1 and SMARCE1, forming the base of the

Article Snippet: Missense variants in other BAF base complex subunits (File S2 sheet “missense_other_BAF”) were reviewed from published literature reports (Table S2), standardized to fit the model transcript and used to analyze spatial proximity in the BAF complex model. Functional analyses of missense variants FLAG-tagged SMARCC2 was obtained from Addgene (plasmid #19142)27 and variants were introduced using the InFusion HD Cloning Kit (Clontech).

Techniques: Binding Assay, Functional Assay, Protein-Protein interactions

Journal: Genetics in medicine : official journal of the American College of Medical Genetics

Article Title: Elucidating the clinical and molecular spectrum of SMARCC2-associated NDD in a cohort of 65 affected individuals.

doi: 10.1016/j.gim.2023.100950

Figure Lengend Snippet: Figure 2 Facial appearance and representative cMRIs of SMARCC2 individuals. Facial features, facial overlay and images from hands and feet of individuals with LGD (A) and non-truncating (B) SMARCC2 variants. Ind-7 and Ind-8 carrying the missense variant c.230C>T p.(Pro77Leu), which leads to SMARCC2 protein loss, were grouped together with the LGD variant individuals. In addition to other craniofacial anomalies, a triangular face with narrow chin is frequently depicted in photos of both groups. Also note the pronounced coarseness of facial characteristics in individuals with non-truncating variants. C. Neuroimaging characteristics of SMARCC2 individuals and a control subject. Ind-02, Ind-09 and Ind-11 exhibit normal corpus callosum appearance. Corpus callosum hypoplasia (empty arrows) or dysplasia with thinning of the corpus callosum splenium (thick arrows), may or may not be accompanied by anterior commissure hypoplasia/ agenesis (arrowheads) and small inferior cerebellar vermis (thin arrows).

Article Snippet: Missense variants in other BAF base complex subunits (File S2 sheet “missense_other_BAF”) were reviewed from published literature reports (Table S2), standardized to fit the model transcript and used to analyze spatial proximity in the BAF complex model. Functional analyses of missense variants FLAG-tagged SMARCC2 was obtained from Addgene (plasmid #19142)27 and variants were introduced using the InFusion HD Cloning Kit (Clontech).

Techniques: Variant Assay, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: AAVone: A cost-effective, single-plasmid solution for efficient AAV production with reduced DNA impurities

doi: 10.1016/j.omtn.2025.102563

Figure Lengend Snippet: Design and testing of the AAVone production system (A) Schematic illustrations of the AAVone, triple-plasmid (pHelper, pTri-plasmid; and mini-pHelper-1.0, mTri-plasmid) systems, dual-plasmid (pDG-like and AAVdual) systems, and the AAVone system. (B) A comparative analysis of AAV productivity using AAVone and alternative systems in adherent HEK-293T cells. For every system compared, the total pDNA amount was maintained at 0.5 μg/well (24-well plate), and the PEImax to pDNA ratio was consistently kept at 3:1. The plasmid molecular ratios for each system is displayed under each dataset. (C) A comparison of vector yields with different serotypes between the AAVone and the pTri-plasmid system in suspension HEK-293T cells. The pTri-plasmid set was transfected at equal plasmid molecular ratios at a total DNA amount of 1 μg/1E6 cells, at a cell density of 3E6 cells/mL. Crude lysate samples were quantified using qPCR with primers targeting Egfp , and data were normalized to the average values of each pTri-plasmid group. (D) Productivity optimization of the AAVone system in suspension cultured HEK-293T cells as assessed by crude lysate titration. Different cell densities, total pDNA, and transfection reagent (PEImax) were examined.

Article Snippet: The mini-pHelper-1.0 (Addgene #230933) was generated by deleting introns of E2 and E4 genes from the pHelper (AAVnerGene, SM000002) parent construct. pAAVdual- CMV - Egfp plasmid (Addgene #230931) was cloned by inserting AAV-CMV- Egfp genome (including ITRs) into mini-pHelper-1.0 at the Pme I site, located between pMB1 ori and VA RNA . mini-pHelper-AAV plasmids were cloned by inserting the rep-cap cassette into the Cla I site of mini-pHelper-1.0, which is located between E4 and VA RNA . pAAVone-AAV2- CMV - Egfp plasmid (Addgene #230930) was achieved by insertion AAV2- CMV - Egfp genome into the mini-pHelper-AAV2 at the Pme I site. pAAVone plasmids with different capsids and transgenes are based on pAAVone-AAV2- CMV - Egfp and generated by replacing the Cap ORF(such as pAAVone-AAV9- CMV - Egfp (Addgene #230928) and pAAVone-AAV-PHP.eB- CMV - Egfp (Addgene #230928) or the GOI (such as pAAVone-AAV9- CMV - Egfp-2.2kb , and -4.7kb) .

Techniques: Plasmid Preparation, Comparison, Suspension, Transfection, Cell Culture, Titration

Journal: Molecular Therapy. Nucleic Acids

Article Title: AAVone: A cost-effective, single-plasmid solution for efficient AAV production with reduced DNA impurities

doi: 10.1016/j.omtn.2025.102563

Figure Lengend Snippet: Characterization of AAV vectors produced by the AAVone and tri-plasmid systems Purified AAV vectors produced in suspension HEK-293T under different production platforms. (A) Purified AAV vectors (∼5E11 particles) from pTri-plasmid, mTri-plasmid, and AAVone were electrophoresed and visualized by Coomassie G-250 staining. (B) Automated electrophoresis analysis (TapeStation) of vector DNAs illustrating full-length genomes (arrow) and partial genomes (bracket). Arrow heads designate specific bands identified by software. (C) Mass photometry analysis of AAV particles. y axis, particle counts; x axis, mass of particles. (D) Transduction activity of vectors packaging the AAV2- CMV-Egfp reporter produced by AAVone or triple-plasmid systems in HeLa cells at a multiplicity of infection (MOI) of 10,000 vg/cell. and EGFP expression was observed 48 h post-transduction (scape bar, 100 μm).

Article Snippet: The mini-pHelper-1.0 (Addgene #230933) was generated by deleting introns of E2 and E4 genes from the pHelper (AAVnerGene, SM000002) parent construct. pAAVdual- CMV - Egfp plasmid (Addgene #230931) was cloned by inserting AAV-CMV- Egfp genome (including ITRs) into mini-pHelper-1.0 at the Pme I site, located between pMB1 ori and VA RNA . mini-pHelper-AAV plasmids were cloned by inserting the rep-cap cassette into the Cla I site of mini-pHelper-1.0, which is located between E4 and VA RNA . pAAVone-AAV2- CMV - Egfp plasmid (Addgene #230930) was achieved by insertion AAV2- CMV - Egfp genome into the mini-pHelper-AAV2 at the Pme I site. pAAVone plasmids with different capsids and transgenes are based on pAAVone-AAV2- CMV - Egfp and generated by replacing the Cap ORF(such as pAAVone-AAV9- CMV - Egfp (Addgene #230928) and pAAVone-AAV-PHP.eB- CMV - Egfp (Addgene #230928) or the GOI (such as pAAVone-AAV9- CMV - Egfp-2.2kb , and -4.7kb) .

Techniques: Produced, Plasmid Preparation, Purification, Suspension, Staining, Electrophoresis, Software, Transduction, Activity Assay, Infection, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: AAVone: A cost-effective, single-plasmid solution for efficient AAV production with reduced DNA impurities

doi: 10.1016/j.omtn.2025.102563

Figure Lengend Snippet: AAVone-packaged AAV2 vectors exhibit similar in vivo efficacies as vectors produce d by triple-plasmid systems (A) Representative fundoscopy images of murine retinas at 4 weeks post-injection. (B) Representative retina cross-sections stained to visualize EGFP (anti-EGFP, green), nuclei (DAPI, blue, and microglia (IBA1, red) at 6 weeks post-injection. Cross-sections of representative whole eye cups (scale bars, 0.5 mM). (C) Quantification of EGFP + cells in the photoreceptor (PR) layer. (D and E) ddPCR quantification of vector genomes (D) and vector transcripts (E) in retina samples at 6 weeks post-injection ( n = 4). (F) Counts of total microglia in retina cross-sections at 6 weeks post-injection. (G) Representative cross-section of a vector-treated retina illustrating microglial (IBA1 + ) infiltration in retinal layers: photoreceptor segment layer (PS); outer nuclear layer (ONL); outer plexiform layer (OPL); inner nuclear layer (INL); inner plexiform layer (IPL); and ganglion cell layer (GCL). IBA1 + cells (red) and DAPI (blue). (H) Percentage of IBA1 + cells in each retinal layer. Values represent means ± SD, ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001, ns = not significant; by one-way ANOVA and Tukey’s multiple comparison.

Article Snippet: The mini-pHelper-1.0 (Addgene #230933) was generated by deleting introns of E2 and E4 genes from the pHelper (AAVnerGene, SM000002) parent construct. pAAVdual- CMV - Egfp plasmid (Addgene #230931) was cloned by inserting AAV-CMV- Egfp genome (including ITRs) into mini-pHelper-1.0 at the Pme I site, located between pMB1 ori and VA RNA . mini-pHelper-AAV plasmids were cloned by inserting the rep-cap cassette into the Cla I site of mini-pHelper-1.0, which is located between E4 and VA RNA . pAAVone-AAV2- CMV - Egfp plasmid (Addgene #230930) was achieved by insertion AAV2- CMV - Egfp genome into the mini-pHelper-AAV2 at the Pme I site. pAAVone plasmids with different capsids and transgenes are based on pAAVone-AAV2- CMV - Egfp and generated by replacing the Cap ORF(such as pAAVone-AAV9- CMV - Egfp (Addgene #230928) and pAAVone-AAV-PHP.eB- CMV - Egfp (Addgene #230928) or the GOI (such as pAAVone-AAV9- CMV - Egfp-2.2kb , and -4.7kb) .

Techniques: In Vivo, Plasmid Preparation, Injection, Staining, Comparison

Journal: Molecular Therapy. Nucleic Acids

Article Title: AAVone: A cost-effective, single-plasmid solution for efficient AAV production with reduced DNA impurities

doi: 10.1016/j.omtn.2025.102563

Figure Lengend Snippet: Abundance of producer plasmid contaminants in AAV vectors generated by tri-plasmid systems and AAVone (A and B) Coverage traces of aligned SMRT reads to the pAAVone (A), pAAV-CMV-EGFP, (B) and AAVone (C) plasmid references. Dashed lines indicate the boundaries of the ITR references. (D) ddPCR quantification of trans plasmid sequences ( rep and cap ), AdV helper plasmid ( VA RNA ), backbone sequences (pMB1 ori and F1 ori ), and rcAAV in vector preparations. Values are means ± SD ( n = 3). ∗ p ≤ 0.05; ∗∗∗∗ p ≤ 0.0001 by two-way ANOVA. Color of asterisks correspond to p values when compared with pTri-plasmid (red), mTri-plasmid (blue), and AAVone (purple) data.

Article Snippet: The mini-pHelper-1.0 (Addgene #230933) was generated by deleting introns of E2 and E4 genes from the pHelper (AAVnerGene, SM000002) parent construct. pAAVdual- CMV - Egfp plasmid (Addgene #230931) was cloned by inserting AAV-CMV- Egfp genome (including ITRs) into mini-pHelper-1.0 at the Pme I site, located between pMB1 ori and VA RNA . mini-pHelper-AAV plasmids were cloned by inserting the rep-cap cassette into the Cla I site of mini-pHelper-1.0, which is located between E4 and VA RNA . pAAVone-AAV2- CMV - Egfp plasmid (Addgene #230930) was achieved by insertion AAV2- CMV - Egfp genome into the mini-pHelper-AAV2 at the Pme I site. pAAVone plasmids with different capsids and transgenes are based on pAAVone-AAV2- CMV - Egfp and generated by replacing the Cap ORF(such as pAAVone-AAV9- CMV - Egfp (Addgene #230928) and pAAVone-AAV-PHP.eB- CMV - Egfp (Addgene #230928) or the GOI (such as pAAVone-AAV9- CMV - Egfp-2.2kb , and -4.7kb) .

Techniques: Plasmid Preparation, Generated