packaging plasmid pcmv vsvg  (Addgene inc)

Journal: BMC Biology

Article Title: CoREST3 exhibits isoform specific expression in Alzheimer’s disease and regulation of HDAC2

doi: 10.1186/s12915-025-02349-x

Figure Lengend Snippet: CoREST3 knockdown significantly increased neurite length and branch points in iPSC derived cortical neurons but did not alter cell viability. RCOR3 gene knockdown was performed via lentiviral transduction of three different shRNA sequences that target different regions of the gene and a scrambled sequence into immature cortical neurons derived from the healthy control iPSC line. A Schematic of plasmid maps used for RCOR3 gene knockdown using shRNA. B Representative brightfield images of neurons on day 7 post-transduction with scrambled or shRNA sequenes. Scale bar = 100 µm. C RCOR3 levels were analysed by RT-qPCR 7 days post infection. Neuronal genes, ( D ) PAX6, ( E ) TUBB3 (encodes β-III-tubulin) and ( F ) MAP2 levels were also analysed by RT-qPCR 7 days post infection. Data is presented using the 2 (−ΔCt) method to the mean of two housekeeping genes. n = 3–5 independent differentiations, n = 2 technical replicates. G Cell viability of neurons was assessed by a resazurin assay on day 7 and presented as the relative fluorescence units (544/590 nm). n = 4 technical replicates, n = 3 independent differentiations. Data are presented as the mean ± SEM. Data were analysed using a One-way ANOVA with a Holm-Sidak for multiple comparisons. H Neurite analysis was performed using the Incucyte Zoom live cell imager. Neurite length (mm/mm 2 ) and neurite branch points (1/mm 2 ) were analysed over time for neurons derived from each cell line and normalised to day 0 to account for interwell variability. Neurite analysis was taken from 3–5 wells of a 96 well plate, per cell line ( n = 3–5 technical replicates) from 4 independent differentiations ( n = 4 biological replicates)

Article Snippet: Briefly, HEK293T cells were transfected with the DNA of lentiviral packaging plasmids vSVG (Addgene, USA, #8454), RSV (Addgene, #12,253), pMDL (Addgene, #12,251), and either the tetracycline transactivator (TTA) vector, M2rtTA (Addgene, #20,342), the NGN2 overexpression vector, TetO- NGN2 -eGFP-Puro plasmid (Addgene, #79,823), or one of the transfer vectors using polyethyleneimine (Sigma-Aldrich, USA, #408,727).

Techniques: Knockdown, Derivative Assay, Transduction, shRNA, Sequencing, Control, Plasmid Preparation, Quantitative RT-PCR, Infection, Resazurin Assay, Fluorescence

Journal: BMC Biology

Article Title: CoREST3 exhibits isoform specific expression in Alzheimer’s disease and regulation of HDAC2

doi: 10.1186/s12915-025-02349-x

Figure Lengend Snippet: CoREST3 exhibited isoform specific regulation of neurite length and branch points in iPSC derived cortical neurons. CoREST3 overexpression was performed via lentiviral transduction of each RCOR3 -variant into immature cortical neurons derived from the healthy control iPSC line. A Schematic of plasmid maps used for RCOR3 overexpression. B Representative brightfield images of neurons on day 7 post-transduction with empty vector or CoREST3 isoform. Scale bar = 100 µm. C RCOR3 levels were analysed by RT-qPCR 7 days post infection. Neuronal genes, ( D ) PAX6, ( E ) TUBB3 (encodes β-III-tubulin) and ( F ) MAP2 levels were also analysed by RT-qPCR 7 days post infection. Data is presented using the 2 (−ΔCt) method to the mean of two housekeeping genes from n = 2 technical replicates, n = 3 independent differentiations. G Cell viability of neurons was assessed by a resazurin assay on day 7 and presented as the relative fluorescence units (544/590 nm) from n = 6 technical replicates, n = 3 independent differentiations. Data are presented as the mean ± SEM. Data were analysed using a One-way ANOVA with a Holm-Sidak correction for multiple comparisons. H Neurite analysis was performed using the Incucyte S3 live cell imager. Neurite length (mm/mm 2 ) and neurite branch points (1/mm 2 ) were analysed over time for neurons derived from each cell line and normalised to day 0 to account for inter-well variability. Neurite analysis was taken from 6 wells of a 96 well plate, per cell line from n = 6 technical replicates, n = 3 independent differentiations

Article Snippet: Briefly, HEK293T cells were transfected with the DNA of lentiviral packaging plasmids vSVG (Addgene, USA, #8454), RSV (Addgene, #12,253), pMDL (Addgene, #12,251), and either the tetracycline transactivator (TTA) vector, M2rtTA (Addgene, #20,342), the NGN2 overexpression vector, TetO- NGN2 -eGFP-Puro plasmid (Addgene, #79,823), or one of the transfer vectors using polyethyleneimine (Sigma-Aldrich, USA, #408,727).

Techniques: Derivative Assay, Over Expression, Transduction, Variant Assay, Control, Plasmid Preparation, Quantitative RT-PCR, Infection, Resazurin Assay, Fluorescence

Journal: BMC Biology

Article Title: CoREST3 exhibits isoform specific expression in Alzheimer’s disease and regulation of HDAC2

doi: 10.1186/s12915-025-02349-x

Figure Lengend Snippet: CoREST3 isoform specific regulation of HDAC2 levels. CoREST3 isoforms and an empty vector control were overexpressed in cortical neurons derived from iPSCs using inducible lentiviral delivery. Samples were harvested 7 days post transduction and western blots were performed targeting CoREST3 ( A ) and was observed as three distinct bands. Total CoREST3 ( B ), Band I ( C ), Band II ( D ) and Band III ( E ) was normalised to total protein and presented as the mean ± SEM, n = 2 technical replicates, n = 3 independent differentiations. Western blots were performed targeting HDAC2 ( F and G ), with data normalised to total protein and presented as the mean ± SEM, n = 2 technical replicates, n = 3 independent differentiations. Data were analysed using a One-way ANOVA with a Holm-Sidak post-hoc, where p < 0.05*, p < 0.01**, p < 0.001*** and p < 0.0001****. Full blots can be found in Additional file 4

Article Snippet: Briefly, HEK293T cells were transfected with the DNA of lentiviral packaging plasmids vSVG (Addgene, USA, #8454), RSV (Addgene, #12,253), pMDL (Addgene, #12,251), and either the tetracycline transactivator (TTA) vector, M2rtTA (Addgene, #20,342), the NGN2 overexpression vector, TetO- NGN2 -eGFP-Puro plasmid (Addgene, #79,823), or one of the transfer vectors using polyethyleneimine (Sigma-Aldrich, USA, #408,727).

Techniques: Plasmid Preparation, Control, Derivative Assay, Transduction, Western Blot