lentiviral dominant negative tcf4 plasmid  (Addgene inc)

Journal: International Journal of Biological Sciences

Article Title: MicroRNA-495 Modulates Neuronal Layer Fate Determination by Targeting Tcf4

doi: 10.7150/ijbs.94739

Figure Lengend Snippet: TCF4 promotes the increased generation of deep-layer neurons. (A) Schematic diagram of TCF4 overexpression clone construction. (B) Western blot validation of TCF4 overexpression clone efficiency. β-actin is as the reference protein. (C-H) pCIG+pCAG (control) and pCIG- Tcf4 +pCAG (TCF4) were electroporated into embryonic brains from mice at E13.5. Brain sections at P3 were immunostained with cortical markers (CUX1 and SOX5). Scale bar: 500 µm (C, D) and 100 µm (E-H, E'-H'). (I) The sections labelled A' to B' were divided into 10 bins to count the distribution of EGFP-positive cells in the cortex. These sections were derived from the electroporated mouse brains, which were from different littermates (control: n=15 sections from 13 brains, TCF4: n=4). (J) Statistical analysis of the percentage of CUX1+/GFP+ cells in (E', F') and SOX5+/GFP+ cells in (G', H') (control: n = 15 sections from 13 brains, TCF4: n=4). Statistical significance was determined using an unpaired two-tailed Student's t-test. *P<0.05, ** P<0.01 and ***P<0.001

Article Snippet: The TCF4 knockdown plasmid was constructed using the pLL3.7 vector (Addgene).

Techniques: Over Expression, Western Blot, Biomarker Discovery, Control, Derivative Assay, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: MicroRNA-495 Modulates Neuronal Layer Fate Determination by Targeting Tcf4

doi: 10.7150/ijbs.94739

Figure Lengend Snippet: TCF4 knockdown restrains the generation of deep-layer neurons. (A) Schematic diagram of TCF4 knockdown clone construction. The designations sh1, sh2, and sh3 refer to pLL3.7-sh1- Tcf4 , pLL3.7-sh2- Tcf4 , and pLL3.7-sh3- Tcf4 , respectively. (B) WB validation of TCF4 knockdown clone efficiency. β-actin is as the reference protein. (C-K) pLL3.7-Scr+pCAG, pLL3.7-sh1- Tcf4 + pCAG and pLL3.7-sh2- Tcf4 + pCAG were electroporated into the embryonic brains of mice at E13.5. Brain sections at P3 were immunostained with cortical markers (CUX1 and SOX5). Scale bar: 500 µm (C, D, E) and 100 µm (F-K, F'-K') . (L) The sections labelled F to H were divided into 10 bins to count the distribution of EGFP-positive cells in the cortex. These sections were derived from the electroporated mouse brains, which were from different littermates (Scr: n=3, sh1-Tcf4: n=3, sh2-Tcf4: n=3). (M) The percentage of CUX1+/GFP+ cells and SOX5+/GFP+ cells in (F-K) (Scr: n=3, sh1-Tcf4: n=3, sh2-Tcf4: n=3). Statistical significance was determined using an unpaired two-tailed Student's t-test. Results are expressed as the mean ±SD. P values are shown as *P<0.05, ** P<0.01.

Article Snippet: The TCF4 knockdown plasmid was constructed using the pLL3.7 vector (Addgene).

Techniques: Knockdown, Biomarker Discovery, Derivative Assay, Two Tailed Test

Journal: Nature communications

Article Title: Transcription Factor 4 loss-of-function is associated with deficits in progenitor proliferation and cortical neuron content.

doi: 10.1038/s41467-022-29942-w

Figure Lengend Snippet: Fig. 8 Reversal of abnormal phenotypes in PTHS organoids after trans-epigenetic correction of TCF4 expression. a Schematic representation of CRISPR-based trans-epigenetic correction of TCF4 expression using constructs for guide RNA (gRNA), transcriptional activation module MPH, and dead Cas9 (see “Methods” for details). b Top: Virus application regimen. Bottom: Brightfield images of PTHS brain organoids at 4 weeks in vitro after correction of TCF4 expression (PTHS + TCF4 gRNA), compared with controls transduced with scrambled gRNA (scr gRNA). c Fluorescence microscopy images of transduced organoids at 2 weeks in vitro after immunostaining for TCF4 (green). Clustered TCF4+ cells (arrowhead) can be seen in aberrant outgrowth in image on the right in PTHS + scr gRNA condition. See Supplementary Fig. 12d, e for quantification of number of TCF4+ cells and mean TCF4 staining pixel intensity in transduced organoids. d Increase in TCF4 expression levels after CRISPR-mediated trans-epigenetic TCF4 correction in organoids at 2 weeks in vitro. N = 3 replicates per group (circles). e–g Expression levels of GADD45G (e), CDKN2A (f), and MAP2 (g) in organoids at 4 weeks in vitro after trans- epigenetic TCF4 expression correction. N = 3 replicates per group (circles). Organoids are from parent–patient pair #4. h Transduced organoids stained for MAP2 (magenta) and SOX2 (green), at two developmental time points. Arrowheads in middle panels: polarized PTHS organoids. High mag insets: clustered abnormally shaped MAP2+ cells in polarized organoid outgrowth. Arrowhead in right panel: neural rosettes. Experiments were conducted with organoids from parent-patient pair #4 (circle symbols in bar graphs). Colors in bar graphs represent parents (orange), PTHS (blue), or genetically manipulated PTHS (light blue) groups. Error bars represent SEM. n.s., not significant; *p < 0.05; **p < 0.01; ***p < 0.001; one-way ANOVA followed by Tukey’s HSD post-hoc test in bar plots. Scale bars are 100 μm. DAPI nuclear staining in blue. See Supplementary Data 1 for sample and effect sizes and exact p-values. Attribution of DNA image in a: Ioana Davies/Shutterstock.com.

Article Snippet: To evaluate the efficiency of the designed TCF4 gRNA sequences at increasing the endogenous expression of the TCF4 gene via trans-epigenetic activation, we transfected SH-SY5Y cells with the pLentiSAMv2 and pLentiMPHv2 (Addgene #89308; http://n2t.net/addgene:89308; RRID:Addgene_89308) plasmids, followed by RT-qPCR to verify the levels of TCF4 transcripts.

Techniques: Expressing, CRISPR, Construct, Activation Assay, Virus, In Vitro, Transduction, Fluorescence, Microscopy, Immunostaining, Staining

Journal: Nature communications

Article Title: Transcription Factor 4 loss-of-function is associated with deficits in progenitor proliferation and cortical neuron content.

doi: 10.1038/s41467-022-29942-w

Figure Lengend Snippet: Fig. 10 Model of dysregulated pathways underlying PTHS pathophysiology. Mechanistic model to explain aberrant cellular phenotypes in PTHS neural structures. Due to TCF4 loss-of-function in PTHS, Wnt signaling activity diminishes, in turn leading to decreased SOX3 expression in NPCs, impairing proliferation. Moreover, we observed that SOX4 is also downregulated in PTHS cells, which we suggest impairs neuronal differentiation and content in the PTHS neural tissue.

Article Snippet: To evaluate the efficiency of the designed TCF4 gRNA sequences at increasing the endogenous expression of the TCF4 gene via trans-epigenetic activation, we transfected SH-SY5Y cells with the pLentiSAMv2 and pLentiMPHv2 (Addgene #89308; http://n2t.net/addgene:89308; RRID:Addgene_89308) plasmids, followed by RT-qPCR to verify the levels of TCF4 transcripts.

Techniques: Activity Assay, Expressing