third generation viral packaging plasids  (Addgene inc)

Journal: Human Genetics and Genomics Advances

Article Title: Bi-allelic variants in BCAT1 impair mitochondrial function and are associated with a candidate neurometabolic disorder

doi: 10.1016/j.xhgg.2025.100525

Figure Lengend Snippet: Loss of BCAT1 impacts hiPSC-derived cortical neuron differentiation (A) Schematic of hiPSC lentiviral transduction for Ngn2-iN and experimental design. (B) Representative western blot analysis of BCAT1 E/K F/L and BCAT1 −/− Ngn2-iNs collected at day 2 and day 14 of differentiation (left). Quantification of vinculin expression in day 14 neurons (right). Results are mean ± SEM ( n = 3 independent experiments per group in all 3 clonal lines) and analyzed by one-way ANOVA with Dunnett’s post hoc test. (C) qRT-PCR analysis of BCAT1 , BCAT2 , and NeuroD1 transcript levels in day 14 Ngn2-iNs normalized to RPLP0 . Results represented as means ± SEM ( n ≥ 3 independent experiments per group in all 3 clonal lines), analyzed by two-way ANOVA with Dunnett’s post hoc analysis test for multiple comparisons. (D) Representative confocal images and (E) fluorescence intensity measurements of developing neurons 2, 7, and 14 days post-differentiation (dpd) showing increased Tuj1 and Map2 expression. Scale bars, 100 μm. Immunocytochemistry was performed in all three clonal lines and two cell culture replicates per time point, with similar results. (F) Reconstructions of sparse-labeled Ngn2-iNs derived from BCAT1 E/K F/L and isogenic control lines. Experiments were performed in all two cell culture replicates per time point. Results are shown in (G). BCAT1 neurons exhibit reduced growth properties and were significantly shorter at 2 dpd (left) compared with 7 dpd (right) (two-way ANOVA with Sidak correction; 2 dpd: ∗∗ p < 0.005; 7 dpi: n.s., not significant). Values show means ± SEM.; BCAT1 E/K F/L ( n = 1; 33 technical tracing replicates in total); control ( n = 1; 29 technical tracing replicates in total); n refers to biologically independent clonal lines. A.U., arbitrary units.

Article Snippet: Ngn2-iN lentiviruses were produced as previously described in HEK293T cells (ATCC, VA) using polyethylenimine (Polysciences, PEI, 23966-2) and third-generation lentiviral packaging plasmids pMDLg/pRRE (Addgene, 12251), pRSV-RV (Addgene, 12253), and VSC-G (Addgene, 12259), along with pLVX-UbC-rtTA-Ngn2:2A (Addgene, 127288).

Techniques: Derivative Assay, Transduction, Western Blot, Expressing, Quantitative RT-PCR, Fluorescence, Immunocytochemistry, Cell Culture, Labeling, Control