puc 3gla uas dscam9 28 lexa  (Addgene inc)

Journal: bioRxiv

Article Title: Stochastic splicing and deterministic inclusion of exon variables promote diversification of Down Syndrome Cell Adhesion Molecule expression

doi: 10.1101/2025.05.21.655277

Figure Lengend Snippet: (A) Schematic of the Drosophila Dscam1 genomic locus with mutually exclusive alternatively spliced exon clusters 4, 6, 9 and 17 highlighted in green, purple, blue and red, respectively. (B) Schematic of dual amplification splicing reporters UAS-Dscam9.1-9.33-LexA (positive control monitoring inclusion of all 33 exon 9 variables in the reading frame with LexA) and single isoform UAS-Dscam9.25-LexA. The reporters contain constitutive exons 7, 8, 10, 11, and all 33 exon 9 variables, as well as an HA epitope tag, a T2A self-cleaving peptide, a FLAG epitope tag and a transcriptional activator LexA. The transgenes are under the control of a UAS element. In a single isoform reporter ( Dscam9.25 in pink), the target variable has a single base pair deletion bringing it in-frame with LexA. (C) Schematic of the dual amplification system. The panneuronal elav induces transcriptional activator GAL4, which binds to the upstream UAS element of the Dscam exon 9 reporter construct. Inclusion of the mutated variable exon 9.25 (pink) in the mature mRNA leads to LexA expression. LexA then activates LexAop-tdTomato , resulting in fluorescent labelling of neurons expressing the specific isoform. (D) Genetic cross to visualize dual-amplification reporters. Flies carrying elav-GAL4 ; LexAop-tdTomato are crossed to UAS-Dscam9.25-LexA , enabling isoform-specific labelling in progeny. (E-P) Ventral views of the third instar larvae VNC showing inclusion patterns of Dscam9.1-9.33 (positive control expressing all 33 exon 9 variables in the reading frame with LexA, E), Dscam9.20 (H), Dscam9.25 (K), and Dscam9.28 (N), visualised using nuclear-localised elav-GAL4;LexAop-tdTomato . Brains were counterstained with DAPI (F, I, L, O) and shown as merged images (G, J, M, P). White arrows highlight cells arranged in parallel horizontal rows within the VNC. The scale bar is 50 µm.

Article Snippet: The sequences for the pUC 3GLA UAS-Dscam9-LexA and pUC 3GLA UAS-Dscam9.28-LexA have been deposited in GenBank under the accession numbers PV611053 and PV611054 , respectively and plasmids are available from Addgene and the European plasmid repository.

Techniques: Amplification, Positive Control, FLAG-tag, Control, Construct, Expressing

Journal: bioRxiv

Article Title: Stochastic splicing and deterministic inclusion of exon variables promote diversification of Down Syndrome Cell Adhesion Molecule expression

doi: 10.1101/2025.05.21.655277

Figure Lengend Snippet: (A-F) Representative third instar larvae central brains of elav-GAL4 visualised by nuclear-localised UAS-Histone2B::YFP (A), and UAS-Dscam9.1-9.33-LexA (positive control expressing all 33 exon 9 variables in the reading frame with LexA, D), counterstained with DAPI (B, E), and merged (C, F). The scale bar is 50 µm. (G-I) Panneuronal elav-GAL4 expression in larval salivary gland, visualised using nuclear-localised UAS-Histone2B::YFP (G), counterstained with DAPI (H), and merged (I). (J-U) Representative salivary glands showing inclusion levels of Dscam9.1-9.33 (J), Dscam9.20 (M), Dscam9.25 (P), and Dscam9.28 (S) visualised using nuclear-localised elav-GAL4;LexAop-tdTomato . Brains were counterstained with DAPI (K, N, Q, T) and shown as merged images (L, O, R, U). White arrows highlight cells in the same focal plane exhibiting various splicing efficiencies. The scale bar is 100 µm. (V) Quantification of relative signal intensity for elav and Dscam9 positive control with all 33 variables in the reading frame with LexA normalised to DAPI signal in individual salivary gland cells. Each dot represents a single nucleus. Statistical significance assessed using Levene’s test for variance is indicated by asterisks (** p ≤ 0.001).

Article Snippet: The sequences for the pUC 3GLA UAS-Dscam9-LexA and pUC 3GLA UAS-Dscam9.28-LexA have been deposited in GenBank under the accession numbers PV611053 and PV611054 , respectively and plasmids are available from Addgene and the European plasmid repository.

Techniques: Positive Control, Expressing

Journal: bioRxiv

Article Title: Stochastic splicing and deterministic inclusion of exon variables promote diversification of Down Syndrome Cell Adhesion Molecule expression

doi: 10.1101/2025.05.21.655277

Figure Lengend Snippet: (A) Schematic of the third instar larval eye disc illustrating a single anterior row of differentiated photoreceptors (pink), each comprising eight cells (R1-R8). The morphogenetic furrow (dotted line) indicates that start of photoreceptor differentiation. (B-D) Immunostaining of a third instar larval eye disc with anti-Dscam (B) and anti-ELAV (C) antibodies and merged (D). The scale bar is 50 µm. (E-G) Eye discs with photoreceptors showing inclusion of Dscam exon 4.1-4.12 variables (positive control expressing all twelve exon 4 variables in the reading frame with GAL4) reporter visualised by nuclear-localised UAS-Histone2B::YFP (E), co-stained with anti-ELAV (F) and merged (G). (H-S) Eye discs with photoreceptors showing inclusion of the Dscam9.1-9.33 variables (H), Dscam9.20 (K), Dscam9.25 (N), and Dscam9.28 (Q). Reporter expression was visualised using nuclear-localised elav-GAL4;LexAop-tdTomato , co-stained with anti-ELAV antibody (I, L, O, R) and merged (J, M, P, S). White arrows indicate cells at the same position across ommatidia, consistently expressing the same isoform. Scale bars are 20 µm and 50 µm, respectively.

Article Snippet: The sequences for the pUC 3GLA UAS-Dscam9-LexA and pUC 3GLA UAS-Dscam9.28-LexA have been deposited in GenBank under the accession numbers PV611053 and PV611054 , respectively and plasmids are available from Addgene and the European plasmid repository.

Techniques: Immunostaining, Positive Control, Expressing, Staining