Journal: Molecular Therapy. Nucleic Acids

Article Title: Subnuclear organization and mislocalization of plasmids reduce transgene expression

doi: 10.1016/j.omtn.2025.102730

Figure Lengend Snippet: Different types of promoters on the same plasmid affect its nuclear redistribution pattern in a transcription-dependent manner (A) Plasmids carrying both Pol II and Pol III promoters redistribute into unique patterns. The nuclei of A549 cells were injected with pU6-shRNA-CMV-GFP-SV40 (250 ng/μL). After 4 h, the cells were processed for in situ hybridization and immunofluorescence for either Nop58 (nucleoli) or SC35 (nuclear speckles). Yellow arrows indicate areas of restricted DAPI staining. The numbers presented in the overlay image refer to the percentage of injected cells with positive FISH signal that show this redistribution pattern (first number) and the percentage of cells showing colocalization with the stained protein marker (second number). (B) Plasmids carrying different combinations of Pol II and Pol III promoters show similar redistribution patterns. A549 cell nuclei were injected with pU6-shRNA-CMV-RFP-SV40, pH1-gRNA-CMV-mCherry, or pACE-tRNA-CMV-mCherry (250 ng/μL). After 4 h, the cells were processed for in situ hybridization and immunofluorescence using FISH probes generated from the plasmid sequences. Yellow arrows indicate areas of restricted DAPI staining. The percentage of microinjected cells with positive FISH signal that display the shown redistribution patterns of plasmids are indicated for each plasmid on the overlay image. (C) Pol II or Pol III transcriptional inhibition resulted in redistribution patterns more similar to plasmids with single promoter types. A549 cells were either untreated or pretreated for 30 min with the Pol II inhibitors actinomycin D (1 μg/mL) and α-amanitin (5 μg/mL) prior to injection or treated with the Pol III inhibitor ML-60218 CAS (66 μM) for 20 h before injection of pU6-shRNA-CMV-GFP-SV40 (250 ng/μL). FISH (red) was performed 4 h after injection, and cells were also stained for SC35 (green). Three representative cells for each treatment are shown. The numbers presented in the overlay image refer to the percentage of injected cells with positive FISH signal that show this redistribution pattern (first number) and the percentage of cells showing colocalization with SC35 (second number). (D) The presence of multiple Pol II promoters on a single plasmid has no effect on redistribution compared to plasmids with a single Pol II promoter. A549 cells were injected with pCMV-GFP-SV40 or pCMV-EGFP (250 ng/μL). After 5 min or 4 h, the cells were processed for FISH (red) and SC35 immunofluorescence (green). The percentage of microinjected cells with positive FISH signal that display the shown redistribution patterns of plasmids are indicated for each plasmid on the overlay image. (E) Injection of a mixture of pU6-shRNA and pCMV-GFP plasmids maintained their Pol II-like and Pol III-like distribution patterns. A549 cells were injected with an equimolar mixture of pU6-shRNA and pCMV-EGFP-SV40 (250 ng/μL). After 5 min or 4 h, the cells were processed for double in situ hybridization using red or green FISH probes generated from unique sequences on each plasmid to differentiate the two DNAs (Pol II and Pol III, respectively). The percentage of microinjected cells with positive FISH signal that display the plasmid redistribution patterns are indicated for each plasmid on the overlay image. For each condition, at least 50 cells were injected, and the injections were repeated three times. Bar, 10 μm.

Article Snippet: Plasmids ACE-Arg-tRNA, ACE-Trp-tRNA, and ACE-Gly-tRNA all have Pol III type 2 promoter-driven tRNA cassettes cloned into a pUC57 backbone. pU6-shRNA-CMV-GFP-SV40 (pGFP-V-RS-scramble) has a CMV-GFP cassette, a U6-scrambled shRNA cassette, and an SV40-neomycin cassette (OriGene, Rockville, MD). pU6-shRNA-CMV-RFP-SV40 (pRFP-C-RS-scramble) was purchased from OriGene, Rockville, MD. pH1v1 has a complete H1 promoter-driven gRNA cassette (Addgene). pCMV-mCherry-SV40-neo has two Pol II cassettes: one expressing mCherry from the CMV immediate-early promoter and enhancer and the other expressing neomycin resistance from the SV40 early promoter and enhancer (Takara Bio #632524, Kusatsu, Shiga, Japan). pH1-gRNA-CMV-mCherry-convergent has a CMV-mCherry cassette and an H1-driven gRNA cassette.

Techniques: Plasmid Preparation, Injection, shRNA, In Situ Hybridization, Immunofluorescence, Staining, Marker, Generated, Inhibition

Journal: Molecular Therapy. Nucleic Acids

Article Title: Subnuclear organization and mislocalization of plasmids reduce transgene expression

doi: 10.1016/j.omtn.2025.102730

Figure Lengend Snippet: Pol II promoter activity is reduced when a Pol III promoter is present on the same plasmid The nuclei of A549 cells were injected with either a dual Pol II-Pol III promoter plasmid or the corresponding Pol II plasmid only. Both plasmids were injected on the same coverslip with the same copy number and injection pressure over 15 min to minimize variation. Cells were imaged hourly for GFP expression, and the number of expressing cells was calculated as a percentage of those injected (mean ± SD). Fluorescent protein expression in each expressing cell was determined fluorescence intensity using the ROI function in ImageJ for all injected cells (mean ± SEM). All images used an exposure of 500 ms. The injections were repeated three times, and each time at least 40 cells were injected for each condition. Statistical analyses were by two-way ANOVA followed by Tukey’s multiple comparisons tests. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. (A) pU6-shRNA-CMV-GFP-SV40 or pCMV-GFP-SV40. (B) pgRNA-CMV-mCherry-Tandem or pCMV-mCherry-Tandem. (C) pACE-tRNA-EF1a-RFP-Tandem or pEF1a-RFP-Tandem. In an additional experiment, a mixture of equal copy numbers of pEF1a-RFP-Tandem and pACE-tRNA plasmids were injected in trans and showed similar expression to that seen with pEF1a-RFP-Tandem alone. Bar, 50 μm.

Article Snippet: Plasmids ACE-Arg-tRNA, ACE-Trp-tRNA, and ACE-Gly-tRNA all have Pol III type 2 promoter-driven tRNA cassettes cloned into a pUC57 backbone. pU6-shRNA-CMV-GFP-SV40 (pGFP-V-RS-scramble) has a CMV-GFP cassette, a U6-scrambled shRNA cassette, and an SV40-neomycin cassette (OriGene, Rockville, MD). pU6-shRNA-CMV-RFP-SV40 (pRFP-C-RS-scramble) was purchased from OriGene, Rockville, MD. pH1v1 has a complete H1 promoter-driven gRNA cassette (Addgene). pCMV-mCherry-SV40-neo has two Pol II cassettes: one expressing mCherry from the CMV immediate-early promoter and enhancer and the other expressing neomycin resistance from the SV40 early promoter and enhancer (Takara Bio #632524, Kusatsu, Shiga, Japan). pH1-gRNA-CMV-mCherry-convergent has a CMV-mCherry cassette and an H1-driven gRNA cassette.

Techniques: Activity Assay, Plasmid Preparation, Injection, Expressing, Fluorescence, shRNA