Journal: Cell systems

Article Title: SEMPER: Stoichiometric expression of mRNA polycistrons by eukaryotic ribosomes for compact, ratio-tunable multi-gene expression.

doi: 10.1016/j.cels.2024.06.001

Figure Lengend Snippet: Figure 3. Utilizing 2-ORF SEMPER constructs to express gas vesicles in mammalian cells (A) Architecture of an mRNA transcript transcribed from transfected SEMPER mARG plasmid DNA. The first ORF, gvpA, encodes the main structural protein, whereas the second ORF, gvpNJKFGW-EmGFP, encodes all necessary accessory proteins strung together with P2A self-cleaving peptides. (B) Flow cytometry distributions of emerald GFP normalized by mCherry values for each TIS combination tested in addition to an IRES-mCherry control. The thick horizontal lines depict the means of the distributions. Individual cells are represented as individual points. A minimum of 4,000 cells are depicted per condition. Error bars indicate mean ± SEM. p value < 0.0001 (Kruskal-Wallis test). p values of multiple comparisons are found in Table S6. (C) BURST images of acoustic contrast due to gas vesicle expression within HEK293T cells. Depicted are HEK293T cells loaded into agarose phantoms 3 days after transfection of SEMPER mARG plasmids or the leading two-plasmid system. The color bar represents the magnitude of BURST signal measured in linear arbitrary units. The floor and ceiling of the images are set to 0 and 5,000, respectively (n = 1, representative of four biological replicates). (D) BURST signal quantification of gas vesicle acoustic contrast for HEK293T samples transfected with SEMPER mARG plasmids or the leading two-plasmid system. Error bars indicate mean ± SEM (n = 4 biological replicates). Statistical analysis was conducted using a one-way ANOVA (p < 0.0001) followed by Fisher’s least significant difference (LSD) post-hoc test with a single pooled variance to compare each treatment group with every other. Pairwise p values are reported in Table S7. (E) Annexin V staining assays to quantify the number of apoptotic cells following expression of mARG vectors. HEK293T cells transfected with pgvpA-IRES- mCherry with the start codon removed in front of gvpA were used to establish a baseline (not treated). A subset of these cells was treated with raptinal to induce apoptosis (raptinal treated). Other cell populations were transfected solely with a fully functional pgvpA-IRES-mCherry (gvpA-IRES-mCherry), the two-vector system (two-vector), or the ACC/ACC SEMPER mARG plasmid (SEMPER mARG). Error bars indicate mean ± SEM (n = 4 biological replicates). Statistical analysis was conducted using a one-way ANOVA (p < 0.0001) followed by Fisher’s LSD post-hoc test with a single pooled variance to compare each treatment group with the not treated control. Pairwise p values are indicated above the bars.

Article Snippet: A DNA-polyethylenimine (PEI) Max transfection mixture was prepared by dissolving 206 mg of PEI-MAX (40 kDa, Polysciences, #24765-2) in 500 mL of tissue culture (TC)-grade phosphate-buffered saline (PBS) to achieve a final concentration of 0.412 mg/mL (10.3 mM).

Techniques: Construct, Transfection, Plasmid Preparation, Flow Cytometry, Control, Expressing, Staining, Functional Assay