Journal: Microbial Cell Factories

Article Title: Generation of diploid Pichia pastoris strains by mating and their application for recombinant protein production

doi: 10.1186/1475-2859-11-91

Figure Lengend Snippet: Vectors for anti-HER2 light-chain and heavy chain expression. ( a ) Plasmid pGLY10969 encodes the light chain of anti-HER2 antibody and mCherry red fluorescent protein in association with the arsenic resistance selection marker and targets the URA6 locus in P. pastoris genome ( b ) Plasmid pGLY10970 encodes the heavy chain of anti-HER2 antibody and yEGFP3 green fluorescent protein in association with nourseothricin resistance selection marker and targets the URA6 locus in P. pastoris genome. ( c ) Plasmid pGLY6830 encodes both light-chain and heavy-chain of anti-HER2 expression cassettes in association with zeocin resistance selection marker and targets to the TRP2 locus in P. pastoris genome. The Spe I restriction enzyme site used to linearize each vector prior to yeast transformation is underlined.

Article Snippet: Green fluorescent protein yEGFP3 [ ] and red fluorescent protein mCherry [ ] were codon optimized and synthesized from Genscript (Piscataway, NJ).

Techniques: Expressing, Plasmid Preparation, Selection, Marker, Transformation Assay

Journal: Microbial Cell Factories

Article Title: Generation of diploid Pichia pastoris strains by mating and their application for recombinant protein production

doi: 10.1186/1475-2859-11-91

Figure Lengend Snippet: Yeast strains used in this study

Article Snippet: Green fluorescent protein yEGFP3 [ ] and red fluorescent protein mCherry [ ] were codon optimized and synthesized from Genscript (Piscataway, NJ).

Techniques: Plasmid Preparation, Glycoproteomics

Journal: Frontiers in Immunology

Article Title: Endocytosis Deficient Murine Xcl1-Fusion Vaccine Enhances Protective Antibody Responses in Mice

doi: 10.3389/fimmu.2019.01086

Figure Lengend Snippet: Xcl1(Δ1) specifically binds cDC1s, but is not actively internalized. (A) BM-derived Flt3L-induced cDC1s (left) and cDC2s (right) from BALB/c mice were analyzed by flow cytometry for binding to Xcl1-mCherry and Xcl1(Δ1)-mCherry vaccibodies with anti-NIP-mCherry as a negative control. mCherry vaccibodies were detected with biotinlyated anti-mCherry mAb and streptavidin-APC-Cy7 to boost the signal. (B) cDC1s from WT or Xcr1 KO mouse splenocytes were gated as Lin − MHCII + CD11c + and CD24 + (cDC1s) or CD11b + (cDC2s) as shown in , and evaluated for binding to mCherry vaccibodies. (C) Internalization of purified mCherry vaccibodies by Flt3L DCs was determined as described in . (D) Internalization of the Xcr1 receptor on Flt3L DCs after incubation with Xcl1- or Xcl1(Δ1)-mCherry vaccibodies for 0, 15, or 30 min at 37°C as determined by ImageStream. The plotted values are relative to background internalization after incubation with anti-NIP-mCherry vaccibodies. (E) Migration of Flt3L DCs in transwell plates after incubation with mCherry vaccibody proteins. Ratio of cDC1 and cDC2 was determined by flow cytometry. (F) In vivo binding of mCherry vaccibody proteins as determined by flow cytometry. Spleens were harvested 1 h after i.v. injection of 25 μg of protein, and splenic DCs were defined as in (B) . (C) Data are pooled from two separate experiments, n = 4, values shown are mean. (D–F) Data shown are mean ± SEM. (D) Data are pooled from three separate experiments, n = 3. (E,F) n = 3. (D–F) Unpaired t- test (two-tailed), * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: As a non-targeted control, a fluorescent mCherry vaccibody construct containing a single chain variable fragment specific for the hapten NIP was included (referred to as anti-NIP-mCherry) , and the previously published human XCL1 (hXCL1) mCherry vaccibody construct was used as a positive control ( ).

Techniques: Derivative Assay, Flow Cytometry, Binding Assay, Negative Control, Purification, Incubation, Migration, In Vivo, Injection, Two Tailed Test

Journal: Molecular Therapy. Nucleic Acids

Article Title: mRNA Encoding a Bispecific Single Domain Antibody Construct Protects against Influenza A Virus Infection in Mice

doi: 10.1016/j.omtn.2020.04.015

Figure Lengend Snippet: Delivery of DOTAP/Cholesterol mRNA Lipid Nanoparticles via Intratracheal Instillation (A) Particle size analysis after incubating DOTAP/cholesterol mRNA lipoplexes in HEPES buffer or bronchoalveolar lavage fluid (BALF). BALB/c mice were i.t. administered with DOTAP/cholesterol particles formulated with different mRNA sequences (mRNA dose of 5 μg). (B) Graph and representative whole-body images showing expression levels of luciferase mRNA (5-methoxyuridine) in lungs of BALB/c mice measured via bioluminescence imaging at 6 h (n = 3 mice). DOTAP/cholesterol nanoparticles containing 1 mol% of the lipophilic dye DiR and packaged with mCherry mRNA (5-methoxyuridine) were i.t. administered, after which nanoparticle uptake (DiR fluorescence) and mRNA expression (mCherry protein) were evaluated in a variety of pulmonary cells subsets (n = 5 mice) at 6 and 24 h post administration. The flow cytometry gating strategy used to discriminate between pulmonary immune cell subsets can be found in the Figure S1 and was adopted from Knight et al. PBS-instilled mice serve as negative controls. (C) Graph shows nanoparticle uptake in CD45 positive cells (immune cells) and CD45 negative cells (nonimmune cells). (D) Nanoparticle uptake in a variety of pulmonary immune cells subsets, including alveolar macrophages, CD103 + dendritic cells (CD103 + DCs), granulocytes, other subsets of monocytes and macrophages that are not encompassed by the other subsets (Mono/macrophage), interstitial macrophages, and CD11b + DCs. (E) Representative flow cytometry plots of nanoparticle uptake and mCherry mRNA expression in alveolar macrophages. The mean percentage of DiR-positive and mCherry-positive cells, together with the mean fluorescence intensity of each signal, are given in each flow plot. (F) 5 μg of FcγRIV VHH-M2e VHH or FcγRIV VHH-RSVF VHH (irrelevant mRNA) formulated in DOTAP/cholesterol particles or 50 μg FcγRIV VHH-M2e VHH protein was instilled i.t. in BALB/c mice. 6, 24, or 48 h after instillation, BALF was isolated and cells were removed from the BALF and the ability of His 6 -tagged proteins to bind to M2e was investigated in a peptide ELISA (see Figure S2 ). De absolute titers were calculated using the standard curve shown in Figure S2 . Graphs show mean ± SEM (n = 3 mice per group). Statistical analyses on datasets were performed by one-way ANOVA followed by Tukey’s post hoc test. Asterisks indicate statistical significance compared to negative control (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001).

Article Snippet: The mRNA constructs encoding for firefly luciferase or the fluorescent mCherry protein were purchased from TriLink (San Diego, CA, USA).

Techniques: Particle Size Analysis, Expressing, Luciferase, Imaging, Fluorescence, Flow Cytometry, Isolation, Peptide ELISA, Negative Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: mRNA Encoding a Bispecific Single Domain Antibody Construct Protects against Influenza A Virus Infection in Mice

doi: 10.1016/j.omtn.2020.04.015

Figure Lengend Snippet: Inflammatory Response to Intratracheally Administered mRNA Nanoparticles (A) Graph depicts the relative distribution of each pulmonary immune cell subset as a percent of total viable cells, for PBS-treated mice, and mice that were i.t. instilled with DiR-labeled mRNA nanoparticles containing mCherry mRNA (mRNA dose of 5 μg), that were either sacrificed at 6 or 24 h post administration (n = 5 mice). (B) Serum samples collected from these mice were screened for the presence of inflammatory cytokine responses. Statistical analyses on datasets were performed by one-way ANOVA followed by Tukey’s post hoc test. Asterisks indicate statistical significance compared to negative control (∗p < 0.05; ∗∗∗p < 0.001).

Article Snippet: The mRNA constructs encoding for firefly luciferase or the fluorescent mCherry protein were purchased from TriLink (San Diego, CA, USA).

Techniques: Labeling, Negative Control