synthetic expression cassette  (Twist Bioscience)

Journal: medRxiv

Article Title: Immune profile and responses of a novel Dengue DNA vaccine encoding EDIII-NS1 consensus design based on Indo-African sequences

doi: 10.1101/2021.09.21.21263883

Figure Lengend Snippet: A) Distribution of DENV serotypes 1-4 from the sites from which samples were received for this study. Serotype distribution for each site is shown as pie charts over time. Total numbers from the site are indicated, each serotype is represented by a different colour. B-E) Genotype assignment based on maximum likelihood phylogenetic trees. Phylogenetic trees DENV1-4 using the maximum likelihood method are shown. All available (dated) complete coding nucleotide sequences (DENV1 n = 1800, DENV2 n = 1395, DENV3 n = 823, DENV4 n = 220) from human host, were used for tree construction. Sylvatic strains EF457905 (for DENV1), EF105379 (for DENV2), KT424097 (for DENV3) and JF262779-80 (for DENV4) were used as outgroups to root the tree. Branches have been collapsed to aid visualization. Greyscale colours show different genotypes. Region, where Indian sequences are present, are colored in blue. The detailed tree structure is shown only for the neighbouring sequences. Amino acid mutations with respect to the recent ancestor are shown near important nodes (marked in blue circles).

Article Snippet: Finally, the synthetic DENV DNA expression cassette was inserted into the pVAX1 expression vector (Invitrogen, CA) between NheI and HindIII under the control of the cytomegalovirus immediate early promoter (Genscript Biotech, USA).

Techniques:

Journal: medRxiv

Article Title: Immune profile and responses of a novel Dengue DNA vaccine encoding EDIII-NS1 consensus design based on Indo-African sequences

doi: 10.1101/2021.09.21.21263883

Figure Lengend Snippet: A) Frequency based representation of all the variable sites, for each serotype within our study sequences. Colours assigned based on the frequency of sequences bearing a mutation at that particular site. B, D, F and H) Frequencies of variants within DENV1, DENV2, DENV3, DENV4 strains of our study sequences, respectively. Amino acid variants residue identified relative to (NC_001477(DENV1), NC_001474 (DENV2), NC_001475 (DENV3), NC_002640 (DENV4). C, E, G and I ) Amino acid variants also spotted on EDIII PDB structure (ribbon). Stick and ball representation on PDB structure indicates variants at the particular position. PDB: 4gt0.1, 4ut6.2.8,4Gsx.1 and 5BIC.1 used to annotate the genetic variants of DENV.

Article Snippet: Finally, the synthetic DENV DNA expression cassette was inserted into the pVAX1 expression vector (Invitrogen, CA) between NheI and HindIII under the control of the cytomegalovirus immediate early promoter (Genscript Biotech, USA).

Techniques: Mutagenesis, Residue

Journal: medRxiv

Article Title: Immune profile and responses of a novel Dengue DNA vaccine encoding EDIII-NS1 consensus design based on Indo-African sequences

doi: 10.1101/2021.09.21.21263883

Figure Lengend Snippet: A, D, G & J) The genetic diversity within EDIII of DENV1, DENV2, DENV3 and DENV4 Indian strain variants spotted on EDIII PDB structure (ribbon). Stick and ball representation on PDB structure indicates variants at the particular position. PDB: 4gt0.1, 4ut6.2.8,4Gsx.1. and 5BIC.1 used to annotate the genetic variants of DENV. B, E, H & K) Frequencies of variants within Indian DENV1, DENV2, DENV3, DENV4 strains, respectively. Amino acid variants residue identified relative to (NC_001477(DENV1), NC_001474 (DENV2), NC_001475 (DENV3), NC_002640 (DENV4). C, F, I and L) Relative frequencies of multiple amino acid substitution at a given position in DENV1, DENV2, DENV3 and DENV4 represented in different colors, respectively,

Article Snippet: Finally, the synthetic DENV DNA expression cassette was inserted into the pVAX1 expression vector (Invitrogen, CA) between NheI and HindIII under the control of the cytomegalovirus immediate early promoter (Genscript Biotech, USA).

Techniques: Residue

Journal: medRxiv

Article Title: Immune profile and responses of a novel Dengue DNA vaccine encoding EDIII-NS1 consensus design based on Indo-African sequences

doi: 10.1101/2021.09.21.21263883

Figure Lengend Snippet: A & B) Schematic representation of DENV DNA vaccine and cloning strategy. Synthetic DENV expression cassette was inserted into the pVAX1 expression vector in between NheI and HindIII under the control of the cytomegalovirus immediate early promoter. C) RT-PCR of RNA extracts from HEK293T cells transfected with DNA DENV vaccine. GAPDH used as an internal expression normalization gene. D-F) Analysis of in vitro expression of ED III and NS1 protein after transfection of 293T cells with DDV or plasmid control by Western blot. 293T cells supernatant and lysates resolved on a gel and probed with anti-DDV immune sera. Blots were stripped then probed with β-actin loading control. G) Immunofluorescence staining of 293T cells transfected with 5ug/well of DDV or plasmid control. Expression of antigen was measured using anti-DDV immune sera. Cell nuclei were counterstained with DAPI.

Article Snippet: Finally, the synthetic DENV DNA expression cassette was inserted into the pVAX1 expression vector (Invitrogen, CA) between NheI and HindIII under the control of the cytomegalovirus immediate early promoter (Genscript Biotech, USA).

Techniques: Cloning, Expressing, Plasmid Preparation, Control, Reverse Transcription Polymerase Chain Reaction, Transfection, In Vitro, Western Blot, Immunofluorescence, Staining

Journal: medRxiv

Article Title: Immune profile and responses of a novel Dengue DNA vaccine encoding EDIII-NS1 consensus design based on Indo-African sequences

doi: 10.1101/2021.09.21.21263883

Figure Lengend Snippet: A) Schedule of vaccination and antibody assays. BALB/c and C57BL/6J mice were immunized by TA injection of 50ug DDV or plasmid vector at day 0,15 and 30. Sera were collected at each time points. B-E) Indirect ELISA reactivity showing O.D. measured at 450nm (in pooled sera) and Serum IgG binding end point titers (in individual animals) of BALB/c (B & C) and C57BL/6J strains (D & E) . Antibody titers were measured by ELISA plates coated with purified recombinant DENV DNA vaccine antigens. F-I) Sera neutralization titers against DENV 1-4 laboratory prototype strains and recent clinical isolates. Sera was collected at 14 days after second booster dose and analysed for neutralization of DENV by FNT assay. F & H) Representative normalized percentage of infection curves are shown from laboratory prototype strains and recent clinical isolates, respectively. FNT50 values were calculated for individual animals and presented in G & I from prototype strains and clinical isolates, respectively. Each point represents an individual animal, while horizontal lines indicate the mean, SD.

Article Snippet: Finally, the synthetic DENV DNA expression cassette was inserted into the pVAX1 expression vector (Invitrogen, CA) between NheI and HindIII under the control of the cytomegalovirus immediate early promoter (Genscript Biotech, USA).

Techniques: Injection, Plasmid Preparation, Indirect ELISA, Binding Assay, Enzyme-linked Immunosorbent Assay, Purification, Recombinant, Neutralization, Infection

Journal: medRxiv

Article Title: Immune profile and responses of a novel Dengue DNA vaccine encoding EDIII-NS1 consensus design based on Indo-African sequences

doi: 10.1101/2021.09.21.21263883

Figure Lengend Snippet: A) Schedule of vaccination and T cell assays. Mice (n□=□6/group) were immunized with 50 µg DENV DNA vaccine. T cell responses were analysed 2 weeks after second booster dose. B) Map of the DDV and predicted potential immunodominant peptides through NETCTL and VAXIJEN. C – F) Antigen specific T responses to pooled EDIII-NS1 peptides were measured by IFN-γ ELISpot assays after vaccination with DDV or plasmid control in BALB/c and C57BL/6J animals. Bars represent the mean□+□SD. PMA/IONOMYCIN was used as a non-specific positive control. G-J) Flow cytometric analysis of Intracellular cytokine staining for IFN-g in C57BL/6J mice splenocytes. G & H) Representative image of Intracellular cytokine staining in CD8+ T cells in DDV or plasmid control groups. I & J) Percentage of IFNγ+ CD8+ and CD4+ T cells in DDV and plasmid control vaccinated animals determined through ICC.

Article Snippet: Finally, the synthetic DENV DNA expression cassette was inserted into the pVAX1 expression vector (Invitrogen, CA) between NheI and HindIII under the control of the cytomegalovirus immediate early promoter (Genscript Biotech, USA).

Techniques: Enzyme-linked Immunospot, Plasmid Preparation, Control, Positive Control, Staining

Journal: medRxiv

Article Title: Immune profile and responses of a novel Dengue DNA vaccine encoding EDIII-NS1 consensus design based on Indo-African sequences

doi: 10.1101/2021.09.21.21263883

Figure Lengend Snippet: A) Th1/Th2 Assay schedule: BALB/c and C57BL/6J mice were immunized by TA injection of 50ug DDV or pVAX1 at day 0,15 and 30. two weeks after the 2 nd dose sera collected and assayed for IgG subclass antibodies. B & C) DENV specific IgG subclasses and the ratio of IgG2a/IgG1 in BALB/c mice immunized with DDV or plasmid control. D & E) DENV specific IgG subclasses and the ratio of IgG2c/IgG1 in C57BL/6J mice immunized with DDV or plasmid control.

Article Snippet: Finally, the synthetic DENV DNA expression cassette was inserted into the pVAX1 expression vector (Invitrogen, CA) between NheI and HindIII under the control of the cytomegalovirus immediate early promoter (Genscript Biotech, USA).

Techniques: Injection, Plasmid Preparation, Control

Journal: medRxiv

Article Title: Immune profile and responses of a novel Dengue DNA vaccine encoding EDIII-NS1 consensus design based on Indo-African sequences

doi: 10.1101/2021.09.21.21263883

Figure Lengend Snippet: A) Schematic representation of the experimental design. Groups of AG129 mice were administered (i.p) BALB/c immune sera in two dosage levels, 100 µl and 300 µl/mouse. Two hours after passive transfer the mice were challenged with a lethal dose of DENV 2 (105 FIU/mouse). All groups were monitored for body weight changes (B), clinical symptoms (C) and Survival (D).

Article Snippet: Finally, the synthetic DENV DNA expression cassette was inserted into the pVAX1 expression vector (Invitrogen, CA) between NheI and HindIII under the control of the cytomegalovirus immediate early promoter (Genscript Biotech, USA).

Techniques:

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Target-Cell-Directed Bioengineering Approaches for Gene Therapy of Hemophilia A

doi: 10.1016/j.omtm.2018.01.004

Figure Lengend Snippet: AAV Vector Delivery of Tissue Optimized Transgene Cassettes (A) Schematics of AAV2/8 transgene designs. Straight lines represent DNA sequence of no known function. (B) AAV2/8-HLP-An53-LCO and AAV2/8-HLP-An53-HCO were delivered intravenously to hemophilia A mice at a dose of 1 × 10 11 vg/kg (n = 4 per group). (C) AAV2/8 HCB-HSQ-LCO, AAV2/8-HCB-ET3-LCO, and AAV2/8-HLP-V3co were delivered intravenously to hemophilia A mice at a dose of 1 × 10 11 vg/kg (n = 3–4 per group). (D) A long-term, dose-finding experiment was performed with varying does of AAV2/8-HCB-MVM-ET3-LCO (n = 3 for all other doses). Data are presented as the mean ± sample SD.

Article Snippet: The synthetic AAV2 expression cassette was constructed by Genscript Biotech and inserted into the pUC57 backbone.

Techniques: Plasmid Preparation, Sequencing

Journal: Molecular Microbiology

Article Title: UAP56 is a conserved crucial component of a divergent mRNA export pathway in Toxoplasma gondii

doi: 10.1111/mmi.13485

Figure Lengend Snippet: Establishment of a conditional Cas9 (ddCas9). A. Western blot showing ddCas9‐FLAG overexpression. Parasites were induced for indicated times with 1µM of Shld1 prior protein extraction. RH‐Δhxgprt strain was used as control (first and second lanes ‐ wild type‐ wt) Aldolase: Loading control. B. i) Nuclear localization of Cas9 in RHddCas9 parasites after addition of 1 µM Shld1 for 24 h. Nuclear and apicoplast DNA staining with DAPI: in blue. Scale bar: 10µm. ii) Co‐localization map, showing in grey the areas where there was co‐localization with an M2 of 0.8. Graph represents the areas where there was a signal for green (green line) and areas where there was signal for blue (blue line). y ‐Axis: intensity level; x ‐axis: distance in microns. C. Plaque assays for parental RHddCas9 and RHddCas9‐ gap40 gRNA strains. Both parasite strains were grown on human foreskin fibroblasts, in the presence or absence of 1 µM of Shld1, as indicated, for 108 h. −, not induced; +, induced. Scale bar: 500 µm. D. Immunofluorescence assay of parental RHddCas9 and RHddCas9‐ gap40 sgRNA strains in the presence or absence of Shld1. Collapsing vacuoles with a significant reduction of GAP40 signal were only observed in induced parasites that expressed the specific sgRNA against gap40 . Scale bar: 8 µm. E. mRNA distribution was analyzed in RHddCas9, RHddCas9‐ gap40 gRNA and RHddCas9‐ uap56 gRNA strains after incubation for 4 h with 1 µM Shld1 and further incubation for 48 h in media. The analysis was performed by fluorescent in situ hybridization (FISH) using oligodT‐Alexa594 as probe, in red. Nuclear and apicoplast DNA was staining with DAPI: in blue. Scale bar: 5 µm. F. mRNA distribution and TgUAP56 protein presence analysis in RHddCas9‐ uap56 gRNA strain after incubation for 4 h with 1 µM Shld1 and further incubation for 48 h in media. i) The analysis was performed by immunofluorescence assay, α‐TgUAP56 in green, combined with fluorescent in situ hybridization (FISH) using oligodT‐Alexa594 as probe, in red. mRNA export blocking was observed in absence of TgUAP56 in induced parasites that expressed the specific sgRNA against uap56 . Nuclear and apicoplast DNA was staining with DAPI: in blue. Scale bar: 5µm. ii) Western blot to analyse TgUAP56 protein levels after 1 µM Shld1 incubation for 24 and 48 h. The protein was detected with anti‐TgUAP56. Aldolase was used as loading control. ddCas9‐FLAG was detected using antibody α‐Flag. −, not induced; +, induced.

Article Snippet: To generate conditional ddCas9 plasmid ( p5RT70DDmycFlagCas9 ) the synthetic cas9 expressing cassette from Lourido's lab (Addgene ID 52694 (Sidik et al ., )) was cloned into plasmid p5RT70ddmycGFPPfMyoAtailTy‐HX (Hettmann et al ., ) using NcoI‐NotI restriction sites.

Techniques: Western Blot, Over Expression, Protein Extraction, Control, Staining, Immunofluorescence, Incubation, In Situ Hybridization, Blocking Assay