Journal: Scientific Reports

Article Title: Virus Like Particles (VLP) as multivalent vaccine candidate against Chikungunya, Japanese Encephalitis, Yellow Fever and Zika Virus

doi: 10.1038/s41598-020-61103-1

Figure Lengend Snippet: Generation and characterization of bicistronic lentiviral vectors expressing flaviviral structural proteins. (A) JEV CprME was cloned into a lentiviral vector that included an IRES sequence followed by the Zika NS2B-3 protease. 293 T cells were transfected with the JEV CprME construct alone or the bicistronic JEV lentiviral construct. Culture supernatants were harvested and VLPs analyzed for JEV E protein and Capsid protein secretion via western blotting. (B) YFV CprME was cloned into a lentiviral vector as above and VLP secretion determined in the culture supernatants by western blotting for E and Capsid protein. (C) CHIKV C-E3-E2-E1 genes were cloned into a lentiviral vector and VLP secretion determined in the culture supernatants by western blotting for E1-E2 protein. Images were analyzed using GENETOOLS gel analysis Software version 4.03 (f). (Syngene, https://www.syngene.com/software/genetools-automatic-image-analysis/ ).

Article Snippet: A codon optimized Zika NS2B3 containing an HA Tag and IRES sequence was synthesized by GenScript Inc. using gene synthesis technology and has been described previously .

Techniques: Expressing, Clone Assay, Plasmid Preparation, Sequencing, Transfection, Construct, Western Blot, Software

Journal: BMC Genomics

Article Title: Insertion/deletion-activated frame-shift fluorescence protein is a sensitive reporter for genomic DNA editing

doi: 10.1186/s12864-019-5963-z

Figure Lengend Snippet: The frame-shift CFP (FsCFP) reporter successfully distinguished genome editing events of different targeting efficiencies, with very low noise signals. See also Additional file : Figure S2 and S3 for the establishment of the detection gatings, and S4 for the intensity of background CFP signal. a Genome editing was performed on a target sequence derived from the RRS gene of Chinese hamster. The corresponding gRNA is not expected to react with the essential RRS gene in the HEK293T cells, which is the host cell for the editing. The sequence of the wildtype gRNA and the mutant gRNA are presented on top, where the two mutated nucleotides in the center of the mutRRS-gRNA are indicated by red color. On the bottom, two representative flow charts show the HEK293T cells transduced with the FsCFP with the RRS-targeting sequence and the wildtype or mutatnt RRSgRNA-Cas9. Among the cells expressing both the mCherryFP and GFP, which are the markers for the FsCFP and the gRNA-Cas9, repectively, a subset of them also exhibited detectable CFP signals. b Similar to A, except that a target sequence derived from human VEGF and the correponding gRNA are used. c Representative flow charts of cells with VEGF-FsCFP and either a non-targeting sequence gRNA (ns-gRNA) and Cas9, or just GFP alone without gRNA and Cas9. In both the cases, the ratio of CFP-positive cells was close to or lower than 0.01%. d Quantification of CFP-positive events detected in mCherryFP and GFP double positive cells with different gRNAs as shown in a , b and c . The mean value was calculated from independent repeats ( n = 4 for RRS and mutRRS; n = 3 for the others). The error of means were shown after the +/− sign

Article Snippet: To construct a backbone sequence for the FsCFP-mCherryFP reporter, a nucleotide sequence containing CFP, IRES, and mCherryFP was synthesized (Genscript, NJ).

Techniques: Sequencing, Derivative Assay, Mutagenesis, Transduction, Expressing

Journal: BMC Genomics

Article Title: Insertion/deletion-activated frame-shift fluorescence protein is a sensitive reporter for genomic DNA editing

doi: 10.1186/s12864-019-5963-z

Figure Lengend Snippet: The application of the frame-shift CFP (FsCFP) reporter in two other cell lines also can effectively detect genome editing events with good quantitative quality and low background signals. See Additional file : Figure S4 and S5 for the background CFP signal of these cells. a The host cell CHO was stably induced with a FsCFP reporter containing target sequence derived from hVEGF. To initiate genome editing, vectors containing Cas9 and either the corresponding gRNA, a mutant gRNA, or a non-specific (ns) gRNA were used, with GFP as a traceable marker, all similar as in Fig. b. On the bottom right corner is the quantification of CFP-positive events detected in mCherryFP and GFP double-positive populations. The mean value were calculated from independent repeats ( n = 3). The error of means were shown. b similar to A, except that MEF were used as host cells, and that n = 5 for the cells with nsgRNA, n = 3 for other cells

Article Snippet: To construct a backbone sequence for the FsCFP-mCherryFP reporter, a nucleotide sequence containing CFP, IRES, and mCherryFP was synthesized (Genscript, NJ).

Techniques: Stable Transfection, Sequencing, Derivative Assay, Mutagenesis, Marker