generation lentiviral plasmid backbone  (Lentigen Inc)

Journal: bioRxiv

Article Title: Complementation of a human disease phenotype in vitro by intercellular mRNA transfer

doi: 10.1101/2024.11.06.622258

Figure Lengend Snippet: A. Schematic of PEX6-Tomato. The Tomato ORF was inserted between the CDS and 3’UTR of human PEX6 and in-frame to PEX6 to produce a PEX6-Tomato fusion protein, as indicated. The construct was expressed under a constitutive ubiquitin promoter. Lengths of each segment are marked in base pairs. B. PEX6-Tomato can rescue a peroxisome mutant phenotype. PEX6mut cells were transiently co-transfected with plasmids expressing either PEX6-Tomato and a GFP-tagged peroxisomal marker, GFP-SKL, or a control construct expressing tdTomato alone along with GFP-SKL. Images were taken 72hrs post transfection. Scale bar: 10 µm. C. Over-expression level of PEX6-Tomato mRNA. Stable PEX6-Tomato HEK293T cells were created by lentiviral infection. PEX6 and PEX6-Tomato gene expression was quantified by RT-qPCR. 18S gene expression served as endogenous control. Data is shown as the mean of three replicates and the corresponding SEMs. ****: P<0.0001

Article Snippet: The entire construct was then transferred to the 3rd generation lentiviral vector pRRLSIN.cPPT.PGK-GFP.WPRE backbone (a gift from Didier Trono, Swiss Federal Institute of Technology Lausanne, Switzerland; Addgene # 12252).

Techniques: Construct, Mutagenesis, Transfection, Expressing, Marker, Control, Over Expression, Infection, Quantitative RT-PCR

Journal: Bioengineered

Article Title: GXMR-CAR containing distinct GXM-specific single-chain variable fragment (scFv) mediated the cell activation against Cryptococcus spp. And had difference in the strength of tonic signaling

doi: 10.1080/21655979.2023.2281059

Figure Lengend Snippet: 2H1-GXMR-CAR and 18B7-GXMR-CAR recognize soluble GXM from C. gattii and C. neoformans. (a) scheme of the GXMR-CAR constructs containing antigen binding domains sourced from clones 18B7 (blue) or 2H1 (red) of anti-GXM monoclonal antibody followed by CD8 molecule as hinge/transmembrane domain (black), CD137 as a costimulatory portion (purple), and CD3ζ (yellow) as signaling transduction domain. The enhanced green fluorescence protein (EGFP) is a marker separated by T2A peptide from GXMR-CAR expression. The constructs were called 18B7-GXMR-CAR and 2H1-GXMRCAR. (b) steps of the protocol of co-transfection of GXMR-CAR plasmids and helper plasmids in HEK-293T cells to produce lentiviral particles carrying the DNA sequence of 2H1-GXMR-CAR or 18B7-GXMR-CAR and the lentiviral particles were obtained from the cell supernatant after 24, 48, and 72h post-transduction. The lentiviral particles were concentrated, the titer was calculated as described in the M&M, and the Jurkat cell line was modified by transduction to express GXMR-CAR constructs. (c) Jurkat cells (1×10 cells/well) distributed in a 48-well plate were incubated with lentiviral particles and performed the spinoculation method for transduction, and after 72h the percentage of GFP+ cells were determined by flow cytometry to calculate the lentiviral particles titer for each GXMR-CAR construct. (d) binding assay between Jurkat cells expressing 18B7-GXMR-CAR or 2H1-GXMR-CAR and soluble GXM (10µg/mL) obtained from C. gattii or C. neoformans. After incubation between soluble GXM and GXMR-CAR Jurkat cells was added an anti-GXM murine monoclonal antibody (clone 18B7) followed by the addition of goat anti-mouse IgG secondary antibody biotin conjugated. The presence of GXM on the cell surface was revelead by incubation with streptavidin-PE, and the mean fluorescence intensity (MFI) was measured by flow cytometry. As a negative control, Jurkat cells modified with lentiviral particles carrying GPF alone (pLenti-MOCK) were considered in all steps. Jurkat cells expressing GXMR-CAR or GFP alone (pLenti-Mock) were gated, and the MFI of the interaction of GXM was represented in histograms.

Article Snippet: The GXMR-CAR constructs specific to the GXM of Cryptococcus spp. were synthesized using GenScript (GenScript, NJ, USA), and the sequence encoding GXMR-CAR was subcloned into a second-generation lentiviral plasmid backbone (pLentiCas9-EGFP, GenScript, NJ, USA) containing the GFP sequence as a reporter.

Techniques: Construct, Binding Assay, Clone Assay, Transduction, Fluorescence, Marker, Expressing, Cotransfection, Sequencing, Modification, Incubation, Flow Cytometry, Negative Control