Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Packaging cells for lentiviral vectors generated using the cumate and coumermycin gene induction systems and nanowell single-cell cloning

doi: 10.1016/j.omtm.2023.02.013

Figure Lengend Snippet: Diagram of constructs used in this study and mechanism of gene regulation by the cumate/coumermycin gene induction system (A) Constructs used to make the 293SF-CymR/λR-GyrB cells. (i) The coding sequence for the repressor of the cumate gene switch (CymR) is controlled by a strong constitutive promoter (CMV5). (ii) The coding sequence for the coumermycin transactivator (λR-GyrB) is controlled by the cumate-inducible CMV5CuO promoter. (B) Transfer vectors used for the production of LV. (i) Conditional SIN LV expressing GFP regulated by the coumermycin-inducible promoter (12xlambda-TPL). (ii) SIN LV expressing GFP regulated by the CMV promoter. (C) Genes used to construct the packaging cell line. (i) The Rev gene is under the regulation of the coumermycin-inducible promoter (13xlambda-TPL). (ii) The Gag/ P ol gene is regulated by the constitutive hybrid CMV enhancer/β-actin promoter (CAG). (iii) The VSV-G gene is regulated the 13xlambda-TPL promoter. (D) Mechanism of gene regulation by the cumate/coumermycin induction system. The 293SF-CymR/λR-GyrB cell line constitutively expresses CymR and it contains the gene for λR-GyrB under the control of CMV5CuO promoter. In the absence of cumate, CymR binds to CMV5CuO and prevents transcription. Addition of cumate releases CymR from the promoter and the gene for λR-GyrB can be transcribed. In the presence of coumermycin, λR-GyrB forms a dimer that binds to several copies of the lambda operators (λOp; in this example, 12 copies are indicated) to activate transcription of the transgene of interest. 5′ LTR and 3′ LTR, long terminal repeats located at the 5′ and 3′ ends of the lentivirus respectively; R, R region of the LTR; U5, U5 region of the LTR; Tet, tetracycline promoter; ψ, encapsidation signal; RRE, Rev responsive element; cPPT, central polypurine track; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; SD and SA, splice donor and acceptor respectively; pA, polyadenylation signal; 12xλTPL, 12xlambda-TPL promoter.

Article Snippet: The transfer vector used to construct an LV expressing GFP regulated by the coumermycin-regulated promoter (pTet07-CSII-12xlambda-TPL-GFPq) was constructed by first ordering the sequence of the coumermycin-regulated promoter (12xlambda fused to the CMV-CuO minimal promoter and adenovirus tripartite leader) from a gene synthesis company (GenScript, Piscataway, NJ).

Techniques: Construct, Sequencing, Expressing, Control, Virus

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Packaging cells for lentiviral vectors generated using the cumate and coumermycin gene induction systems and nanowell single-cell cloning

doi: 10.1016/j.omtm.2023.02.013

Figure Lengend Snippet: Induction level of the coumermycin/cumate gene expression system Suspension culture of clones of 293SF-CymR/λR-GyrB (7-2, 7-3, 7-10, 8-59,15-35, and 15-51) were transduced at an MOI of 5 TU with an LV producing GFP regulated by the coumermycin-inducible promoter (LV-12xlambda-TPL-GFP). The next day, the cells were induced by the addition of cumate and coumermycin, and, at 3 days post-transduction (2 days post induction), the level of GFP expression was analyzed by flow cytometry. The relative fluorescent index of the cell population in the absence (off) and in the presence of inducers (on) is indicated. 293SF cells (without the gene induction system) were transduced in parallel. The induction level after induction (On/Off ratio of fluorescence index) is indicated above the bars for each clone. The data are the mean ± SD of two independent experiments.

Article Snippet: The transfer vector used to construct an LV expressing GFP regulated by the coumermycin-regulated promoter (pTet07-CSII-12xlambda-TPL-GFPq) was constructed by first ordering the sequence of the coumermycin-regulated promoter (12xlambda fused to the CMV-CuO minimal promoter and adenovirus tripartite leader) from a gene synthesis company (GenScript, Piscataway, NJ).

Techniques: Gene Expression, Suspension, Clone Assay, Transduction, Expressing, Flow Cytometry, Fluorescence