Chloroquine lentivirus production

Discussion in 'Health Canada Drug Database' started by nikro00, 19-Mar-2020.

  1. Lellopito XenForo Moderator

    Chloroquine lentivirus production


    Transfections allow for transient expression of a gene of interest in a target cell line and can be useful for short term studies of protein function. We specifically use this protocol with Lenti-X 293T cells, a cell line optimized for production of lentiviral vectors.

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    Required for boosting lentiviral production e.g. chloroquine phosphate, sodium butyrate, caffeine. • Faster — time to virus collection is 24 hours less than other reagents, and reverse transfection time is almost 2 days less than other reagents with comparable viral titers. • Easier — eliminates the need for coating of plates or fl asks. Figure 1. Lentiviral production using Lipofectamine 3000 reagent. A lentiviral construct containing the gene of interest along with lentiviral packaging mix is cotransfected into 293T or 293FT cells using Lipofectamine 3000 reagent. Lentivirus production and transduction. Scrambled shRNA #RHS6848 and specific Nurr1 shRNA cloned into pLKO.1 lentiviral vector were purchased from GE Dharmacon. Knockdown of Nurr1 expression levels was verified by quantitative real-time PCR.

    Last Upload: June 10, 2016 Day 0: Seed Lenti-X 293T cells (this cell line is optimized for production of lentiviral vectors) Day 1 (pm): Transfect Cells Day 2 (am): 18h post transfection - Remove media, replace with fresh media Day 3 or more (am): Observe fluorescence, harvest cells, or perform your experiment *Pro-Tips* Different brands and lots of FBS can promote or inhibit transfection. This approach can be adapted for different cell lines and different transfection reagents.

    Chloroquine lentivirus production

    Production of lentiviral vectors, Improve Lentiviral Production Using Lipofectamine 3000.

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  5. Leave Chloroquine on the cells for more than 24 hours due to cytotoxicity. 4Pipette up and down and carefully add don’t add too quickly or you’ll wash the cells off the plate! the 1mL of precipitated DNA to cells and incubate overnight. in 3% CO. 2. incubator 10-12 up to 24 hours from adding chloroquine. 24 Hours has been done and is fine.

    • Lentivirus Protocol.
    • Chloroquine modulates inflammatory autoimmune responses..
    • Chloroquine use in transfection. - Tissue and Cell Culture.

    Background. During the past twelve years, lentiviral LV vectors have emerged as valuable tools for transgene delivery because of their ability to transduce nondividing cells and their capacity to sustain long-term transgene expression in target cells in vitro and in vivo. However, despite significant progress, the production and concentration of high-titer, high-quality LV vector stocks is. If kept for extended periods of time, chloroquine is toxic. The solution in the plates looks fuzzy, due to a very fine, dust-like precipitation of the transfection mixture. Note It is important to do all media changes with extreme gentleness, as the cells have been sensitized by the chloroquine, and can easily detach from the plate. We specifically use this protocol with Lenti-X 293T cells, a cell line optimized for production of lentiviral vectors. This approach can be adapted for different cell lines and different transfection reagents. Last Upload June 10, 2016

     
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