Chloroquine gene transfection

Discussion in 'Chloroquine 150 Mg' started by Aloff, 24-Feb-2020.

  1. Black_mooN User

    Chloroquine gene transfection


    Detailed information on how Wiley uses cookies can be found in our Privacy Policy. This protocol describes a general method for transfecting mammalian cells using linear polyethylenimine.

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    Pro-Tip* There can be batch to batch variation when making the PEI working stock, therefore the ratio of μg DNAμg PEI needs to be empirically determined. Once a batch of PEI is prepared, transfect cells with a fluorescent plasmid using a variety of ratios. Check the cells 1-2 days after transfection to determine what ratio gives the highest percentage of GFP positive cells. Treatments may modify cell membrane structure to enhance uptake of DNA. Chloroquine, which is generally applied to cells with the DNA, appears to enhance transfection efficiency by inhibiting DNA degradation by lysosomes 15. Sodium butyrate treatment following transfection has been reported to result in higher levels of transient expression 16. A pH-responsive peptide fragment modelled on the influenza virus haemagglutinin INF7-SGSC can promote the transfectional activity of polyL-lysine pLL/DNA complexes against 293 cells.

    We specifically use this protocol with Lenti-X 293T cells, a cell line optimized for production of lentiviral vectors. 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.

    Chloroquine gene transfection

    Chloroquine use in transfection. - Tissue and Cell Culture, ProFection Mammalian Transfection System - Promega

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  6. Determine DNA concentration by performing A260/A280 readings on a spectrophotometer or by using the Quant-iT™ DNA Assays Kits Q33130, Q33120. Plasmid DNA or siRNA used in transfection has degraded or is of poor quality Ensure that the plasmid DNA or siRNA used for transfection is of high quality.

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    Chloroquine, which buffers lysosomes, enhanced the transfection efficiency of N‐quaternized PEIs and polylysine by 2–3‐fold. In contrast, chloroquine did not improve the transfection efficiency of PEI. The measured average pH environment of PEI vectors was 6.1, indicating that they successfully avoid trafficking to acidic lysosomes. Introduction. This protocol describes a general method for transfecting mammalian cells using linear polyethylenimine. 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. Chloroquine has been frequently used in cell culture gene transfection studies Cotton et al. 1992, Wagner et al. 1992, Lucas et al. 1995, but has not been reported yet to be successful for enabling gene transfection in vivo. As mentioned above, the mechanisms of action of chloroquine seems to be related to elevating the intravesicular pH of lysosomes and endosomes, thereby providing a suboptimal pH environment for the various lysosomal enzymes including those involved in DNA degradation.

     
  7. shamuey New Member

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  9. scanlab.name New Member

    EFFECT OF CHLOROQUINE ON TRANSDUCTION OF CELL LINES AND. In this study, the effect of chloroquine on transduction and associated toxicity of baboon CD34 cells and a variety of cell lines was studied. Materials and Methods. Cell lines. To study the effects of chloroquine on retroviral transduction of cell lines, three cell lines were chosen Hela cells, D17 and 208 F cells.

    Retrovirus Production and Infection