Generation of foamy virus vectors and transduction of hematopoietic stem cells
Generation of foamy virus vectors and transduction of hematopoietic stem cells
Foamy virus (F V ), or spumavirus, is a nonpathogenic retrovirus developed as an integrating viral vector. Several potential advantages of F V vectors include a broad host range, robust packaging capabilities, and stable pre-integration complexes containing double-stranded D N A. The F V vectors have been shown to be effective in transducing stem cells, including various hematopoietic stem cells. F V is particularly efficient for the transduction of stem cells, including various hematopoietic stem cells Author: T. Friedman et al.
Operation method
F V Carrier generation and hematopoietic stem cell transduction Move F V Carrier generation and hematopoietic stem cell transduction Materials reagents Use of double-distilled water and sterilization techniques. Bovine Serum Albumin (B S A ) Fragment V CaCl2 (2.0m ol/L) Dissolve 29.4 g CaCl2 in IOOml H2O. Filter to remove bacteria. Store at 4-C. Cells 293 cells many different subclones (Graham e ta l.1977) are suitable. Select a known subclone with high transfection efficiency. Freeze multiple tubes to ensure reproducibility. Do not allow cells to grow through before transfection. A frozen tube can be resuscitated and maintained in growth medium for 4 to 6 weeks for transfection. After this time, resuscitate a new tube. Chloroquine (100 mmol/L) Dissolve 0.52 g of chloroquine phosphate in IOml of water. Filter and store at 4°C. Cytokines (Xf target cell specific) Stem Cell Factor (SCF) Flt3-Ligand (FL) Thrombopoietin (T P O ) DlO DlO is DMEM with 10% heat-inactivated FBS, 100 units/m l penicillin G and IOOfigM l streptomycin. Dimethyl sulfoxide (DMS 〇, 5% ) DM EM medium Fetal Bovine Serum (FBS), heat-inactivated (56°C, 30 m i n ) Hematopoietic cells Human CD34+ cells from spinal cord, peripheral blood, or umbilical cord blood can be isolated using commercially available reagents and methods (MiltenyiBiotec, Auburn, California) , using magnetic beads. Murine hematopoietic cells could be obtained from bone marrow and Miltenyi system-deficient lines. Enrichment of stem cells is facilitated by pre-treatment of mice with 5-fluorouracil (5FU). This optimized procedure is suitable for the transduction of hematopoietic regenerative cells in non-tissue culture 6-well plates. If a different plate or dish area is used, adjust the cell density and reagent volume accordingly. 2 X HEPES Salt Dissolve 8.18 g NaCl and 5.96 g HEPES in 400 ml of water. adjust pH to 7.10 with 0.5 mol/L NaOH, adjust final volume to 500 ml with water, and filter. The final solution was 280 mmol/L NaCl and 50 mmol/L HEPES. Store at 4°C. Human fibronectin fragment C H-296 (RetroNectin, Takara Shuzo, Otsu, Japan). Thaw freeze-dried RetroNectin to a concentration of lmg/m l in sterile water. Filtrate with 0.22-called 1-filter membrane. Store at 20°C. Phosphate buffer (P B S ), calcium and magnesium ion free Phosphoric acid mixture Plasmid DNA Aφ plasmid vector (containing the target gene) Helper plasmids: p C i G S A ^ , p C i P S , p C i E S Plasmid DNA can be purified by centrifugation using the QIAGEN kit or CsCl gradient, extracted with phenol and chloroform, ethanol precipitated and resuspended in TE (pH 8.0). Plasmids can be heat treated at 68°C for 30m in to destroy bacteria and prevent contamination. Details of the four plasmid F V vector generation systems are detailed in Figure 1. Sodium butyrate (500 m m o l /L ) Dissolve 5.5 g of sodium butyrate in 100 ml of DME. Filter and store at 20°C. Transduction Medium Concentrated vector in DM EM containing 20 % FBS and containing the following cytokines (specific for target cells): Stem Cell Factor (SCF), Flt3-Ligand (FL), Thrombopoietin (TPO), each at a concentration of l0 ng/ml. TE: I X (formulation below) and 0.1 X solution 10 mmol/L Tris-HCl (pH 8. 0) lmmol/L EDTA Instrument Centrifuge (B e c k m a n S W 28 turntable and tubes) Millipore Filters (0.45fzm and 0.22fxm; 0.45p m Stericup/Steritop Filters, DuraporeP V D F , S C H V U 02R E ) Plates (6-well plates) SlideA-Lyzer reagent kit (10K m .w . cutoff; PierceBiotechnology, 66450) Tissue Culture Dish (IO cm ) 37°C water bath method F V Carrier generation 2. Precipitate the transfected cells with calcium phosphate-DNA. a. Prepare a 9. 2m l D N A solution by combining the following: The initial collection was combined. Microscopic observation ensured that all cells were collected. For more product details, please visit Aladdin Scientific website.
Mix 4.95 ml 1.0 mol/L N a H 2P04 (12 g of monovalent salt dissolved in IOOml of water), 10.05 ml l. 0 mol/L Na2 H P 04 (26.8 g of di-phosphate dissolved in 100m l of water) and 85m l of water. Filter and store at 4-C.
Centrifuge tubes (2 x 250m l or several 50m l tubes)
1. 24 h before transfection, inoculate 3. 25X106 293 cells with IOml of DLO in tissue culture dishes (IO cm) in a total of 23 plates, and incubate overnight.
1 .15 ml 2. 0mol/L CaCl2
Transduced cells can be grown in liquid medium for stem cell cloning reactions or animal transplantation experiments.