Lentiviral transduction of hematopoietic cells
Lentiviral transduction of hematopoietic cells
Most hematopoietic cells, including hematopoietic stem cells and terminally differentiated cells such as primary T cells and macrophages, are undifferentiated or self-renewed slowly.
self-renewal is slow. For these cells, most non-viral or retroviral transfection methods are not feasible. Lentiviral vectors are suitable for transfection of undifferentiated cells and for long-lasting maintenance of transgene expression. Lentiviral vectors with various genes have successfully transfected many hematopoietic cells. Lentiviral vectors can be used for many reproduction manipulations such as short hairpin R N A (sh R N A ) and long-lasting expression of functional genes. They will also have great potential in gene therapy.
Author: T. Friedman et al, Translated by W. Qin et al. This experiment is from "Gene Transfer".
Operation method
Transfection of hematopoietic cells with lentiviral vectors Move Transfection of hematopoietic cells with lentiviral vectors Materials for transfection of hematopoietic cells with lentiviral vectors reagents Anti-CD; M antibody-conjugated magnetic beads (if transducing early hematopoietic stem cells; Miltenyi Biotec In c .) Bovine serum albumin (BSA; 2% in P B S ) Dissolve 2 g of BSA (fragment V) in 100 ml of PBS. Filter through a 0.2um filter. Store at 4°C. CaCl2 (2mol/L) Dissolve 29.4 g CaCl2 (J.T. Baker) in 70 ml of water. Replenish to 100 ml. Filter through a 0.2 nm filter. Cells 293T (human embryonic kidney cells containing S V 4 0 large T antigen) H T 1080 (human fibrosarcoma cell line) Transduced cells (target cells) Formaldehyde (3. 7% in P B S) Dilute 37 % formaldehyde solution (Sigma-Aldrich) with PBS at a ratio of I : 10. 2 X HEPES buffer (H BS; 0.05m ol/L HEPES, 0.28m ol/L NaCl, and I.5 mm ol/L Na2 HPO4 , pH 7. 12). Dissolve 2. 38 g of HEPES, 3. 28 g of NaCl, and 42. 6 mg of Na2 HPO4 in 200 ml of water. Filter through a 0. 2umn filter. 293T and HT 1080 Cell Culture Base High sugar (4500 mg/L) DMEM medium with 10% fetal bovine serum (FBS), 100ug/ml penicillin, 100ug/ml streptomycin Target cell culture medium Depending on the cell type and experimental design, the appropriate medium, cytokines and other nutrients are selected. For example, the CD34+ transduction medium used in this method is: IMDM, containing 20 % BIT9500 (Stem Cell Technology, Vancouver, B.C. Canada), 100 ng/ml Stem Cell Factor (SCF), 100 ng/ml Flt3-lig, and 100 ng/ml Thrombopoietin (TPO). Phosphate buffer solution (P B S , p H 7. 4) 137 mm ol/L NaCl 2.7 mm ol/L KCl 8.lm m ol/L Na2 H P 0 4 I . 5 mmol/L KH2P 〇 4 Plasmid DNA Plasmid DNA, i.e. pHIV-GFP, pCgp, pCMV-reu, pCMV-G, containing the target transgenes, is purified using the QIAGEN Plasmid Kit according to the instructions. Polyglutamine (4 mg/ml) Dissolve 40 mg of hexadimethrine bromide in IOml of water. Filtrate through a 0.2um filter. Store at 4°C. RetroNectin (Takara Mirus Bio Inc. > Madison, Wisconsin) Prepare 25fig/ml RetroNectin according to instructions. Store at 4°C. TE 79/10 Dilute TE 79 (lOmmol/L Tris-HCl and lm m ol/L EDTA, pH 7. 9 ) with water at a ratio of I : 10. Filter through a 0.2um filter. Instrumentation Centrifuge (1X3. 5in polyallomer [Beckman]) Bacteria are sterilized by high pressure (15 psi, 15-20 min) before use. Fluorescence Activated Cell Separator (FACS) Incubator 37°C Non-tissue culture treated plates (4 8-well and 2 4-well) Culture plates (12 wells) Syringe (30 ml) Needle Filter (0.2um) Cell Culture M (IOmm) Ultracentrifuge with SW 28 rotor head (Beckman) Methods Generation of lentiviral vectors: packaging of vectors 2- Prepare I m l of phospho-D N A suspension per IOOm m plate of cells as follows: a- Prepare two sterile tubes labeled Tube〗 and Tube 2 for each transfected plate. b- Add 0. 5m l 2 X H B S to tube 1 . c. Add TE 79/10 to tube 2. The volume of TE 79/10 is equal to 440W minus the volume of DNA solution. d - Add 15/xg of transfer vector containing the transgene, 15ugpCgp, 5ugpCMV-rew, and 5ugpCMV-G, to Tube 2 and mix well. e- Add 60ul of 2mol/LCaCl2 to tube 2 and mix well. f- Add liquid from tube 2 to tube 1 drop by drop and mix lightly. g . Incubate at room temperature for 30 min. 3- Suction beating or vortexing to mix the precipitate. 4 . Add I m l suspension to the IOOm m plate containing the cells. Add slowly and shake the medium gently. Return the plate to the incubator at 3 7 °C for 4 h and discard the precipitate. S - Replace old medium with 6 m l of fresh medium. Add 60ul of 0.6m o l / L sodium butyrate. Incubate in incubator. After 6.48 h, collect the supernatant and freeze at 80°C or perform a concentration step. Concentration of the vector 7.900 g centrifuge I O m in supernatant (freshly collected or thawed from the refrigerator) to remove cellular debris. 8.0.2 um needle filters are used to filter the supernatant. 9 . Transfer the supernatant to an autoclaved centrifuge tube and ultracentrifuge for I.5 h at 4°C with a BeCkm anS W 28 turntable at 24,500 r/m in to concentrate the supernatant. 10- Discard the supernatant and resuspend the precipitate in an appropriate amount of medium, e.g., if 100-fold concentration is required, 300 ul of medium should be added to 30 ml of the original solution. 1 1 . Dispense the concentrated vector in 10 to 154 portions and store at 80°C. Determination of the titer of the carrier 12- Inoculate at 5X104/well in 12-well plates, complete medium, incubate at 37°C with 5% C02 overnight. 13- Add each dilution gradient of vector and 4ul/m l of polyglutamine to the cells. Continue incubation for 48 h. 14-Digest the cells. Centrifuge, discard supernatant and resuspend cells with 300ul of PB S containing 3. 7 % formaldehyde. 15- Percentage of E G F P positive cells analyzed by FA CS. The titer is expressed as transfection units (TU) per milliliter of concentrated vector, i.e., TU/ml. Lentiviral Vector Transfection of Target Cells For transfection of cultured hematopoietic cells 16 - Inoculate exponentially growing cells in 2 4-well plates with Im l medium at 2XIO5 cells/well. 17- Add different volumes of concentrated vector depending on the cell type. For the K 562 cell line (C M L leukemia cell line), 100% transduction is achieved at a susceptibility index (m oi ) of 10. 18 . Add g/ml of polyglutamine. Incubate cells at 37°C. 19- After overnight incubation, the cells were centrifuged, the supernatant was discarded and resuspended with fresh medium and incubated again. 20 . After 48 h of transfection, the transfection efficiency was calculated by FA CS analysis (see Troubleshooting). Transfection of Early Stage Hematopoietic Stem Cells 21 - Procedure for isolation and purification of CD34+ hematopoietic stem cells from umbilical cord blood or bone marrow using immunomagnetic beads conjugated with anti-CD34+ antibody. 22. 48 h before transfection, culture C D 34+ cells in C D 34+ transfection medium. 2 3 . Coat 48-well culture plates with 0.2m l 25pg/ml RetroNectin (approx. 5/ug/cm2 ) for 2 h at room temperature. 2 4 . Discard R etro N ectin and add 0-2m l of PB S block with 2% B SA. The sample was incubated for 30 min at room temperature. 25 - After washing the plate with PB S, adjust the lentiviral vector to the appropriate infection index (range 5~40) with normal IMD M medium, final volume 200^1, and add to the coated plate. Incubate at 26.37°C for 2 h. Discard the vector supernatant and wash the plates with PBS. 2 7 . Add pre-stimulated CD34+ cells to the wells at a density of I X l0.5 cells/well with 0.2 ml of growth medium and incubate at 37°C. After 28-overnight incubation, cells were centrifuged, resuspended in Im l medium, and transferred to 2 4-well plates and incubated in an incubator. 2 9 . After 6 d of transfection, FA CS analyzed the transfection efficiency. For more product details, please visit Aladdin Scientific website.
I. 293T cells were grown in complete medium, 37°C oven, 5 % C O 2 . Twenty-four hours prior to transfection, exponentially growing 293T cells were inoculated in Io o m m dishes at a density of 4 X 106 cells/plate. The cell density at the time of transfection was approximately 80%.
