Retroviral-Mediated Transduction: A Method to Introduce Target Gene into Cancer Cells

Protokol

1. Transduction

1. Retroviral plasmid transfection
   1. Freshly thaw HEK293 cells the afternoon before in a water bath set to 37 °C. Spin the cryotube containing the HEK cells at 435 x g at room temperature for 3 min. Remove the supernatant via a vacuum and suspend the cell pellet in 1 mL of DMEM supplemented with 10% FBS, penicillin, streptomycin, and glutamine.
   2. Count the cells and add the appropriate volume to a 10 cm treated dish (~4 x 10⁶ HEK293T cells). Add 14 mL of the DMEM 10 media to the dish and mix it well by sliding up and down for an equal distribution. Place the dish in a 37 °C, 5% CO₂ incubator overnight.
   3. On the next morning, check the confluence of the cells under an inverted light microscope (50% or more works well).
   4. Combine the DNA (desired retroviral plasmid), PclEco (packaging plasmid), 2 M CaCl₂, and H₂0 in a 1.5 mL tube (= 500 µL) using a micropipette in the following amounts: 7.5 µg of DNA (BCR-ABL1-YFP), 7.5 µg of pCLeco, 62 µL of 2 M CaCl₂, and water to a final volume of 500 µL. Add 500 µL of 2x HBS to another 1.5 mL tube.
   5. Add the DNA mix dropwise to the 1.5 mL tube containing 500 µL of 2x HBS (280 mM NaCl, 100 mM HEPES, and 1.5 mM Na₂HPO₄) while mixing. Achieve this by setting a vortex to the lowest speed and hold the HBS tube on it for constant mixing while adding the transfection mix.
   6. Allow the transfection mix to incubate for 20–30 min at room temperature. During the incubation, add 25 nM chloroquine to the HEK cells and place them back in the incubator. After the incubation, add the transfection mix dropwise to the HEK cells and, then, gently swirl the media to mix the transfection mix throughout the plate.
   7. Incubate the cells with the mix for ~8 h in a 37 °C, 5% CO₂ incubator. Change the media on these cells by very slowly adding 14 mL of fresh DMEM 10 media. Pipetting slowly against the wall of the dish helps to prevent any stripping of the HEK cells. Incubate the cells overnight in the 37 °C, 5% CO₂ incubator.
   8. Collect the viral supernatant with a 10 mL syringe, draw the supernatant into the syringe, and attach the 0.45 µm syringe filter. Plunge the viral supernatant into a new collection tube. Take the first collection of viral supernatant ~16 h later.
2. Fibronectin viral concentration and transduction
   1. Add 2 mL of a 0.1 mg recombinant human fibronectin fragment in 1x PBS to untreated 6-well culture plates. Prepare as much fibronectin as needed, which depends on the number of cells and genotypes. One well can sufficiently transduce 10 million c-Kit+ cells. Incubate the plate overnight at 4 °C.
   2. The next day, remove the fibronectin from the wells with a pipette, pipette 2 mL of sterile 2% BSA in 1x PBS to block the plate, and incubate for 30 min at room temperature. Remove the BSA via a vacuum and wash thoroughly by pipetting 2 mL of 1x PBS then remove it via a vacuum.
   3. Immediately add freshly collected and filtered (at 0.45 µm) viral supernatant up to 5 mL. Centrifuge at 435 x g at 32 °C for 2 h. Remove the supernatant via a vacuum and repeat this step with additional freshly collected viral supernatant and spin again. Remove the supernatant via a vacuum and wash the wells by adding 2 mL of 1x PBS; then, remove it via a vacuum.
   4. Add the c-Kit+ mouse cells that were cultured overnight by spinning down the cells and resuspending the pellet in complete IMDM ,for culture. Culture these cells overnight in 2 x 10⁶ cells/1 mL of cell IMDM complete media by placing them in an incubator at 37 °C and 5% CO₂) to the viral coated wells by mixing the cell suspensions well and pipetting them into the now viral concentrated fibronectin plate. Spin at 1,200 x g at 25 °C for 90 min. Put the cells in a 37 °C, 5% CO2 incubator.
   5. 48 h post transduction, pellet the cells by centrifugation at 1,200 x g at 4 °C for 5 min and resuspend them in FACS buffer #1 (1x PBS + 0.5% BSA) to 6 x 10⁶/mL. Determine the percentage of BCR-ABL1 positive cells by measuring the percentage of YFP positive using flow cytometry.
   6. Acquire events with the standard procedure. First, gate the live cells based on forward and side scatter. Then, gate the YFP positive live cells excluding the YFP-negative cells determined by negative control (Figure 1).

Sonuçlar

Figure 1: FACS showing the frequency of YFP+ cells from the blood of a transplanted and a WT mouse (negative control). The top panels show a scatter plot of the cells and the gating. The lower panels show histograms of YFP vs. side scatter. Cells with an MFI of >10³ were considered YFP+. Similar gating was used for bone marrow cell...

Malzemeler

Name	Company	Catalog Number	Comments
DMEM	Cellgro (corning)	15-013-CV
RetroNectin Recombinant Human Fibronectin Fragment	Takara	T100B
FBS	Atlanta biological	S11150
Penn/Strep	Cellgro (corning)	30-002-CI
L-Glutamine	Cellgro (corning)	25-005-CL	5 mg/mL stock in water
HEPES	Sigma	H7006
PBS	Corning	21040CV
Na2HPO4.7H2O	Sigma	S9390
Calcium Chloride	Invitrogen	K278001
2x HBS	Invitrogen	K278002
EDTA	Ambion	AM9261
BSA	Sigma	A7906
Human Long-Term Culture Initiating Cell Assay	Stemp Cell Technologies
TC-10 automated cell counter	Bio-RAD
ROSACreERT2/c-Fos fl/fl Dusp1-/-	Made in house
ROSACreERT2/c-Fosfl/fl	Made in house
ROSACreERT2	Jackson Laboratory
C57Bl/6	Jackson Laboratory
CML-CD34+ and Normal CD34+ cells	University Hospital, University of Cincinnati
LSR II (FACS analyzer)	BD
Fortessa I (FACS analyzer)	BD
FACSAriaII (FACS Sorter)	BD
NAPCO series 8000 WJ CO2 incubator	Thermo scientific
Swing bucket rotor cetrifuge 5810R	Eppendorf
1/2 cc Lo-Dose u-100 insulin syringe 28 G1/2	Becton Dickinson	Becton Dickinson
Hydrocortisone Sodium Hemisuccinate	Stem Cell Technologies	7904
GM-CSF	Prospec	CYT-221
G-SCF	Prospec	CYT-220
Human IL-3	Prospec	CYT-210
human SCF	Prospec	CYT-255
Erythropoiein	Amgen	5513-267-10
BD Perm/Wash (permeabilization and wash solution for phospho flow)	BD	554723
BD Cytofix/Cytoperm (Fixing and permeabilization solution)	BD	554714
BD Pharm Lyse	BD	555899
70 μm nylon cell stariner	Becton Dickinson	352350
0.45 μM acro disc filter	PALL	2016-10