Retroviral-Mediated Transduction: A Method to Introduce Target Gene into Cancer Cells
Published: April 30, 2023
Abstract
Source: Kesarwani, M., et al. Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence. J. Vis. Exp. (2019).
This video describes the protocol for genetic validation by transferring target genes using retroviruses in chronic myeloid leukemia cell lines. The retroviruses act as a mediator to transfer genetic material into the leukemia cell through the process called transduction, which enables long term expression of target gene.This method can be applied to any target cells for genetic validation and therapeutic development.
Protocol
1. Transduction Retroviral plasmid transfection 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. Count the cells and add the appropriate volume to a 10 cm treated dish (~4 x…
Representative Results
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…
Disclosures
The authors have nothing to disclose.
Materials
| 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 |
Cite This Article
Retroviral-Mediated Transduction: A Method to Introduce Target Gene into Cancer Cells. J. Vis. Exp. (Pending Publication), e20276, doi: (2023).