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)