- Helper dependent protocol
Retrovirus using Phoenix Lines. This is one of two transfection
protocols you can find in this web page set.
viral supernatants produced by these methods might,depending upon
your retroviral insert, contain potentially hazardous recombinant
virus. The user of these systems must exercise due caution in the
production, use and storage of recombinant retroviral virions, especially
those with amphotropic and polytropic host ranges. This consideration
should be applied to all genes expressed as amphotropic and polytropic
pseudotyped retroviral vectors. Appropriate NIH and other regional
guidelines should be followed in the use of these recombinant retrovirus
production systems. The user is strongly advised NOT to create retroviruses
capable of expressing known oncogenes in amphotropic or polytropic
host range viruses.
Retroviral Producer Line Protocol
0 | Day 1 | Day 2 |
Day 3 | Day 4 | Day
5 | X-Gal Staining}
0: Preparation of Phoenix Retrovirus Producer cells for Transfection.
hours prior to transfection, plate Phoenix cells at 1.5-2 million
cells per 6 cm plate in producer cell growth media.
adding cells, set plates in incubator and gently shake forward
and backward, then side to side, 3-4 times. This distributes cells
evenly about the plate. Do not disturb cells for several hours
while they attach to plate.
are allowed to attach for 10 hours to 24 hours on 10 or 15 cm
2/3 confluence, a 10 cm plate should provide 5 million cells,
a 15 cm plate will contain about 7.5-15 million cells. It is at
this subconfluent stage that cells are most transfectable and
will survive the rigours of transfection best, giving the highest
titer virus possible.
DMEM, 10% FCS, 1% Penicillin-Streptomycin, 1% Glutamine.
let cells reach confluence. This will reduce transfection
efficiency in the short term. For maximally healthy cells
a split of 1:4 or 1:5, of a 70-80% confluent 10 cm plate,
into a new plate every 2-3 days should provide optimal cell
conditions. Local media and serum conditions will vary.
of Phoenix™ cells every few months in Hygromycin (at 300 ug/ml)
and Diptheria Toxin (1 ug/ml) for one week is recommended.
Cells can be analyzed and sorted by FACS for expression of
CD8 (a proxy measure of gag-pol in this cell line) and for
surface expression of envelope protein with 83A25 antibody
(see Chesboro et al (ref)).
million cells on a 60 mm plate is a good starting point for
seeding Phoenix cells prior to transfection; it is important
to titer slightly up and slightly down to maximize transfection
efficiencies. Efficiencies of 50-60% as determined by X-gal
staining of the Phoenix cells should be achieved. We have
found that the highest transfection efficiencies are obtained
with Phoenix cells that are 70-80% confluent at the time of
the DNA in HBS for application to cells. 1. About 5 minutes prior
to transfection, add chloroquine to each plate to 25uM (chloroquine
stock is 50 mM; for 3 mL media + 1 mL DNA, add 2u1).
acts to inhibit lysosomal DNases by neutralizing vesicle pH.
delivered by Ca2PO4 transfection is thought to transit through
a 15 mL tube, add (per 6 cm plate, with all reagents at room temperature):
ug DNA (DNA is added in a drop to side of tube).
u1 dd H2O (wash the DNA to bottom of tube with water).
ul 2M CaC12 (from Mallinkrodt) -mix thoroughly with finger
ul total volume.
volume and DNA/reagent amounts if necessary.
0.5mL 2xHBS quickly then bubble vigourously with automatic pipettor
(keep eject button depressed) for 3 - 15 sec (actual length of
bubbling time depends on each batch of 2xHBS).
HBS/DNA solution dropwise onto media (gently and quickly) by spreading
across cells in media.
the cells under a microscope; you should observe evenly distributed
VERY small black particles.
plate(s) in 37 C incubator; rock plates foreward and backward/back
and forth a few times to evenly distribute DNA/CaPO4 particles.
for Calcium Phospahte Coprecipitation Transfection
Make a stock solution of Na2HPO4 dibasic (5.25 g in 500 ml
Make 2 x HBS: 8.0 g NaCl 6.5 g HEPES (sodium salt) 10 ml Na2HPO4
pH to 7.0 using NaOH or HCl. Bring volume up to 500 mls. Check
pH again. The pH is very important, it must be exactly 7.0
HEPES is from Sigma (catalog # H-7006)
CaCl2 is from Mallinkrodt (catalog # 4160)
Because pH is so important make 3 batches pH 6.95, pH 7.00,
pH 7.05. Test each solution and use the one that yields the
All reagents should be at room temperature prior to use.
e-mail any questions to Angelica Trejo: email@example.com
2: 24 hours post-transfection
media and prepare target cells
media to 3 mL fresh DMEM, 10% FCS
is more stable if incubation is carried out at 32 Celsius,
although 37 is okay.
not leave chloroquine on cells more than 24 hours. It is toxic.
for Titering if necessary
NIH 3T3 cells at 200,000 per 6 cm plate in DMEM, 10% CS (1% PentStrep,
1 % Glut).
using suspension cells, they should be growing in log phase at
time of infection (see below--for Jurkats, ideal density at time
of infection is 5E+5 /mL).
3: 48 hours post-transfection - infecting cells with retroviral supernatent
supernatant from transfected Phoenix cells into 15 mL tubes and
centrifuge at 1500 rpm for 5 minutes to pellet cell debris.
through 0.45 um filter removes cells as well.
can be frozen at -80 C for later infection, although titer
drops by one-half for each freeze-thaw cycle.
Phoenix cells with X-gal to gauge transfection efficiency
only works if you used a lacZ or other stainable reporter
your virus does not contain such a marker, you can dope the
transfection with about 1/10 molar plasmid of a marker that
will not interfere with your experiment).
1 mL media from each 3T3 plate.
3 uL polybrene (polybrene is lOOOx at 5 mg/ml) to each 3T3 plate;
with gentle and thorough shaking.
1 mL viral supernatant to each 3T3 plate and place at 32/37
Celsius with gentle shaking.
for 8-24 hours, spin cells, and wash away virus supernatent.
5x10^5 suspension cells, resuspend cell pellet in 1 mL virus
+ 1 uL lOOOx polybrene.
cells, especially some B cells and T cells are sensitive to
may be necessary to titrate polybrene to lower levels.
4: Remove Virus Supernatent.
hours post-infection: change media on 3T3s to fresh DMEM, 10%
CS and place at 37 Celsius.
suspension cells, spin out of media and resuspend in 2 mL
at 37 Celsius.
5: 24- 48 hours post-infection
are now ready to assay for biochemical event of interest.
actual reverse transcription and integration take place within
24-36 hours, depending on cell growth kinetics.
can start to be observed at 24 hours, usually maxing out at
there on in continued retroviral expression might drop over
a period of weeks to months, depending on cell line, site
of integration, relative toxicity of insert and a whole bunchof
things nobody really understands.
on your own from here. However, if you did add a reporter enzyme,
such as lacZ, you can stain 3T3s or supsension cells with X-gal
or prepare them for FACS-Gal.
(Glutaraldehyde is 25% stock/500x, from Sigma)
3 mL ferri/ferrocyanide solution
uL X-Gal (40mg/mL in DMSO--store at -20oC in the dark).
MgCl2 in ddH2O
the solutions from exposure to light and store at 4oC.
nonadherent cells add FCS to 1-5%
media from adherent cells or spin down nonadherent cells in 15mL
2 mL fixative to 6cm plate of adherent cells or resupsend nonadherent
cells in 1 mL fixative. Leave for 1 min.
adherent cells, remove and wash 3xPBS (first two washes are
quick, third is for 3 min).
nonadherent cells, quench fixative by adding 5-lOmL PBS/1-5%
FCS to conical and spin down again.
3mL staining solution onto adherent cells or resuspend nonadherent
cells in 1 mL staining solution and place in well of 24well plate.
staining will occur 24 hours later. If longer, remove staining
solution from cells at 24 hours and re-layer/resupsuspend in ferri/ferrocyanide
solution without X-Gal.
be left on cells for 2 min and washed 3xfast with PBS.
is much more difficult to prepare:
4% paraformaldehyde stock: in fume hood, dissolve 8g powder
in 150mL of 0.1M sodium phosphate pH 7.3 (66mM NA2HPO4
or 33mM NAH2PO4), stirring and heating at 60oC
10N NaOH at rate of 1 drop/min until solution clears.
Bring up volume to 200mL with 0.1 M sodium phosphate pH
7.3. Store at 4 C for up to 1 month.
50mL 4% paraformaldehyde solution with 49.2 mL 0.1M sodium
phosphate pH 7.3 and 0.8mL 25m% glutaraldebyde. Store
at 4 for up to 1 week.