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2.
Collect virus-containing medium from the
Φ
NX
cells transfected with a
retroviral vector.
3.
Centrifuge the supernatant at 1000
g
for 5 min to spindown cell debris and filter
through a 0.45-
µ
m filter to remove cells as well. Add polybrene to a final concen-
tration of 4
µ
g/mL and filter medium through a 0.45-
µ
m filter.
4.
Prepare serial dilution of a virus-containing media using a fresh culture medium
containing 4
µ
g/mL of polybrene (six 10-fold serial dilutions are usually prepared).
5.
Infect target cells by adding virus-containing medium to the wells.
6.
Change the media with virus to a normal one and add appropriate antibiotic
(depending on the drug selection marker) 24 h postinfection. The optimal con-
centration should be determined for each cell line. Puromycin selection takes less
than 1 wk at 0.5-1.5
µ
g/mL while G418 takes 2-3 wk at 0.1-1.0 mg/mL.
7.
The titer of virus corresponds to the number of colonies present at the given
dilution multiplied by the dilution factor. For example, the presence of 4 colonies
in the 10
5
dilution would represent a viral titer of 4
10
5
(
see
Notes
).
×
3.6. Measurement of Transduction Using a Reporter Gene (LacZ)
In order to measure an efficacy of retroviral transduction, a retroviral vector
containing a reporter gene (alkaline phosphatase,
-galactosidase, luciferase,
or green fluorescent protein) should be utilized in parallel experiments. Trans-
fection and infection of retrovirus containing a reporter gene can be carried out
together. The following procedure describes the procedure for detection of cells
transduced by the retroviral vector containing the
β
β
-galactosidase gene.
1.
Remove media from adherent cells.
2.
Add 2 mL of fixative solution to a 60-mm plate of adherent cells at 4
°
C for 5 min.
3.
Remove medium and wash 3 times with PBS.
4.
Transfer 1 mL of prepared staining solution into cells.
5.
Optimal staining will occur 12-18 h later.
4. Notes
1.
The viral supernatants produced by these methods might contain potentially haz-
ardous recombinant virus. The user of these systems must exercise caution in the
production, use, and storage of recombinant retroviral virions, especially those
with amphotropic host ranges. This consideration should be applied to all genes
expressed as amphotropic and polytropic retroviral vectors. The user is strongly
advised NOT to create retroviruses capable of expressing known oncogenes in
amphotropic or polytropic host-range viruses. NIH guidelines require that
retroviral production and transduction be performed in a Biosafety Level 2 facility.
2.
For efficient transfection using calcium phosphate method, pH of 2XHBS solu-
tion should be 7.0-7.05. Because pH is so important it would be useful to make
and to test 2XHBS with pH 6.95, pH 7.0, and pH 7.05.
3.
We found frequent rearrangements (mainly large deletions) in the retroviral vec-
tor when the ligation mixture was transformed into DH5
α
cells. Thus, it is neces-
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