-80C Box 10 D-1, LN2 Gretchen Box)
Regular Medium
DMEM
10% Fetal Bovine Serum – heat inactivated
10mM Hepes (1:100 of 1 M stock)
2 mM Glutamine (1:100 of 200 mM stock)
[100 U/ml Penicillin – 100 ug/ml Streptomycin (1:100 of stock)
10ug/ml Gentamicin (1:1000 of 10mg/ml stock)]
Recipe for induction medium

DMEM

FBS
Sigma Aldrich
Gemini

L-Glutamine 200mM Gibco

PenStrep 100x Gibco

Gentamicin 10mg/ml Gibco

ZnCl2

CdSO4
100mM made in lab
1mM made in lab
D6429
100-106 heat-
25030-081
15140-122
15710-064
6/10/2009 inact.: >1h at 56°C

NaBiCarb

Hepes
7.5%
1mM made in lab made in lab
Jun 12 pH 7.3
Pseudoviruses (Glycerol Stocks in GG Bacteria 4 (-80 9C2) final conc.
10%
2mM
1x
10µg/ml
100µM
1µM
0.15%
50mM
JC-M1SV196-TK-eGFP-pUC (amp)
JC-M1SV196 TK inducible promotor expressing eGFP in place of TAg; Grow in CMT4 cells which express TAg with heavy metal media. From the DiMaio lab
SV40-776--TK-eGFP-pUC (amp)
SV40 776 TK inducible promotor expressing eGFP in place of TAg; Grow in CMT4 cells which express TAg with heavy metal media. From the DiMaio lab
Digest viral genome in backbone
Digest for 2 hours at 37°Cwith the enzyme of interest, e.g. KpnI/EcoRI:
Use 10µg of virus genome in the respective backbone, e.g. puc19/pBR322:
5µl 10x buffer
10µg plasmid of interest
3µl enzyme of interest
Add H
2
O to a total volume of 50µl
Run sample on a 1% agarose gel (add 1µl EtBr stock solution/20ml gel)
Excise band of interest and purify DNA with a gel purification kit, e.g. Qiagen Gel Extraction Kit

Ligation
Ligate sample o/n at 16°C:
All Purified sample
5µl 10x buffer
5µl T4 ligase
Add H
2
O to a total volume of 50µl
Precipitate or purify DNA sample with a PCR product purification kit, e.g.by Qiagen and elute to final concentration of
100ng/µl
Transfection
Add 1µg of ligated DNA OPTI-MEM medium to a final volume of 800µl
Mix 55µl Lipofectamine Reagent with 745µl OPTI-MEM for a final volume of 800µl
Mix the two together and incubate for 15-30 minutes at room temperature
In the meantime: wash cell layer twice with PBS and once with OPTI-MEM
Add 6.4ml OPTI-MEM to the mixture for a final volume of 8ml (to be used on a T75 flask – scale up for T150)
Add transfection mix to the cells and let incubate for 5-6 hours
Change medium to induction medium
Harvest when pronounced CPE appears and go to up to 5 rounds of infection for propagation of the virus
Infection
Grow CMT4 cells to about 80% confluence in tissue culture flasks (T150). Aspirate medium and replace with fresh medium. Add Pava at MOI 0.05 to 0.5 (typically about 1.2x10
5 – 1.2x106 PFU/flask) to the medium and gently shake the flask. Incubate a t 37°C for 5-6 hours and change to induction medium
Virus harvest
Observe the cytopathic effect of the virus. When about 50% of the cells show CPE scrape the cells into the medium
(using a cell scraper). Pour the medium with the cells into a 50ml blue cap tube. Spin for 510 minutes at 1000rpm at 4°C
(cold room). Pour the supernatant into bleach or Lysol and save the pellet. If there is remaining liquid briefly spin the tube again and carefully remove the remaining supernatant (avoid aspirating, because you might risk losing the pellet).
Resuspend the cell pellet in 0.45 ml/flask of cold Tris (10mM) and disperse the pellet using a p1000 pipet. Transfer mix into a screwcap freezing vial.
Place the vial on ice for at least 30 minutes vortexing the mixture every 5 minutes. Spin the cells in a microfuge at maximum speed for 10 minutes at 4°C. Transfer the supernatant to 0.1ml/flask of 10%DMEM/100mM Hepes (pH 7.3) into another screwcap tube. Let the extract sit on ice for 5 minutes and spin again in a microfuge at maximum speed for about
10 minutes at 4°C to sediment remaining debris and salt. Again, transfer the supernatant to a fresh tube and aliquot your virus stock. At this point the titer should be 10 7 -10 9 PFU/ml. Avoid repeated freeze-thawing.
Alternatives
If you need a stopping point in the prep you can aspirate the medium and store the flask cells at 80°C before scraping.
Then add the appropriate amount of Tris buffer and do 3 freeze/thaw cycles at 80°C/37°C making sure not to leave the cells at 37°C longer than only until thawed.
After the prep you can filter the virus through a syringe filter to remove remaining debris. However, your titer will decrease by approximately 1log
10
.

Things to note
CMT4 cells derived from African green monkey CV-1 cells. SV40-TAg is expressed from the inducible metallothionein promoter.
CMT4 cells should never reach 100% confluence if possible. They will start detaching. You can passage CMT4 in induction medium, but they will always look rather unhappy. Always include a mock control to distinguish between actual
CPE and toxic effects from the induction medium.
If you want to propagate reporter viruses other than Pava you might want to passage transfected or infected CMT4 cells for a few weeks. In my hands, the JC reporter viruses all need more than 2 weeks for efficient production