Ron Lab


 

Production of VSV-G retroviral virus by CaCl2 Transfection

Starting Material –             293T Cells
                                    Plasmids:             pJK3
                                                            pCMV-tat-HIV
                                                            pL-VSV-G
                                                            Specific DNA (pBabe plasmid)

Production of the virus in 293T Cells:

Day 0 – Count the cells and plate 2 x 106 293T cells in 100-mm dish

Day 1 – Transfect 293T cells with the following amount of plasmids with the CaCl2 transfection protocol


Low DNA method
            0.4 mg pCMV-tat-HIV
            3 mg pJK3
            1.5 mg pL-VSV-G
            4.5 mg specific plasmid (pBabe)

If low titre use following amounts:
            1.2 mg pCMV-tat-HIV
            6mg pJK3
            3mg pL-VSV-G
            10 mg specific plasmid (pBabe)


Dilute plasmids in H2O up to a volume of 900 mL.  Add 100 mL 2.5 M CaCl2.  Add 1 mL 2x HBS. Mix and add dropwise to the media of 293T dish.
Do the transfection in the afternoon and let it stand overnight.  From now and onward the cells are not exposed to the light when working in the hood (the virus is light sensitive).

Day 2 – At first hour in the morning remove the media and discard it* (VERY IMPORTANT: add bleach to everything that has to be discarded and expose to UV light).  Add 4 mL of fresh media and put cells back into the incubator.

Day 3 – At first hour in the morning collect the media (first viral collection) and store in foil at 4 deg C.  Add 5 mL of media to the cells and incubate for another day.

Day 4 – At first hour in the morning collect media (second viral collection) and discard the 293T cells*.  Pool the first and second viral media (total 10 mL) and filter through a 0.4 mm filter.  The virus can be kept at 4 deg C in foil or can be used right away to infect cells.  Usually, I have a titer of ~1-5 x 106 cfu in 10 ml of viral stock media

Infection of CHO.K1 cells

Day 0 – Count the cells and plate 1 x 106 CHO.K1 cells into a 100 mm dish

Day 1 – Infect cells by adding virus stock media + 1x polybrene final (10 mg/ml polybrene =1000x) in total volume of 10 mL.  After ~8 h remove the media and discard it*, add fresh media to the cells.  Incubate infected cells at least 2 or 3 times the length of a cell cycle (this period allows for the interaction and expression of the viral genes), in the case of CHO.K1 incubate for 2 days.

Day 3 – Add puromycin to the media if the virus has a puro marker or analyze the cells by ACS if the marker is GFP.  For CHO.K1 cells add 8 mg/mL puromycin.  The infected cells can be split at this point and a fraction of them can be plated (1:10, 1:20).

Day 5 – After 2 days in selection discard the media and add fresh media with puromycin.  After ~10 days of puromycin selection count the colonies (at this time the colonies should be obvious).

Calculate the titer as follows:

CFU/mL = no colonies/(virus volume (mL) x replication factor x factor of infected cells plated)

CFU = colony forming units
Virus volume = amount of viral media added to infect cells in mL

Replication factor = number of divisions that occurred between the time of infection and the time of puromycin addition (that is determined by counting the cells at the time of infection and counting the cells at the time of puromycin addition).

Fraction of infected cells plated = this applies only if the cells are split and a fraction of them are plated, but if all of the infected cells are used this number equals 1. 

Solutions

2X HBS                                                    500ml
HEPES (Acid)                5g
NaCl                                    8g
Dextrose                        1g
KCl                                    3.7g
Na2HPO4(7H2O)            10ml of Na2HPO4(7H2O) *Stock Solution (see below)

- Add Purified H2O up to ca. 450ml
- Adjust pH to exactly 7.1

Aliquot into sterile tubes and keep frozen at –20oC.
We then keep one working tube at 4oC.

*Na2HPO4(7H2O) Stock Solution :

Na2HPO4(7H2O)             0.94 g of Na2HPO4(7H2O)   in 50ml HPLC Purified H2O

2.5M CaCl2:
100ml
CaCl2                                    27.75g anhydrous CaCl2
Or
36.75g CaCl2(2H2O)

-We typically aliquot into sterile 15 ml tubes (10 ml in each) and store at –20oC (although keeping at–20oC is probably not necessary – this is primarly to keep it in a fairly germ free environment and to keep it together with the 2X HBS).

 

pJK3- Bam HI cutà ~4-6kb + ~2.3kb + ~0.300 (visible on burned in gel)
pCMV-tat- Bam HI cutà ~3 + 1kb + ~0.400 (visible on burned in gel)
pVSVG  EcoR1 cutà ~1.9 (x2?) + 1.5 +0.9 + ~400 (visible on burned in gel)
:::Desktop:IRE1:BAC- hIRE assays:e213 Flag_hIRE_Tev const:101201a1vsvg helpers.tif:::Desktop:IRE1:BAC- hIRE assays:e213 Flag_hIRE_Tev const:101201a1vsvg helpersdk.tif

 

Heather P. Harding, updated 9 December 2010