Virus Preparation for the TVA System

 Select a vector, either RCASBP(A) or other ALV-A vector

 Clone your gene of interest into the selected vector 

Purify the plasmid DNA using a method that will yield pure DNA (Cesium chloride gradients, Qiagen Maxiprep, etc.) 

Prepare either chicken embryo fibroblast cells or DF1 chicken fibroblasts for transfection.  The protocol below is for DF 1 cells.

 Maintain DF1 cells in growth medium (DMEM with high glucose, 10% fetal bovine serum, 2 mM L-glutamine, 10 units/ ml penicillin, 10 ug/ml streptomycin) at 37^oC and 5% CO₂.

 Transfection using Superfect (calcium phosphate and Lipfectamin Plus reagents may also be used)

One day before transfections, pass DF1 cells to 60 mm tissue culture dishes so that they will be 30-80% confluent at the time of transfection 

Mix 150 ul of DMEM (without serum, penicillin or streptomycin) and 5 ug of pure viral DNA¹ (such as RCAS-lacZ); add 30 ug of Superfect (Qiagen); vortex mixture for 10 seconds; leave the mixture at room temperature for 5 to 10 minutes; add 1 ml of growth medium and pipet up and down three times 

    ¹Caution should be taken in preparing and transferring viral plasmid DNA.  The use of filtered tips is strongly recommended to avoid the possibility of contamination from another viral plasmid

  Aspirate the medium from the DF1 TC dishes; add the transfection mix; swirl to ensure that the cell layer is covered; incubate for 2-3 hours at 37^oC

  Aspirate the transfection mixture; rinse the TC dish once with growth medium; add 3 ml of growth medium; return to 37^oC incubator

  Pass the cells 24 to 48 hours later; transient expression may be assayed 48 to 72 hours after transfection

  Continue to pass cells for 5 to 7 days until all of the cells are presumably infected; freeze cells in 10% DMSO and at least 20% fetal bovine serum; for long-term storage, store cells in a liquid nitrogen storage tank; store frozen culture supernatants at –80^oC (see below).

  Virus Collection

  To obtain high titer viruses for stocks, collect virus from cultures grown in minimum amounts of medium (5 ml/100 mm tissue culture dish) and approximately 24 hours after cells become confluent.  Harvests can be done every 12 hours, adding 5 ml of medium each time.  Typically, plates can be passed from 1 to 2 plates, resuming virus collection 18-24 hours after passage.

Remove cell debris by low-speed centrifugation and store supernatant aliquots at –80^oC.  There is a typical loss of up to one log of viral titer after one cycle of freezing and thawing.

  ALV-derived viruses mutate frequently and mutant viruses that have lost the gene of interest commonly show a growth advantage.  Following multiple rounds of infections, culture supernatants may have many mutant viruses but due to viral interference, only a limited number of new infections occur in producer cells.  Viral mutations can be minimized by passing/propagating the producer cells.  New producer cells can be generated by infecting DF1 cells with stocks from frozen culture supernatants collected from early passages following transfection.

Concentrating Viruses

Expand virus-producer cells into 5-10 15-cm dishes depending up the amount of virus needed.

When they are confluent, switch to DMEM/1% FBS, 20 ml/dish; grow overnight.

Collect supernatant.  Remove cell debris by a low speed spin (3000 rpm,10 minutes, 4C).

Spin in a ultracentrifuge for 1.5 hr. at 26K (50,000 g), 4^oC.

Discard all but 1/100 of the supernatant.  Resuspend the pellet by vortexing for 2 min at a medium speed.

Viral Titer Determination

 Viral titer assays can be done on avian DF1 cells or mammalian cells stably expressing tv-a (e.g. NIH3T3-tv-a or 293T-tv-a).  If using DF1 cells, viral supernatants should be filtered using a 0.8 um filter to avoid contamination from detached DF1 producer cells

 One day before assay, pass cells to 60 mm tissue culture dishes so that they will be 30% confluent at the time of infection.

 Make a series of 10-fold dilutions of the viral supernatant (from 10^o to 10⁹) in growth medium.

 Add 1 ml of each dilution to the 60 mm TC dish containing DF-1 cells; make duplicate plates for each dilution .

 Incubate for 3 hours at 37^oC.

 Add 2 ml of growth medium to the dishes; allow cells to grow for 4 to 7 days; assay for gene expression by an immunofluorescence assay or other methods.