Occasionally DNA recombination occurs between the replication-defective retroviral DNA construct and the genome of the packaging cell line that encodes viral proteins resulting in the generation of wild-type retrovirus. Wildtype retrovirus is capable of replicating on its own and infecting unrelated cells making lineage experiments impossible to interpret.
1. 3T3 Fibroblast cells (American Type Culture Collection, Accession Number CCL 92).
2. 3T3 Growth Media (store at 4°C): The following are added to DMEM (formulation containing 1000 mg/L glucose and 2 mM l-glutamine) to give the final indicated concentrations: 10% FBS; 100 U/mL penicillin; 1000 |g/mL streptomycin.
3. 8 mg/mL Polybrene stock solution in PBS.
4. 0.45-|im Disposable sterile syringe filters with cellulose acetate membrane.
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