Viral vector service – Cloning
Adeno-associated virus (AAV) and Lentivirus (LV) can be generated and cloned upon request. We use standard cloning techniques; restriction-ligation approaches and Infusion Cloning. All newly made plasmid DNAs will be sequenced and tested for fluorescence activity.
Viral vector service – Purification AAV
For AAV replication we use helper plasmids that provide all the necessary adenoviral genes. Depending on the target cell type different serotypes of rAAV can be purified (serotypes 1, 2, 5, 6, 7, 8, 9, rh10; synthetic serotype PHP.B, PHP.eB, PHP.B4). For the purification of new AAVs we use heparin columns for AAV2 particles or Iodixanol gradient centrifugation and FPLC for all other serotypes. We routinely test purified rAAV viruses for their physical and infective titer. On request we can also perform qPCR to determine the titer of the virus.
Methods:
- Generation of endotoxinfree plasmid DNA for viral production
- Cloning of new AAV or LV vector (simple or complex cloning strategies)
- AAV purification (Heparin columns or gradient centrifugation and FPLC)
- SDS-PAGE Coomassie staining and viral transduction test in primary neurons
- Viral titer determination using qPCR
For the generation of Lentiviruses (LV) please contact the working group of Alexander Pfeifer (Institut für Pharmakologie).