Most scientists use adeno-associated viruses (AAV) to study gene function and to develop gene therapies. AAV is a small, non-pathogenic virus that can infect both dividing and non-dividing cells. AAV expresses genes efficiently in a wide variety of cell types, making it an ideal tool for studying gene function. You can also check out addgene AAV guide to know more about AAV.
To build a AAV expression system, you will need the following:
1. A plasmid containing your gene of interest under the control of an AAV promoter.
2. An AAV vector expressing the gene of interest.
3. A helper virus (e.g., adenovirus or herpes simplex virus) to provide the necessary proteins for AAV replication.
4. Cell lines that are susceptible to infection by the helper virus and AAV vector.
5. Molecular biology reagents and equipment for plasmid construction and DNA transfection.
The first step is to construct a plasmid containing your gene of interest under the control of an AAV promoter. The most commonly used promoters are the CMV immediate early promoter (CMV IE), the chicken beta-actin promoter (CBA), and the hybrid promoter (hCMV/CBA).
Next, you will need to transfect your plasmid into an AAV vector. The most commonly used AAV vectors are pAAV-MCS, pAAV-RC, and pAAV-CMV. These vectors contain the necessary sequences for AAV replication and packaging.