The goal of this module is to provide DNA constructs and viral vectors for expressing genes in tissues of the visual system, enabling precise molecular manipulation of proteins involved in visual function. The Gene Delivery Module is the only campus facility that provides viral vectors for introducing genes in vivo.
The viral vectors help deliver genes to the area of interest using viruses as carriers — Adeno Associated Virus (AAV) or Lentivirus (LV). These viruses are replication incompetent viruses. They can infect target cells and transmit target genes; however, they cannot replicate within target cells because the viral structure genes are absent. These recombinant viruses have reduced or no immune response in the host and have cloning capacity of up to 4.7 kb for AAV and up to 10 kb for LV. The recombinant viral vectors are packaged by helper virus free approaches, e.g. triple plasmids transfection into HEK293T cells to generate AAV vectors. The triple plasmids are transgene plasmid, RC (rep & cap) plasmid and helper plasmid. Individual researchers submit their own transgene plasmids, in which gene of interest and AAV inverted terminal repeats (ITRs) are included. The GDM provides the RC plasmids and helper plasmids required to generate the serotypes of viral vectors that meet the individual researcher’s needs.
Once the transgene plasmid (100-200 μg) has been submitted to the GDM, the GDM will start culturing a large amount of HEK293T cells; packaging and purifying viral vectors with triple transfection, iodixanol gradient ultracentrifugation and affinity column purification; and at last, measure the viral titers by fluorescent probe labeled quantitative PCR. It takes around three to four weeks to complete generating 1-2 purified viral vectors with the higher titer of 10^11 to 10^14 vg/ml (viral genomes per ml) and a total volume of 150-200 μl for in vivo use. These recombinant viral vectors have reduced or no immune response in the host. These viral vectors generated in the GDM are expected to provide good efficiency. The transgene expression depends on the promoter used, the target cells and the serotype used.
Mei Li, M.D. Ph.D
Gene Delivery Research Specialist
Vision Science Core, Gene Delivery Module
University of California, Berkeley
School of Optometry
592 Minor Hall
Berkeley, CA 94720