Figure 1

Illustration of a typical process to develop site-specific integration of transgene expressing human cell lines. In this process, two plasmids are essential. One plasmid contains the gene of interest and GFP gene with associated regulatory elements flanked by two copies of the AAV ITR. This construct contains the cis-acting IEE sequence from the P5 promoter, which is required for optimal site-specific integration. Another plasmid contains AAV rep gene providing an integration enzyme. HEK 293 cells were co-transfected with the two plasmids at ratio of 50: 1 using PEI. Transfected cells are subcloned into 96-well plates three times and screened by green fluorescence to establish the GFP expression human cell lines. The fluorescence-positive single cell clones were chosen for progressive expansions and further clone evaluations.