Figure 3. Differentiation of SPC25+ from SPC25- PrC cells with genetic manipulation. (A) The DU145 and LNCap cell lines were transduced with 2 AAVs. The first AAV carries a luciferase and a mCherry fluorescent reporter under the control of a cytomegalovirus (CMV) promotor. The luciferase and mCherry reporters are connected by a p2A sequence to allow co-expression of 2 genes by one promoter with similar efficiency. Transduction of the cells with this AAV makes the cells red fluorescent to be sortable by flow cytometry and traceable in vivo by bioluminescence assay. The second AAV carries a nuclear green fluorescent protein (nGFP) reporter under the control of a SPC25 promoter. Transduction of the cells with this AAV makes the SPC25+ cells green fluorescent in the nuclei to be sortable by flow cytometry. Co-transduction of the cells with these 2 AAVs resulted in two populations of interest. Population 1, red fluorescent (expressing mCherry but not nGFP) cells represent SPC25- cells. Population 2, yellow fluorescent (expressing both mCherry and nGFP) cells represent SPC25+ cells. (B) The flow cytometry analysis on infected DU145 and LNCap cells. (C) The purified transduced cells were examined for fluorescence in culture. (D) RT-qPCR for SPC25 in different cell fractions. (E) Flow cytometry for CD133 in SPC25+ and SPC25- fractions. *p<0.05. N=5. Scale bars were 20 µm.