Strategy for generating transgenic mir-302s-expressing mirPS cell lines, using retrovirus-based pLNCX2-rT-SpRNAi vector transfection. A retroviral delivery approach was used to integrate a cytomegalovirus (CMV) promoter-driven SpRNAi-RGFP transgene into the tested cell genomes for steady expression of a manually redesigned mir-302 pre-miRNA cluster (mir-302s). Mir-302s was placed in the intron of the SpRNAi-RGFP transgene and generated as a part of the transgene transcript RNA (pre-mRNA), containing RGFP protein-coding exons and noncoding introns. The introns were spliced out of pre-mRNA and further excised into small miRNA-like mir-302 molecules capable of triggering targeted gene silencing, while the RGFP exons were ligated together to form a mature mRNA for synthesis of a red fluorescent marker protein, RGFP. The presence of RGFP served as an indicator for mir-302s expression and processing.
