Lentiviral Vectors and Protocols for Creation of Stable hESC Lines for Fluorescent Tracking and Drug Resistance Selection of Cardiomyocytes
Key findings
Suite of lentiviral vectors for fluorescent tracking and drug-based purification of hESC-derived cardiomyocytes. Core architecture uses the PGK promoter (−528 to −13 bp) for ubiquitous nuclear H2B-fluorescent reporters, αMHC for cardiomyocyte-selective expression, and Rex-1 for hESC-stage selection. Dual selection (G418/Blasticidin + Puromycin at day 12) yielded >96% pure cardiomyocyte populations. Key finding: unsorted lentiviral populations progressively silence transgene expression from >80% to <5% positive cells by passage 6, necessitating FACS enrichment or clonal selection for stable long-term reporter lines.