Chromatin Insulator Elements Block Transgene Silencing in Engineered Human Embryonic Stem Cell Lines at a Defined Chromosome 13 Locus

Lineage reporters of human embryonic stem cell (hESC) lines are useful for differentiation studies and drug screening. Previously, we created reporter lines driven by an elongation factor 1 alpha (EF1α) promoter at a chromosome 13q32.3 locus in the hESC line WA09 and an abnormal hESC line BG01V in a site-specific manner. Expression of reporters in these lines was maintained in long-term culture at undifferentiated state. However, when these cells were differentiated into specific lineages, reduction in reporter expression was observed, indicating transgene silencing. To develop an efficient and reliable genetic engineering strategy in hESCs, we used chromatin insulator elements to flank single-copy transgenes and integrated the combined expression constructs via PhiC31/R4 integrase-mediated recombination technology to the chromosome 13 locus precisely. Two copies of cHS4 double-insulator sequences were placed adjacent to both 5′ and 3′ of the promoter reporter constructs. The green fluorescent protein (GF...