Insulated piggyBac and FRT vectors for engineering transgenic homozygous and heterozygous eHAP cells.

Transgene expression in eHAP cells, a haploid cell line commonly used to generate gene knockouts, is difficult due to its low transfection efficiency and poor expression of integrated transgenes. To enable simple and reliable transgene expression, we engineered insulated integrating plasmids that sustain high levels of transgene expression in eHAP cells, and that can be used in other cell lines. These vectors are compatible with FLP-FRT and piggyBac integration, they flank a gene-of-interest bilaterally with tandem cHS4 core insulators, and co-express nuclear-localized blue fluorescent protein for identification of high expressing cells. We further demonstrate that transgenic haploid eHAP cells can be fused to form transgenic heterozygous diploid cells. This method creates diploid cells carrying the transgenic material of the haploid progenitors and allows for engineering of cells with defined heterozygous genotypes. These tools expand the range of experiments that can be performed in eHAP cells and other cultured cells.