Wang G., Yang L., Grishin D., Rios X., Ye L.Y., Hu Y., Li K., Zhang D., Church G.M., Pu W.T.Applications of piggyBac transposons for genome manipulation in stem cells. A piggyBac-based toolkit for inducible genome editing in mammalian cells. Schertzer M.D., Thulson E., Braceros K.C.A., Lee D.M., Hinkle E.R., Murphy R.M., Kim S.O., Vitucci E.C.M., Calabrese J.M.YAP inhibition promotes endothelial cell differentiation from pluripotent stem cell through EC master transcription factor FLI1. Quan Y., Shan X., Hu M., Jin P., Ma J., Fan J., Yang J., Zhang H., Fan X., Gong Y., et al.Genetic engineering of human pluripotent stem cells using PiggyBac transposon system.

Enzymatic assembly of DNA molecules up to several hundred kilobases. Gibson D.G., Young L., Chuang R.Y., Venter J.C., Hutchison C.A., 3rd, Smith H.O.

A rapid and highly efficient method for the isolation, purification, and passaging of human-induced pluripotent stem cells. At the same time, we did not do multiple genes (more than two) overexpression at the same time, or the donor cassette removal which could refer to others ( Wang et al., 2017). However, we did not perform the gene overexpression in other stem cells, such as cancer stem cells. PiggyBac transposon system is broadly used tool that allows DNA cargos inserted into genome in “AATT” sequence with high efficiency, which has been applied in gene overexpression, knockdown, genome editing in various mammalian cells, including stem cell ( Schertzer et al., 2019 Sun et al., 2021). Compared to the other methods to overexpress of specific genes, such as plasmid DNA transfection, retroviral- or lentiviral-based infection, electroporation, the PiggyBac transposon system is technically simpler and higher efficiency, it has a large cargo capacity (up to 10 KB) with multiple genes expression at the same time, and the donor cassette can be excised using excision-only PiggyBac transposase in scarless fashion if we do not need them later. The piggyBac transposon system provide us a technically easy approach to overexpression of specific genes in hPSC. The aim of this protocol is to construction inducible gene(s) overexpression to study the function of gene in hPSCs, such as differentiation regulation of hPSCs.

Phusion High-Fidelity DNA Polymerase with dNTPs Chemicals, peptides, and recombinant proteins

Snapgene p2a