Next Generation Sequencing of human trophoblast stem cells expressing a doxycycline-inducible dominant-negative MAML1 construct.
Mastermind-like 1 (MAML1) functions as a co-activator in the NOTCH signaling pathway. A truncated version of its wild type form, the so-called dominant-negative MAML1 (DN-MAML1) , lacks the transactivation domains that are needed for stimulation of gene expression. The aim of the study was to characterize the effects of NOTCH signaling pathway inhibition in human trophoblast stem cells (TSCs) by overexpression of DN-MAML1. Overall design: To examine gene expression patterns of human TSCs with wild-type or inhibited NOTCH signaling, TSCs were genetically modified by piggyBac-transposase-dependent transfection, that stably integrated a doxycyclin-inducible DN-MAML1 construct into the genome. The transfection was performed using lipofection. Three clonal TSC lines expressing DN-MAML1 were cultured further on fibronectin-coated cell culture dishes in a culture medium promoting trophoblast stemness. The transcriptomes of TSCs overexpressing DN-MAML1 by 6 days of doxycycline stimulation were compared with non-stimulated TSCs, that still have wild-type NOTCH signaling activity. As a control for possible off-target-effects, three TSC lines were also cultured with or without doxycycline for 6 days. The analysis comprise in total 12 samples: three genetically modified TSC lines and three wild-type TSC lines, cultured with and without doxycycline, respectively.