Computationally defined and in vitro validated putative genomic safe harbour loci for transgene expression in human cells

Selection of the target site is an inherent question for any project aiming for directed transgene integration. Genomic safe harbour (GSH) loci have been proposed as safe sites in the human genome for transgene integration. Although several sites have been characterised for transgene integration in the literature, most of these do not meet criteria set out for a GSH, and the limited set that do have not been characterised extensively. Here, we conducted a computational analysis using publicly available data to identify 25 unique putative GSH loci that reside in active chromosomal compartments. We validated stable transgene expression and minimal disruption of the native transcriptome in three GSH sites in vitro using human embryonic stem cells (hESCs) and their differentiated progeny. Furthermore, for easily targeted transgene expression, we have engineered constitutive landing pad expression constructs into the three validated GSH in hESCs.

Methods
This dataset contains high content imaging data of Pansio-1, Olônne-18, and Keppel-19 H9 Clover hESC clones differentiated into neuronal, hepatic, and cardiac cell types and stained for lineage marker TUJ1, HNF4alpha, and cardiac Troponin-T. The cells were imaged on CellCarrier Ultra 96-well plates using a PerkinElmer Opera Phenix confocal imager with a 20x objective. 61 fields were imaged for each well and the channels Alexa 488, Alexa 594, and HOECHST 33342 were recorded. Image analysis was conducted on PerkinElmer Columbus software to look for overlap of Clover transgene and positive lineage markers.