Generation of WT1-tdTomato knock-in cynomolgus monkey embryonic stem cell line, WT1-205 using CRISPR/CAS9-based gene targeting.

The WT1 gene is crucial for developing intermediate subtypes, including the kidney, gonad, and adrenal cortex. In this study, we generated a tdTomato knock-in cynomolgus embryonic stem cell line (cyESC) by inserting the tdTomato gene at the WT1 stop codon using CRISPR/Cas9 technology. The construct included a PGK-Neo selection cassette (LSL), which was excised by Cre recombinase. Differentiation into kidney- and gonadal-like cells showed tdTomato fluorescence co-localized with GATA4 and PAX2, markers for gonadal and kidney cells. WT1-positive cells also expressed related intermediate genes. This knock-in line provides a valuable tool for studying monkey intermediate mesodermal development.