Fig. 1: PRDM14-Venus knock-in reporters allow PRDM14 detection in hESCs and hPGCLCs.

a Scheme of CRISPR/Cas9-mediated PRDM14 locus targeting to generate T2A-Venus, AID-Venus or JAZ-Venus reporter versions. 5′ and 3′ arms—homology sequences, T2A—self-cleaving peptide, AID—auxin-inducible degron, Venus—fluorescent gene, Rox—sequences for site-specific recombination recognised by the Dre enzyme, PGK-Puro—puromycin resistance gene under the control of PGK promoter, ΔTK—truncated thymidine kinase gene, MC1-DTA—diphtheria toxin fragment A gene under the control of MC1 promoter. Also see Fig. 3a. b, c Flow cytometry analysis showing Venus fluorescence in targeted hESCs and hPGCLCs compared with negative control. Note that Venus fluorescence predominantly coincides with NANOS3-tdTomato signal, which marks hPGCLCs. d qPCR analysis on sorted PRDM14-T2A-Venus⁺AP⁺ and double-negative cells from D4 EBs. Venus⁺AP⁺ population shows specific expression of germ cell markers. Data show results from three technical replicates (also see Source Data file). e IF analysis (representative of >10 experiments) of PRDM14-AID-Venus in competent hESCs showing co-localisation of PRDM14 and Venus fluorescence. Nuclei were counterstained by DAPI. Scale bar is 100 μm.