Extended Data Fig. 8: Characterization of knockout human reporter cell lines.
From: Recapitulating the human segmentation clock with pluripotent stem cells
a, Overview of knockout reporter cell line generation for HES7, DLL3, LFNG and MESP2 genes. Positions of the sgRNAs used in this study are shown. sgRNAs were designed to target at or near regions of known pathogenic mutations, particularly those resulting in frameshifts and premature termination. Sequence analysis of iPS cell clones used in this study indicating insertion or deletion mutations generated by Cas9. Predicted effects on the protein sequence are listed below the sequence alignments. b, Damping rate of oscillation amplitude in knockout human PSMs. The signal of all area was averaged and detrended (± 100-min window). See also Fig. 3d for quantification of shown data, n = 3. c, Summary of results of oscillation and synchronization assays. See Fig. 3a–d for details. d, Flow cytometric evaluation of DLL1 expression at PSM stage of healthy control and knockout human iPS cell lines. Blue, isotype control; red, DLL1-APC. PSM induction efficiency is high in all analysed samples; slight reduction of DLL1 induction efficiency in LFNG-knockout cell lines. Representative results of three independent experiments of two different knockout lines for each gene are shown (HES7 KO #1 and #8, DLL3 KO #2 and #6, LFNG KO #2 and #12, and MESP2 KO #7 and #11); n = 3. e, Scatter plot of transcriptome analysis of wild-type and knockout cell lines at iPS cell and PSM stages. Positions of expression values for MESP2, DLL3, LFNG and HES7 are highlighted with coloured arrows. Data are averages of two biological replicates, n = 2.
