Figure 5.

Generation of an FP Reporter Line by Targeting FOXA2

(A) Donor DNA used for homologous recombination. Locations of primer sets for genotyping are marked with arrows.

(B) Targeting efficiency of the designed gRNA is verified in HEK 293FT cells after transient transfection of the gRNA and Cas9 expression vectors. Sequencing of the PCR-amplified genomic DNA surrounding the targeting site identifies overlapped peaks, which represents non-homologous end-joining repair after correct DNA targeting.

(C) Genotyping experiments show that seven out of nine randomly picked hESC colonies are successfully targeted. C, wild-type control; F1/R1, primer pair amplifies wild-type allele; F1/R2, primer pair amplifies homologous recombined allele.

(D) The heterozygous FOXA2-2A-eGFP line (colony #1) shows uniform GFP expression 12 days after SHH exposure with the AD differentiation paradigm, recapitulating endogenous FOXA2 expression. Scale bar, 50 μm.

(E) Colonies #3, #4, and #5 are differentiated for 12 days with the AD differentiation paradigm in the presence or absence of SHH. FOXA2 (48 kDa) is only expressed in the SHH-treated groups. FOXA2-2A-eGFP fusion protein (75 kDa) is expressed in colonies #4 and #5, and a large population of FOXA2-2A-eGFP is cleaved at the 2A site as shown by slightly slower shifting bands compared with the endogenous FOXA2.

(F and G) Removal of LDN193189 or SB431542 in the AD differentiation protocol does not affect FP specification as shown by similar GFP expression in a FOXA2-2A-eGFP reporter line induced by SHH. Scale bars, 50 μm.

(H) FOXA2-2A-eGFP reporter line is differentiated under EB conditions for 20 days without extra patterning morphogens. No GFP expression is observed under the fluorescence microscope. Scale bars, 50 μm.

(I) FOXA2-2A-eGFP reporter line is differentiated under EB conditions for 20 days and SHH is applied from day 10 to day 20. Only a small population of GFP-positive cells is seen under the fluorescence microscope. Scale bars, 50 μm.

(J) Confocal image shows that the FOXA2-positive cells derived from the EB differentiated cells are SOX2 negative, indicating a notochord fate rather than FP. Scale bar, 50 μm.

See also Figure S4.