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. Author manuscript; available in PMC: 2021 Jun 1.
Published in final edited form as: J Cell Physiol. 2019 Dec 16;235(6):5241–5255. doi: 10.1002/jcp.29411

Figure 1: Generation of a Noto-2A-mCherry reporter line in H9-hESC:

Figure 1:

(A-B) CRISPR mediated targeting of a Cherry reporter into the 3’UTR locus of the NOTO gene. H9-hESCs were genetically engineered to carry a cherry fluorescent protein reporter into the NOTO gene. In the targeting vector, the stop codon was removed and replaced with a viral 2A peptide followed by a cherry reporter coding sequence. This allows the gene product of NOTO to function properly and at the same time give accurate reporter expression to the endogenous gene. (C) After successful targeting and clonal selection, the Neo cassette is removed from different clonal lines by transient expression of Cre recombinase. (D) Characterization of NOTO-2A-mCherry knock-in in different clones of H9-hESCs. Precise mCherry integrated colonies present PCR products in selected clones.