Figure 3
From: A platform for rapid generation of single and multiplexed reporters in human iPSC lines
Generation of knock-in lines at lineage specific genes.
(a) Experimental strategy of generating MAP2-Nanoluc-KI. The designed ZFNs cut at the C-term of MAP2 gene before the stop codon and the left and right arms of MAP2 for homologous recombination were designed to be ~ 1 kb located before and after the stop codon, respectively. Testing primer sets for “Left” (MAP2 Left arm integration test), “Right” (MAP2 Right arm integration test) and “ORF” (WT MAP2 ORF test) were also illustrated. (b) Experimental strategy of generating GFAP-Nanoluc-KI. The designed ZFNs cut after the stop codon of GFAP ORF. The left and right arms of GFAP for homologous recombination were designed to be ~ 1 kb located before and after the stop codon, respectively. Testing primer sets for “Left” (GFAP Left arm integration test), “Right” (GFAP Right arm integration test) and “ORF” (WT GFAP ORF test) were also illustrated. (c) Validation of MAP2-Nanoluc-KI clone by junction PCR (upper) and sequencing (lower). (d) Validation of GFAP-Nanoluc-KI clone by junction PCR (upper) and sequencing (lower).
