Skip to main content
. 2023 Dec 18;12(24):2860. doi: 10.3390/cells12242860

Figure 1.

Figure 1

Generation of LMX1A-Cre/AAVS1-BFP tracing lines. (A) Schematic illustration of the wild type LMX1A and PPP1R12C (AAVS1) loci and targeting strategy. Exons are indicated as black rectangles. left: LMX1A targeting, the two gRNAs targeting the 3′UTR of LMX1A are indicated in blue with PAM region in red. The P2A-Cre-pA and Neo-pA expression cassettes are flanked by the homologous arms (HA) corresponding to exon 8 immediately upstream of the stop codon and 3′UTR (grey), respectively. Right: AAVS1 targeting, the two gRNAs targeting intron 1 of AAVS1 were indicated in blue with PAM region in red. The homologous arms (HA), indicated in grey, flank a PAC-pA selection cassette and BFP-pA fluorescent tag gene. (B) Mechanism of BFP activation driven by LMX1A expression. (C) Genomic PCR screening strategy for detecting the targeted clones at the AAVS1 and LMX1A locus, respectively. (D) Examples of agarose gels showing amplicons for the WT and HR allele. (E) Targeting efficiency for AAVS1 and LMX1A locus, respectively. (F) Bright field and fluorescent images of day 10 mDA differentiation cultures showing BFP expression in live cells. Scale bar 50 μm. (G) Representative bright field images of iPSC colonies and (H) immunostaining of pluripotency markers OCT4 and TRA1-81 with DAPI counterstain for the parental KOLF2 and A17L25 and A17L35 LMX1ACre-BFP lines. Scale bar 50 μm. Karyotyping results are indicated on the left below the corresponding iPSC lines.