Extended Data Fig. 8: Generation of an inducible apoptosis ACTC1 “kill switch” hPSC line.
From: Regenerating human skeletal muscle forms an emerging niche in vivo to support PAX7 cells
Related to Fig. 7a. a. Vector Maps of ACTC1 iCasp9 and Multiplex CRISPR/Cas9 (VectorBuilder) used for transgene insertion. Table of gRNA sequences, tracer sequence, 3′ and 5′ modifications for improved RNA stability were purchased from IDT. b. Gel shows all conditions used to knockin iCasp9 vector into parental H1 and H9 lines, N = 17. Lipo-Stem protocol from TF: Thermofisher, and nucleofection protocols from LZ: Lonza and SK: Skarns et al., were used. 5′ genomic DNA to 3′ genomic DNA spanning the region with the knockin was PCR’d. Asterisk indicates correct band size. c. After FACS sorted single cell plating and clonal expansion, Clones were collected and PCR’d for iCasp9 insertion, N = 108. Two heterozygous clones (C9 and C25) were identified (asterisk). Clones were then PCR’d for sequencing. d. Sequencing results (Retrogen) of parental, C9, and C25 clones show 100% alignment to expected sequence. Inset shows DNA sequence spanning the 3′ end of ACTC1 gene, T2A readthrough, and start of the iCasp9 transgene.
