Figure 3.

Successful genome editing in fetal cardiac cells after TPGD for acquiring genome-edited fetuses (TPGD-GEF); DNA/lipid complex solutions containing plasmids encoding Cas9 and gRNA targeted to enhanced green fluorescent protein (eGFP) cDNA were injected into tail-veins of pregnant dams (at E12.5) containing EGFP transgenic fetuses. The white dashed boxes in (a) and (b) indicate heart. The heart exhibited strong fluorescence in wild-type (intact) fetuses (a), whereas fluorescence was greatly reduced in some fetuses of experimental group (b). Sequence analyses of PCR products (corresponding to the 5’ region of the eGFP sequence) from fetuses (b) revealed overlapping electrophoretograms (indicated by arrows) immediately upstream of the protospacer adjacent motif (PAM; c). These results indicate the presence of genome-edited and unedited sequences in fetuses with reduced fluorescence in heart tissues.