Fig. 2

Developmental and genetic analysis of F1 PB-Cas9-RFP-FatI cattle expressing RFP and Cas9 proteins. A Representative images showing Development Day 4 (a and a') and Day 7 (b and b') embryos fertilized using frozen/thawed sperm from a F0 transgenic male bull (#R2). RFP expression was monitored, and the successful gene expression was confirmed early in development. B RFP expression in primary cells from F1 PB-Cas9-RFP-FatI cattle. Image demonstrating RFP expression in primary cells derived from all transgenic calves, which confirms the persistence of transgene expression. C Cas9 sequence locus amplified by PCR, showing transgene integration in the genome of the calves, verifying that genetic modification was successfully inherited. D Western blot results showing the presence of Cas9 protein in samples from all calves, which confirms ongoing expression of Cas9 for intended gene editing functions