Fig. 1

Generation and analysis of transgenic cattle via PiggyBac (PB)-mediated all-in-one vectors. A Schematic representation of the microinjection process for generating transgenic cattle. Two PB-mediated all-in-one vectors, PB-Cas9-RFP-FatI and PB-Cas9-GFP-sgPRNP, were microinjected into in vitro fertilized embryos along with a transposase vector to facilitate genomic integration. B Production of gene-edited calves and fluorescence observation in ear tissue-derived fibroblasts. C Integration of transgene in transgenic cattle. Genomic analysis showed successful transgene integration in all PB-Cas9-RFP-FatI cattle (a), while in PB-Cas9-GFP-sgPRNP cattle, integration was confirmed in calves #G1, #G3, #G4, #G6, and #G7 (b). D Cas9 mRNA expression levels in transgenic calves. end-point PCR results indicated Cas9 mRNA expression only in calves where transgene integration was confirmed (a: end-point PCR of Cas9 in PB-Cas9-RFP-FatI cattle, b: end-point PCR of GAPDH in PB-Cas9-RFP-FatI cattle, c: end-point PCR of Cas9 in PB-Cas9-GFP-sgPRNP cattle, d: end-point PCR of GAPDH in PB-Cas9-GFP-sgPRNP cattle). E Quantification of fluorescence-positive cells among transgenic cattle cells. The ratio of fluorescence-positive cells, indicative of successful gene editing and expression, was measured, demonstrating the effectiveness of the CRISPR/Cas9 system in creating transgenic models. (M, marker; WT, wild type; NC, negative control; PC, positive control)