204 POTENTIAL OF GREEN FLUORESCENT PROTEIN LOCUS FOR GENE EDITING IN DNA TRANSPOSON-PRODUCED TRANSGENIC CATTLE

Abstract

Recently, we published on the efficient production of transgenic cattle using the DNA transposon system (Yum et al. 2016 Sci. Rep. 6, 27185). In that study, 8 transgenic cattle were born following transposon-mediated gene delivery system (Sleeping Beauty and Piggybac transposon) via microinjection of zygotes. In the analysis of their genomic stability using next-generation sequencing, there was no significant difference in the number of genomic variants between transgenic and nontransgenic cattle. In this study, we have described current status of those transgenic cattle in term of health, germ-line transmission, and application. All the transgenic cattle have grown up to date (the oldest being 30 months old, the youngest being 12 months old) without any health issue. In general blood analysis, there were not any significant changes between transgenic cattle and wild type. Because the transgene (green fluorescent protein; GFP) expression is constitutively active and has strong expression, it could be visualised without fluorescence equipment. One of transgenic male cattle reached puberty and semen was collected. Over 200 frozen semen straws were produced and some were used for IVF. In every IVF replication, around 80% blastocysts expressed the GFP. Over 36 GFP blastocysts were frozen for embryo transfer in the future, and we are planning to crossbreed for generating homozygotic transgenic cattle. Another application is to use the GFP locus to gene-edit the transgenic cattle, as long-term expression of transgene did not affect their health. In 1 cell stage, embryos produced using GFP frozen-thawed semen, single guide RNA for GFP, Cas9, together with donor DNA that included RFP and homology arms to link the double-strand break of single guide RNA target site, were co-injected and RFP was observed. Knockout/-in for editing GFP locus using CRISPR-Cas9 might be a valuable approach for the next generation of transgenic models by microinjection. In conclusion, we demonstrated that transgenic cattle via transposon are healthy to date and germ-line competence was confirmed. The GFP locus will be used as the target region for future gene engineering via genome-editing technology. Finally, all those animals could be a valuable agricultural and veterinary science resource for studying the effects of gene manipulation on disease resistance and food production. This work was supported by BK21 PLUS Program for Creative Veterinary Science and Seoul Milk Coop (SNU 550–20160004).