Recently, we published a microinjection method for generating transgenic cattle using the DNA transposon system and their analysis by next-generation sequencing (Yum et al. Sci Rep. 2016 Jun 21;6:27185). In that study, we generated transgenic cattle using two different types of DNA transposon system, sleeping beauty (SB) and piggybac (PB), carrying Yellow fluorescent protein with SB (SB-YFP, female) and green fluorescent protein with PB (PB-GFP, male) under the control of the ubiquitous CAG promoter, respectively. The female and male founder cattle have been grown up to date (the female age: 40 months old, the male age: 33 months old) without any health issues. In genomic instability and blood analysis, there was no significant differences between wild type and founder cattle. In the present study, we confirmed germ-line transmission of the transposon-mediated transgene integrations and ubiquitous and persistent expression of transgene in second generation of offspring (F1). The F1 was born without any assistance and expressed GFP in the eyes without UV light. The ubiquitous expression of GFP was detected in skin fibroblast from the ear tissue and confirmed by genomic DNA PCR, which suggest that the transgene from the PB-GFP was successfully transmitted. Unfortunately, no transgene from SB-YFP were identified. To confirm the transgene integration site, the genomic DNA from blood was extracted and performed next-generation sequencing (NGS). The GFP gene was integrated in chromosome 4 (two copies), and 6. As results, a total of two copies of paternal transgene transmitted into the F1. All the integrated position was not related with coding region and there was no significant difference in genomic variants between transgenic and non-transgenic cattle. To our knowledge, this is the first report of germ-line transmission through non-viral transgenic founder cattle. Those transgenic cattle will be valuable resource to many fields of biomedical research and agricultural science.

The CRISPR/Cas9 system is proved to be a powerful tool for knock-out and knock-in in various species. By introducing genetic materials of two components (Cas9 and small guide (sg) RNA) into cells or pronuclear of the fertilized embryo, gene editing occurs. Some studies reported that efficiency of gene editing would be increased as Cas9 was integrated into cells or animals since Cas9 is indispensable in the CRISPR/Cas9 system. Accordingly, the production of Cas9 expressing cattle may provide the broadly used gene editing platform in cattle. For this study, Cas9 and RFP genes were cloned into PiggyBac (PB) transposon system. PB-Cas9-RFP and transposase were microinjected into 1436 in vitro fertilized embryos and 241 blastocysts were formed. Blastocysts with RFP expression accounting for 14.1% of total formed blastocysts were selected and transferred into 5 recipient cow. After gestation periods, four transgenic cattle were delivered without any veterinary assistance. From a transgenic cattle, ear skin tissue was collected for primary culture. On those primary cells, sgRNAs in DNA form for various genes such as PRNP, RB1 and BLG were transfected as 2ug of sgRNA per 5x105 cells using Nucleo factor system (Neon®, invitrogen, program#16). As expected, every group of each sgRNA delivered was confirmed to be mutated by T7E1assay. Those data demonstrated that for the first time, transgenic cattle with Cas9 expression were born, grown up to date and will be avaluable resource for genome-editing in cattle. This work was supported by BK21PLUS Program for Creative Veterinary Science and Seoul Milk Coop (SNU550-20160004).