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In recent years, CRISPR/Cas9 has become known as a powerful tool for generating genetically modified cells or animal models with its site-specific gene editing capability. Because myostatin (MSTN) is associated with muscle differentiation and growth, ablation of MSTN would be ideal for studying phenotypes associated with muscle growth in beef cattle. The aim of this study is to produce Korean beef cattle with MSTN mutation through microinjection of CRISPR/Cas9. For this study, ovaries of Korean beef cattle were obtained from slaughterhouses; 122 immature oocytes were aspirated from 8 Korean beef cattle. The oocytes were matured in tissue culture medium 199-based medium for 22h and inseminated with frozen-thawed semen to produce zygotes. All 122 zygotes were directly injected with 200ng of Cas9 mRNA and 100ng of sgRNA for MSTN after IVF and were cultured for 7 days in two-step chemically defined medium. Each embryo from the individual cattle was cultured separately to distinguish its lineage. After 7 d, 29 (23.8±8.9) blastocysts were recovered, and all blastocysts were assessed with T7 endonuclease (T7E1) mutation assay to check for mutations at the MSTN locus. Based on T7E1, 26 out of 29 blastocysts (89.7±10.5%) had heterozygous mutation in the MSTN gene. After analysis, additional blastocysts were produced and transferred into 14 surrogates. All surrogates were diagnosed for pregnancy via rectal palpation and ultrasonography at Day 55, at which time normal fetuses and embryonic sacs were confirmed in 10 surrogates. In conclusion, these data demonstrate that CRISPR/Cas9 could be employed as an efficient method to genetically modify the MSTN gene in Korean beef cattle.

Interferon tau (IFNT), a type I interferon, is known as a key signal molecule during pregnancy in ruminants because of the necessity in maternal recognition of pregnancy. It is produced in trophectoderm cells of the elongation bovine conceptus at Day 13-21, and peak production is at Day 15 to 17 of pregnancy. In addition, other studies show that it can affect embryonic development and quality. In this study, the effect of IFNT knockout in donor cells to bovine cloned embryonic development by somatic cell nuclear transfer (SCNT) was investigated. To proceed with this experiment, immature oocytes from ovaries at a local slaughterhouse were matured in vitro for 22h. To prepare the donor cell with IFNT knockout, somatic cells were transfected with Cas9 and single guide RNA targeting IFNT, and several single derived colonies with high proliferation were isolated for mutation assay. Finally, one colony that had mono-allelic mutation (4bp deletion) was selected and used as the donor cell for SCNT. A donor cell was injected into an enucleated oocyte. Reconstructed oocytes with the donor cell were fused by electrical shock, activated by chemical stimulation, and cultured for 7 days in chemically defined medium. For this study, control (n=94) and IFNT knockout groups (n=140) were compared with 4 replications. The results showed no significant difference between control and IFNT knockout groups not only in cleavage rate, but also in blastocyst formation rate (control: 15.7±8.3%, IFNT knockout group: 26.3±13.1%). In addition, the number of blastocyst cell was not different between control (88.2±27.0) and IFNT knockout group (88.0±21.1). Some IFNT mutated blastocysts from SCNT were randomly selected for confirmation of the deletion of IFNT, and all samples were positive for mutation. In conclusion, these data demonstrated that the disruption of IFNT did not affect embryonic development. In future study, we would transfer these embryos and check the effect of IFNT during pregnancy status. This work was supported by BK21 PLUS Program for Creative Veterinary Science, the National Research Foundation of Korea (2017R1A2B3004972), and the Technology Development Program (S2566872) by MSS.

The p53 gene is a tumour suppressor gene with many roles in cellular reprogramming, senescence, or cancers. Knockdown of p53 in somatic cells improves generation of induced pluripotent stem cells and developmental competence of cloned embryos. In this study, we hypothesised that disruption of p53 in bovine somatic cells using CRISPR-Cas9 may improve reprogramming and result in increased blastocyst formation. For the study, ovary-derived immature bovine cumulus-oocyte complexes were matured in vitro. To prepare the donor cells with disruption of p53, primary cells from the skin tissue were transfected with spCas9 and single guide RNA for bovine p53. Two days later, genomic DNA was extracted and the mutation was evaluated on the target region using the T7E1 enzyme. Several single cell colonies were isolated to investigate the mutation and sequencing for mutation positive colonies was performed. One colony with high proliferative activity and heterozygotic mutation (−7bp deletion) was selected and used as the donor cell for somatic cell nuclear transfer. The donor cell was microinjected into an enucleated oocyte. Reconstructed oocytes were fused by electrical pulse, activated by chemical stimulation, and cultured in chemically defined media for 7 days at 5% CO2 and 5% O2. Data were subjected to ANOVA and least significant different test to determine differences between experimental groups by using SAS software (SAS Institute Inc., Cary, NC, USA). Statistical significance was determined when the P-value was less than 0.05. Blastocyst formation rate (13.5±7.6%) from the mutated cell line was significantly lower than wild type cells (43.9±12.1%). Moreover, total cell number (115.4±13.3) of the blastocysts from knockout cells was significantly decreased compared with the control group (143.6±22.5). Some randomly selected blastocysts derived from mutated cells were subjected to mutation assay to confirm deletion of p53. As expected, all the samples were positive to mutation. These data did not support our hypothesis that p53 disruption could improve blastocyst formation competence of bovine cloned embryos. In the future, the reason for impaired embryonic development of clones of p53 deleted somatic cells will be further investigated. This work was supported by BK21 PLUS Program for Creative Veterinary Science, the National Research Foundation of Korea (2017R1A2B3004972), and the Technology Development Program (S2566872) by MSS.

A large number of oocytes and adequate recipient conditions are required to produce genetically modified rats by embryo transfer. In this study, we investigated the effect of a synthetic gonadotropin-releasing hormone (GnRH) agonist, buserelin, on (1) superovulatory response in donor rats, and (2) oestrous cycle synchrony in recipient female Sprague Dawley rats at 8 weeks of age. First, for superovulation, donors were divided into 2 groups: control and buserelin injection group (42 μg mL−1). Superovulation was performed in both groups by injection of pregnant mare’s serum gonadotropin (PMSG, 150 IU) at Day −3 and human chorionic gonadotropin (hCG, 150 IU) at Day −1 (Day 0 = oocyte collection). In addition, the buserelin group was given 42 μg mL−1 buserelin on Day −4. Oviduct and ampulla were removed surgically to collect the oocytes. As results, more ovulations were detected in the 42 μg mL−1 buserelin group (6.18 ± 1.10 oocyte/rat, n = 240) than in the control group (5.06 ± 0.82 oocyte/rat; n = 124; t-test P = 0.038). Second, 2 groups were set up to investigate the effect of administration of buserelin on synchronizing the oestrus cycle of recipients. Because the oestrous cycle of the rat was 4 to 5 days, we examined the difference of vaginal impedance between the 2 groups on Day 4 after injection and checked whether there was a significant difference in the synchronization of the oestrous cycle. An impedance value >3.0 kΩ was considered as an indication for proestrus stage; therefore, we analysed the data >3.0 kΩ in both groups. In results, the incidence of proestrus was significantly increased in buserelin injection group (n = 128) than in the control group (n = 70). In addition, there was a significant difference in mean impedance in the 2 groups (buserelin injection group: 4.25 ± 3.79 kΩ; control group: 1.43 ± 1.20 kΩ; P

The CRISPR/Cas9 system has proved to be a powerful tool for knockout and knock-in in various species. When 2 components—Cas9 and single guide (sg)RNA—are delivered into cells or embryos, the events of gene editing occur. Because Cas9 is essential for gene editing in the CRISPR/Cas9 system, some studies have reported the production of Cas9-expressing animals, such as mice, which could be used to increase gene editing efficiency in subsequent experiments. In previous reports, we successfully produced 4 Cas9-expressing cattle via microinjection (Hahn et al. 2016 Reprod. Fertil. Dev. 29, 211). Primary cells from these calves had Cas9 activity because transfection of only sgRNA resulted in gene deletion. The aim of this study was to analyse the blood of the transgenic cattle to investigate the effect of Cas9 expression on health. Two of 4 transgenic calves died; one had severe ruminant tympany, failed to respond to treatment, and died at 4 months of age, and the other died at 5 months of age due to accidental ingestion of a needle from a feed bunk. Blood samples were obtained from the surviving 2 transgenic cattle (1 male and 1 female) at 7 and 12 months for blood analysis. Five milliliters of whole blood samples was collected from the jugular vein. Portions were used for CBC (Hemavet 950, Drew Scientific, Miami Lakes, FL, USA) and for serum chemistry analysis (BS-400, Mindray, Shenzhen, China). Average values for white blood cells (9600 and 1057/mm3), neutrophils (4590 and 3870/mm3), lymphocytes (4020 and 5910/mm3), red blood cells (732,000 and 798,000/mm3), hemoglobin (9.5 and 10.2 g dL−1), packed cell volume (24.3 and 25.3%), platelet (439,000 and 327,500/mm3), AST (76 and 104 IU), ALP (140 and 133 IU), BUN (7.5 and 10.5 mg dL−1), and creatinine (1.3 and 1.0 mg dL−1) of male and female transgenic calves were within the reference range. Additionally, there was no difference in general health information, including body temperature and feeding. In conclusion, we demonstrated that continuous Cas9 expression in transgenic cattle did not affect health status of the surviving calves in terms of blood analysis. They have grown up without any health issues and are currently 14 (female) and 15 (male) months old. In the near future, we will evaluate their germline transmission by natural breeding or in vitro fertilization. This work was supported by BK21 PLUS Program for Creative Veterinary Science, NRF (NRF-2017R1A2B3004972), and Seoul Milk Coop (SNU 550–20160004).

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).

A genome-editing technology, CRISPR/Cas9 system is proved to be a powerful tool for knockout and knock-in in various species. When 2 components [Cas9 and single guide (sg) RNA] are delivered into cells or embryos, the events of gene editing occur. Because Cas9 is essential for every gene editing based on the CRISPR/Cas9 system, some studies reported that efficiency of gene editing would be increased as Cas9 was integrated into cells or animals. Accordingly, if the Cas9-expressing cattle is born, it would be broadly used for gene editing in cattle. For this study, the Cas9 and RFP genes were cloned into the PiggyBac transposon system. PiggyBac-Cas9-RFP and transposase were microinjected into 1436 IVF embryos and 241 blastocysts were formed. Blastocysts with RFP expression accounted for 14.1% of total formed blastocysts. Five blastocysts were selected and transferred into 5 recipient cow (1 embryo per recipient). After gestation periods, 4 transgenic cattle were delivered without any veterinary assistance. From transgenic cattle, ear skin tissue was collected for primary culture. On those primary cells, sgRNA in DNA form for various genes such as PRNP, RB1, and BLG were transfected with 2 μg of sgRNA per 5 × 105 cells using electroporation. As expected, every group of each sgRNA delivered was confirmed to be mutated by T7E1 assay. The data demonstrated that, for the first time, transgenic cattle with Cas9 expression were born, grown up to date (age = 5 months) and will be a valuable resource for genome editing in cattle. This work was supported by BK21 PLUS Program for Creative Veterinary Science and Seoul Milk Coop (SNU 550–20160004).

Site-specific recombinases (SSR), such as Cre and Flp recombinases, which enable DNA excision, insertion, and translocation, have been used for conditional target gene expression in mouse and other vertebrates. In this study, we evaluated another SSR, Dre-recombinase (Dre), which is functionally similar to Cre recombinase in porcine fibroblasts and embryos. For this study, 2 fragment DNA constructs (rox GFP-polyA and rox RFP-polyA) were combined with piggybac transposition expression vector (Kim et al. 2011 J. Vet. Med. Sci.) using a multisite gateway cloning system (MultiSite Gateway® Pro, Invitrogen, Carlsbad, CA, USA). The expression vector carrying rox-flanked green fluorescent protein (GFP) followed by red fluorescent protein (RFP) and transposase were transfected into kidney-derived porcine cells by nucleofection (Neon® Transfection System, Invitrogen). A GFP-expressing cell line, which was not expressing RFP, was established. And then rox-flanked GFP were removed by Dre transfection and RFP was expressed in the kidney cells. At the cellular level, this excision was confirmed by site-specific RT-PCR and sequencing. The rox-flanked GFP cells were reconstructed with enucleated oocytes and then the cloned embryos were cultured in porcine zygote medium-5. Dre was micro-injected into 1 of the 2-cell-stage blastomeres. After 6 days, RFP expression was observed on the part of embryos after microinjection. In conclusion, the data demonstrated that, like other SSR, Dre might be applied in conditional target gene expression for generating porcine biomedical models.