Seok-Hwan Song

Coagulansin-A has beneficial effects on the development of bovine embryos in vitro via HSP70 induction

Treatment with the steroidal lactone, coagulansin-A, improves bovine oocyte maturation and embryo development in vitro by inducing heat shock protein 70 (HSP70), which reduces the levels of reactive oxygen species (ROS), DNA damage and inflammation.

Polydatin improves the developmental competence of bovine embryos in vitro via induction of sirtuin 1 (Sirt1)

The aim of the present study was to investigate the beneficial effect of polydatin (PD), the glycoside form of resveratrol, on embryo development in vitro. Oocytes were aspirated from ovaries of Korean Hanwoo cows and cultured until Day 8 in a humidified atmosphere of 5% CO2 in air at 38.5°C. Protein and gene expression levels were determined through confocal microscopy and reverse transcription-polymerase chain reaction respectively, whereas the number of total and apoptotic cells in Day 8 blastocysts was determined using Hoechst 33342 staining and terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling. Of the different concentrations of PD (0.5, 1.0 and 2.0µM) added to the IVM medium, only 1.0µM PD significantly improved blastocyst development. Immunofluorescence analysis confirmed that protein levels of sirtuin 1 (Sirt1) increased significantly (P<0.05) after PD treatment, whereas levels of reactive oxygen species (ROS) were significantly (P<0.05) decreased, as evidenced by reductions in 8-oxoguanine immunoreactivity. Similarly, protein levels of nuclear factor (NF)-κB and cyclo-oxygenase (COX)-2 were significantly (P<0.05) lower in the PD-treated group than in the control group. Treatment with 1.0µM PD reduced gene expression of BCL2-associated X protein, inducible nitric oxide synthase, COX2 and Nfkb, but increased the expression of Sirt1, supporting the immunofluorescence data. PD possesses antioxidant activity and is useful for embryo development in vitro. We conclude that supplementation of IVM medium with PD improves embryo developmental competence via Sirt1.

Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. The introduced genetic material may integrate into the embryonic genome and generate transgenic animals with foreign genetic information following transfer of the injected embryos to foster mothers. Following the success in mice, PN injection has been applied successfully in many other animal species. Recently, PN injection has been successfully employed to introduce reagents with gene-modifying activities, such as the CRISPR/Cas9 system, to achieve site-specific genetic modifications in several laboratory and farm animal species. In addition to mastering the special set of microinjection skills to produce genetically modified animals by PN injection, researchers must understand the reproduction physiology and behavior of the target species, because each species presents unique challenges. For example, golden Syrian hamster embryos have unique handling requirements in vitro such that PN injection techniques were not possible in this species until recent breakthroughs by our group. With our species-modified PN injection protocol, we have succeeded in producing several gene knockout (KO) and knockin (KI) hamsters, which have been used successfully to model human diseases. Here we describe the PN injection procedure for delivering the CRISPR/Cas9 complex to the zygotes of the hamster, the embryo handling conditions, embryo transfer procedures, and husbandry required to produce genetically modified hamsters.

Characterization of an N-Terminal Non-Core Domain of RAG1 Gene Disrupted Syrian Hamster Model Generated by CRISPR Cas9

The accumulating evidence demonstrates that Syrian hamsters have advantages as models for various diseases. To develop a Syrian hamster (Mesocricetus auratus) model of human immunodeficiency caused by RAG1 gene mutations, we employed the CRISPR/Cas9 system and introduced an 86-nucleotide frameshift deletion in the hamster RAG1 gene encoding part of the N-terminal non-core domain of RAG1. Histological and immunohistochemical analyses demonstrated that these hamsters (referred herein as RAG1-86nt hamsters) had atrophic spleen and thymus, and developed significantly less white pulp and were almost completely devoid of splenic lymphoid follicles. The RAG1-nt86 hamsters had barely detectable CD3+ and CD4+ T cells. The expression of B and T lymphocyte-specific genes (CD3γ and CD4 for T cell-specific) and (CD22 and FCMR for B cell-specific) was dramatically reduced, whereas the expression of macrophage-specific (CD68) and natural killer (NK) cell-specific (CD94 and KLRG1) marker genes was increased in the spleen of RAG1-nt86 hamsters compared to wildtype hamsters. Interestingly, despite the impaired development of B and T lymphocytes, the RAG1-86nt hamsters still developed neutralizing antibodies against human adenovirus type C6 (HAdV-C6) upon intranasal infection and were capable of clearing the infectious viruses, albeit with slower kinetics. Therefore, the RAG1-86nt hamster reported herein (similar to the hypomorphic RAG1 mutations in humans that cause Omenn syndrome), may provide a useful model for studying the pathogenesis of the specific RAG1-mutation-induced human immunodeficiency, the host immune response to adenovirus infection and other pathogens as well as for evaluation of cell and gene therapies for treatment of this subset of RAG1 mutation patients.

210 GENERATION OF APOBEC3CH AND APOBEC3H DOUBLE-KNOCKOUT CATS BY SITE-SPECIFIC GENE TARGETING

The domestic cat (Felis catus) is a useful animal model for biomedical research because it shares many devastating diseases with humans. For example, feline immunodeficiency virus-1 is structurally and functionally similar to human immunodeficiency virus-1 (HIV-1). In vitro, the replication of HIV-1 in cat cells is restricted by the feline APOBEC3H (fA3H) and APOBEC3CH (fA3CH). Accordingly, we hypothesised that cats could be used to study HIV-1 infection in humans if fA3H and fA3CH were knocked out. However, due to limited availability of genomic editing tools in the cat, genetic modification has not been widely reported in this species. Here, we show that the fA3H/fA3CH locus could be knocked out in cat using the CRISPR/Cas9 system. Taking advantage of the fact that the fA3H and fA3CH genes share their last four exons (exons 2-5 for fA3H and exons 5-8 for fA3CH), we PCR-amplified and analysed the partial sequence of fA3H and fA3CH and designed a single guide (sg) RNA targeting exon 3 of fA3H (exon 6 of fA3CH) to achieve double knockout of these 2 genes. After transfecting cells with a sgRNA/Cas9 expression DNA vector or co-transfecting the sgRNA along with Cas9 mRNA, which were prepared by in vitro transcription into cat embryonic fibroblast cells, in vitro gene-targeting analysis revealed that the CRISPR/Cas9 system could introduce indels with high efficiency: 23 and 41% of cat fibroblast cells were targeted in the fA3H/fA3CH locus when Cas9 was introduced in the DNA and mRNA forms, respectively. We chose to perform cytoplasmic microinjection injections 6h post-IVF with 10 pL of injection solution containing 50ngµL-1 for sgRNA and 100ngµL-1 for Cas9 mRNA at a volume ratio of 1:1. We achieved a 95.5% embryo survival rate and a 22.9% blastocyst formation rate. High gene-targeting efficiency (62.5%, 15/24) from single blastocyst was achieved in targeting the fA3H/fA3CH locus by PCR-RFLP assay. We transferred 45 injected embryos to the uterine tubes of 4 pseudo-pregnant queens within 2h of injection. Two of the recipients were determined to be pregnant by ultrasound on Day 30 after embryo transfer, and gave birth to 6 live kittens on Day 65. Genotyping analysis revealed that 2 kittens were successfully targeted in the fA3H/fA3CH locus: kitten 1 was biallelically targeted, whereas kitten 5 was monoallelically targeted. Sanger sequencing of the PCR products subcloned into TA cloning vectors showed that kitten 1 carried the same 1-nucleotide deletion on both alleles, and that kitten 5 also carried the same 1-nucleotide deletion monoallelically. Therefore, we conclude that the fA3H/fA3CH locus in the 2 kittens is specifically targeted by the CRISPR/Cas9 system. To our knowledge, this is the first report of successful site-specific genetic modification in the domestic cat achieved using the CRISPR/Cas9 system and the production of live fA3H/fA3CH double-knockout kittens to study the HIV-1 infected diseases.

81 IMPROVEMENT OF DEVELOPMENTAL COMPETENCE OF BOVINE IN VITRO-PRODUCED EMBRYOS BY ADDING 2-METHOXYSTYPANDRONE IN MATURATION MEDIA

The 2-methoxystypandrone (2-MS) is a naphthoquinone isolated from Polygonum cuspidatum. The objective of this study was to investigate the effects of 2-MS on oocyte maturation, blastocyst development, and embryo quality in terms of cell number and gene expression in vitro. A total of 2364 oocytes were cultured in TCM-199 supplemented with 10% fetal bovine serum, 1μgmL-1 oestradiol-17β, 10μgmL-1 FSH, 10ngmL-1 epidermal growth factor, 0.6mM cysteine, and 0.2mM sodium pyruvate and supplemented with different concentrations of 2-MS as following: 1.5μM (n=458), 1.0 (n=493), 0.5 (n=468), 0.1 (n=470), and 0μM (control, n=475) followed by IVF and then culture in CR1-aa medium supplemented with 44μgmL-1 sodium pyruvate, 14.6μgmL-1 glutamine, 10μLmL-1 penicillin-streptomycin, 3mgmL-1 BSA, and 310μgmL-1 glutathione for the first 3 days, and then the BSA was replaced with 10% FBS until Day 8. The differences in embryo development between experimental groups were analysed by one-way ANOVA. The Duncans multiple range tests were used to test the differences between the treatments. The level of statistical significance was set at P<0.05. The results showed that the addition of 2-MS at 1.0mM significantly improved (P<0.05) the percentage of MII oocytes, which were identified by aceto-orcein staining, compared with that in the control (76.5v. 65.4%, respectively), and remarkably (P<0.05), improved blastocyst development rates (45.29%) compared with control (32.21%). Additionally, TUNEL assay demonstrated that treatment with 1.0μM of 2-MS significantly improved the embryo quality by increasing total number of cells and reducing DNA damage. Immunofluorescent analysis showed that the protein levels of nuclear factor-kappa B (NFkB), inhibitor of kappa B kinase β (IkKβ), 8-oxoguanine, and cyclooxygenase-2 (COX2) declined significantly (P<0.05) after 2-MS treatment compared with the control. These results were confirmed by qRT-PCR, which showed a significant decrease in the mRNA levels of NFkB, IkKβ, COX2, inducible nitric oxide synthase (iNOS), BCL2-associated X protein (BAX), caspase-3, and Janus kinase2 (JAK2) after 2-MS treatment; however, the mRNA level of the anti-apoptotic gene B-cell lymphoma2 (BCL2) was significantly higher than that in the control. In conclusion, the addition of 2-MS at the indicated concentration dramatically improves the developmental competence of bovine in vitro-produced embryos.

Efficient Production of Bovine Tetraploid Embryos by Electrofusion of InVitro Produced Two-Cell Stage Embryos

Tetraploid complementation has been used to improve the production of cloned animals. The main objective of this study was to improve the efficiency of bovine tetraploid embryo production and the development potential of bovine tetraploid embryos into blastocysts. We assessed early embryonic cleavage timing and established the defined time quantum to collect synchronous 2-cell stage bovine embryos for electrofusion. Different electrofusion protocols were also tested. The second aim was to monitor ploidy transition by fluorescence visualization to shed lights on the nuclear fusion process of the cytoplasmic membrane-fused 2-cell stage bovine embryos. Karyotypes of day 8 blastocysts (day 0: day of electrofusion) were determined by karyotyping analysis. We found that electrofusion of in vitro produced bovine 2-cell stage embryos results in both tetraploid and diploid embryos and that blastocyst formation rates from fused 2-cell stage embryos are affected by the number of electrofusion pulses and the timing of how soon the 2-cell stage embryos are formed post insemination. We identified two distinct nuclear configurations after electrofusion of 2-cell stage embryos, each of which is uniquely related to the formation of tetraploid or diploid embryos. Based on the observation that tetraploid and diploid embryos derived from fused 2-cell stage embryos undergo different timings to become 2-cell stage embryos again, diploid and tetraploid 2-cell embryos can be readily separated after electrofusion. Finally, our study established an experimental protocol for the effective production of bovine tetraploid embryos by electrofusion of 2-cell embryos.