Porcine Reproductive and Respiratory Syndrome (PRRS) is the most economically important disease in swine in North America, Europe and Asia. PRRS is caused via infection of the pulmonary alveolar macrophages (PAMs) with the PRRS virus (PRRSV) causing respiratory illness and high fever in young growing pigs that predisposes them to secondary bacterial infections. PRRSV also causes severe reproductive failure in sows and boars. Although research is ongoing, PRRSV continues to elude a successful vaccine. In 2014, piglets were born with a gene edit in exon 7 of the Cluster of differentiation 163 (CD163) gene introduced by using the CRISPR/Cas9 site-directed nucleases system. The resulting litters of pigs were either challenged with multiple PRRSV isolates at 3 weeks of age or bred at maturity for a challenge with pregnant sows. The challenges demonstrated that the pigs were completely resistant to infectivity to both Type 1 and 2 isolates as measured by clinical signs, viremia, antibody response and lung histopathology. In a follow-up study, pregnant CD163-/- pigs were also challenged with PRRSV to determine if absence of CD163 in the dam should be sufficient to protect the CD163+/- fetuses that have functional CD163 protein. The wild-type sow and fetuses were actively infected with the PRRSV and one sow aborted. The CD163-/- sows carrying both the CD163-/- and CD163+/- fetuses were all negative for PRRSV nucleic acid and showed no sign of fetal or placental failure. The results of this study clearly demonstrate that the absence of CD163 in the sow is sufficient to protect a PRRSV-susceptible CD163+/- fetus. Gene editing of CD163 in pigs, via CRISPR/Cas9, successfully blocked PRRSV infectivity in young growing pigs and pregnant sows and their fetuses. This is a great example of the potential of utilizing gene editing to improve animal agriculture.

Importance of the in vitro model of tissues or organs is now evident in tissue engineering and cell biology research. Till now, two-dimensional culture systems have been using for in vitro cell culture, and have contributed to cell function studies despite their limitations. Three-dimensional (3D) culture has been utilized in cell biology research because it appears to mimic morphology and physiology of cells in living tissues and organs, unlike conventional monolayer cell culture. In our laboratory, we are developing 3D culture systems of bovine endometrial cells as a tool for the analysis of uterine endometrial functions. Among them, this lecture introduces spheroid culture and Matrigel culture. 1. Spheroid culture; Spheroids are a spherical mass composed of cells and extracellular matrices (ECMs). We have regenerated multicellular spheroids composed of bovine endometrial stromal and epithelial cells using ascorbate (1). Expression of MMPs, which are key enzymes for the tissue remodeling of the endometrium, were analyzed using the spheroid. E2, P4 and type-I IFN did not affect the gene expression of MMPs in the spheroid. However, treatment of type-I IFN increased the clearance of MMPs in the supernatant. These results suggest that IFN indirectly regulates endometrial tissue remodeling through clearance of MMPs. 2. Matrigel culture; It is reported that cells form lumens automatically by culturing cells in Matrigel (2). Matrigel is a solubilized basement membrane extracted derived from EHS mouse sarcoma cells. The bovine endometrial epithelial cells cultured in 15% Matrigel formed a circular or elliptical gland-like structure. Gene expressions of glandular epithelial specific factors (FOXA2, SERPINA14 and GRP) were significantly high in the Matrigel, compared to the monolayer cultured cells, except FOXA2. Further, SERPINA14 expression was affected by neither P4 nor IFN. However, when epithelial cells in Matrigel were co-culture with stromal cells, SERPINA14 expression increased significantly in the treatment of both P4 and IFN. These results suggest that bovine endometrial epithelial cells cultured in Matrigel show properties similar to the glandular epithelial cells in vivo, and regulated by the factors produced by the stromal cells. Finally, by using these 3D culture systems, it becomes possible to clarify not only factors regulating embryo elongation and implantation but also regulation of their expression. It will be able to reveal the mechanism of the embryo elongation and implantation to contribute to the improvement of the embryo transplantation technique. (1) Yamauchi N, Yamada O, Takahashi T, Imai K, Sato T, Ito A, Hashizume K. A three-dimensional cell culture model for bovine endometrium: regeneration of a multicellular spheroid using ascorbate. Placenta. 2003; 24(2-3):258-69. (2) Eritja N, Llobet D, Domingo M, Santacana M, Yeramian A, Matias-Guiu X, Dolcet X. A novel three-dimensional culture system of polarized epithelial cells to study endometrial carcinogenesis. Am J Pathol 2010; 176:2722-2731.

One of the major hallmarks of uterine diseases is disruption of ovarian steroid hormone control of uterine cell proliferation and differentiation. Estrogen (E2) stimulates proliferation of uterine epithelial cells while progesterone (P4) is inhibitory to E2-mediated proliferation of the epithelium. Mitogen inducible gene 6 (Mig-6) is an important mediator of P4 signaling to inhibit E2 signaling in the uterus. Uterine-specific knockout of Mig-6 caused endometrial P4 resistance and infertility. Levels of ErbB2 (also known as HER2) and phospho-ERK1/2 are significantly higher in Mig-6 knockout mice as well as infertile women with endometriosis. To determine the interplay between Mig-6 and the Erbb2 signaling pathway in the uterus, we generated mice with Mig-6 and Erbb2 conditionally ablated in progesterone receptor-positive cells (Pgrcre/+ Mig-6f/f Erbb2f/f; Mig-6d/d Erbb2d/d). Mig-6d/d mice were infertile whereas control and Mig-6d/d Erbb2d/d mice exhibited normal fecundity. The uterine horns of Mig-6d/d mice had no implantation sites, whereas control and Mig-6d/d Erbb2d/d mice had averaged implantation sites. Additionally, aberrant increment of epithelial proliferation in uterus of Mig-6d/d mice did not show in Mig-6d/d Erbb2d/d mice uterus at pre-implantation stage. Microarray analysis revealed that almost altered genes in Mig-6d/d mice were recovered their expression levels in Mig-6d/d Erbb2d/d mice. The altered pathways such as cell-cycle control, DNA replication, and modification processes by Mig-6 ablation were rescued in Mig-6d/d Erbb2d/d mice. The infertility seen in Mig-6d/d mice is recovered in Mig-6d/d Erbb2d/d mice. These results suggest that Mig-6 mediates a critical P4 function to inhibit E2 signaling by inhibiting ErbB2 signaling. As MIG-6 is a mediator of P4 signaling, the activity of which can suppress unopposed-E2 signaling, our studies provide a potential new drug target for the intervention of female infertility.

Recent transcriptome analyses have shown that long non-coding RNAs (ncRNAs) play prevalent roles in transcriptional regulation. We have reported that promoter-associated ncRNAs (pancRNAs) activate the partner gene expression via local epigenetic changes. Here, we identify thousands of genes under the pancRNA-mediated transcriptional regulation in five mammalian species in common. In the mouse, 1) pancRNA-partnered genes show tissue-specific expression pattern, 2) expression of pancRNAs significantly enriched H3K4me3 and H3K27ac marks towards the partner gene expression, 3) H3K4me1 marks the pancRNA-partnered genes regardless of their expression level, and 4) C- or G-skewed motifs were exclusively overrepresented between -200 and -1 bp relative to the transcription start sites of pancRNA-partnered genes. More importantly, the comparative transcriptome analysis among five different mammalian species using a total of 25 counterpart tissues showed that overall pancRNA expression profile exhibited extremely high species-specificity compared to that of mRNA, suggesting that a significant number of pancRNAs contributed to the enhancement of a set of partner genes' expression in a sequence-specific manner. We conclude that the gain and/or loss of gene-activation-associated pancRNA repertories, caused by formation or disorganization of the genomic GC-skewed structure, finely shapes tissue-specific pattern of gene expression according to a given species.

Mitochondrial and mitochondrial DNA (mtDNA) is maternally inherited in humans and most animals. The degradation of sperm-borne mitochondria after fertilization assures normal preimplantation embryo development and may prevent mitochondrial diseases derived from heteroplasmy. Although it has been known that ubiquitin-proteasome system (UPS) is the major degradation pathway of post-fertilization sperm mitochondria in mammals, it is unclear how the UPS, which is able to get rid of single protein molecule at a time, can eliminate whole sperm mitochondrial organelle. We considered that the autophagy receptors [sequestosome 1(SQSTM1), microtubule-associated protein 1 light chain 3 (LC3), and gamma-aminobutyric acid receptor-associated protein (GABARAP)] and the non-traditional mitophagy pathways involving UPS and the ubiquitin-binding protein dislocase, valosin-containing protein (VCP) may act independently or in concert during post-fertilization sperm mitophagy. We found that the association of SQSTM1 with sperm mitochondria was displayed in both pig and rhesus monkey zygotes after fertilization. Sperm mitochondrial proteins [mitochondrial trifunctional enzyme subunit alpha (HADHA), mitochondrial aconitase 2 (ACO2), and mitochondrial ATP synthase H+ transporting F1 complex β-subunit (ATP5B)] co-purified with the synthetic, SQSTM1-derived, ubiquitin-binding UBA domain were identified. Also, the accumulation of GABARAP-positive protein aggregates was observed around sperm mitochondrial sheaths in fertilized oocytes, which reflects autophagosome formation. Furthermore, the inhibition of VCP delayed the process of sperm mitophagy and completely blocked it when embryos were co-injected with autophagy-targeting antibodies, such as anti-SQSTM1 and/or anti-GABARAP. Thus, both SQSTM1-dependent autophagy pathway and VCP-mediated proteasomal proteolysis facilitate post-fertilization sperm mitophagy in mammals. This explains how the proteolytic pathway can coordinate autophagy pathway to degrade the sperm mitochondrial sheath inside the fertilized oocyte.

The deleted in azoospermia like (DAZL) gene has been identified in many vertebrate species. DAZL shows high homology with deleted in azoospermia (DAZ) genes that identified only in humans, great apes and Old World monkeys, and boule homolog (BOLL) that identified in many vertebrate species. These genes encode RNA binding proteins (RBP), which regulate the post-transcriptional functions of several genes. In humans, DAZ copies are linked to Y chromosome, while DAZL and BOLL are linked to chromosomes 3 and 2, respectively. DAZ copies has been reported to express in prenatal and postnatal germ cells, particularly in the premeiotic spermatogonia. BOLL has been reported to express during the meiotic G2/M transition in germ cells. DAZL has been reported to express in all stages of germ cells. Compared to humans and mice, the detailed functionalities of DAZL is not clear in many vertebrate species. In our studies, we use chickens as an animal model to examine the expression profiling of DAZL gene in germ cells right from the early embryonic development to the adult. Also, we are studying the effects of small interfering RNA (siRNA) mediated knockdown of DAZL and Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR associated protein 9 (CRISPR/Cas9) mediated knockout of DAZL in the chicken primordial germ cells (PGCs). In the chicken, DAZL is linked to chromosome 2 (2p1.3-p1.2), and encodes a 289 amino acids protein. By in situ hybridization, we detected a strong expression of DAZL in the germ plasm of chicken oocytes. Later, the expression of DAZL was strongly detected in all stages of intrauterine development and post-ovipositional development especially in the PGC specifying cells. Moreover, the expression of DAZL was strong and constant in the male and female germ cells until adult stage. The siRNA mediated knockdown of DAZL significantly reduced the PGCs proliferation and increased the apoptosis in vitro. We examined the knockout efficiency of DAZL using CRISPR/Cas9 technique in chicken DF1 fibroblast cell line, prior to test in the PGCs. The results of T7 endonuclease I (T7E1) assay and subsequent sequencing indicates clear mutations on the DAZL gene in DF1 cells, and the method could be applicable to cause mutations on the DAZL gene in PGCs. In conclusion, chicken DAZL express in all stages of germ cells as a germ line marker, and alteration in the gene expression causes germ cells impairment.

Transplantation of stem cells, such as mesenchymal stem cells (MSCs), is a promising strategy for treating several types of intractable disorders. Mechanistically, it could not only replace damaged cells by direct contribution, but also establish an anti-inflammatory or immunomodulatory microenvironment. However, the cellular mechanisms underlying molecular and biological properties of stem cells during ex vivo expansion and also after transplantation in pathological environments remain largely elusive. We recently developed the cyanoacrylamide-based coumarin derivatives (Fluorescent real-time thiol tracer; FreSHtracer*) reversibly react with glutathione for monitoring of glutathione levels in living stem cells. These probes revealed that glutathione levels are heterogeneous among subcellular organelles and among individual cells and show dynamic changes and heterogeneity in repopulating stem cells depending on oxidative-stress or culture conditions. Importantly, a subpopulation of stem cells with high-glutathione levels exhibited increased self-renewal and migration activities in vitro and showed improved therapeutic efficiency in treating asthma. Furthermore, employing a novel combination of longitudinal intravital confocal fluorescence imaging and microcystoscopy in living animals, we investigated the distributions and properties of transplanted multipotent MSCs derived from human embryonic stem cells at single-cell resolution in real-time by performing confocal imaging of bladder tissues in a rat model of IC/BPS for up to 6 months post-transplantation. These novel real-time monitoring strategies demonstrate the novel molecular insight for maintaining stem cell functions and also enhance understanding of the in vivo behaviors of the engrafted stem cells, which is crucial to determine the efficacy and safety of stem cell-based therapies. This strategy may facilitate the translation of various stem cell-based approaches into clinical practice.

Bisphenol A (BPA) is a common industrial chemical that has been used extensively to make certain plastics and resins since the 1960s. As a potential endocrine disruptors, BPA has been investigated for its impact on fertility/reproduction in animals and humans. However, the molecular mechanisms of BPA action and standard method for detecting BPA-related health hazards are unclear. Considering in-vitro experimental model, we investigated the effects of BPA (0.0001 to 100 μM) exposure on mouse spermatozoa. We revealed that BPA affects several sperm functions by triggering the mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and protein kinase-A (PKA) activity. High doses of this chemical was also likely for the activation of protein tyrosine phosphorylation in a PKA-dependent signaling consequently induced a precocious acrosome reaction. Simultaneously, BPA has been found to decrease the rate of fertilization and early embryonic development. In addition, BPA induced differential protein expression in spermatozoa were responsible for the pathogenesis of many diseases. Considering in vivo experimental model, we deliberate the effects of gestational BPA exposure (TDI, NOAEL, and LOAEL doses) on both ejaculated and capacitated spermatozoa in F1 adult mice. We confirmed that BPA affects several sperm function in F1 male. These effects appeared to be caused by reduced numbers of stage VIII seminiferous epithelial cells in testis and decreased PKA activity and tyrosine phosphorylation (non-capacitated) in spermatozoa. We also noticed that BPA decreased average litter size as well as compromise the rates of cleavage and blastocyst formation. Proteins differentially expressed in both capacitated/ejaculated spermatozoa play a critical role in energy metabolism, stress responses, and fertility, finally predispose to the development of several diseases. On the basis of these results, we suggest that BPA alter spermatozoa function and the proteomic profile, ultimately affecting their fertility potential. Therefore, it is of critical public health significance to reevaluate the levels of BPA exposure that are currently deemed to be acceptable.

Stem cells have special properties, such as self-renewal, proliferation, and the multilineage differentiation. Generally, stem cells are categorized into embryonic stem cells (ESCs), adult stem cells (ASCs), and induced pluripotent stem cells (iPSCs). Mesenchymal stem cells (MSCs) are a type of ASCs with a multipotent property. MSCs are easily isolated from various tissues and organs in the human body and can differentiation into multiple lineages, such as bone, cartilage, fat, and muscles. Compared to ESCs and iPSCs, MSCs possess less proliferation and differentiation capacities, therefore, a much scientific concern is concerned toward promoting the proliferation and the differentiation potency of MSCs. There are various methods to achieve this goal such as the treatment of various types of small molecules or culturing on specific peptides. Producing of high-quality MSCs with enhanced proliferation and differentiation capacities will definitely be a useful tool for stem cell-mediated tissue regeneration and the further clinical application.

Induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (iMSCs) serve as a unique source for cell therapy. We investigated whether exosomes from iMSCs promote the proliferation of human keratinocytes (HaCaT) and human dermal fibroblasts (HDFs). iPSCs were established from human Wharton’s jelly MSCs and were allowed to differentiate into iMSCs. Exosomes were collected from the culture supernatant of MSCs (MSC-exo) and iMSCs (iMSC-exo), and their characteristics were investigated. Both exosome types possessed basic characteristics of exosomes and were taken up by skin cells in vitro and in vivo. A significant increase in HaCaT proliferation was observed with iMSC-exo, although both exosomes increased the viability and cell cycle progression in HaCaT and HDFs. No significant difference was observed in the closure of wound scratch and the expression of reparative genes between cells treated with the two exosome types. Both exosomes enhanced the secretion of collagen in HaCaT and HDFs; however, an increase in fibronectin level was observed only in HaCaT, and this effect was better with iMSC-exo treatment. Only iMSC-exo increased the phosphorylation of extracellular signal-regulated kinase (ERK)-1/2. Our results indicate that iMSC-exo promote the proliferation of skin cells by stimulating ERK1/2 and highlight the application of iMSCs for producing exosomes.

The CRISPR/Cas9 system is widely applied in genome engineering due to its simplicity and versatility. Although this has revolutionized genome-editing technology, knock-in animal generation via homology directed repair (HDR) is not as efficient as non-homologous end-joining DNA-repair-dependent knockout. Although its double-strand break activity may vary, Cas9 derived from Streptococcus pyogenens allows robust design of single-guide RNAs (sgRNAs) within the target sequence; However, prescreening for different sgRNA activities delays the process of transgenic animal generation. To overcome this limitation, multiple sets of different sgRNAs were examined for their knock-in efficiency. We discovered profound advantages associated with single-stranded oligo-donor-mediated HDR processes using overlapping sgRNAs (sharing at least five base pairs of the target sites) as compared with using non-overlapping sgRNAs for knock-in mouse generation. Studies utilizing cell lines revealed shorter sequence deletions near target mutations using overlapping sgRNAs as compared with those observed using non-overlapping sgRNAs, which may favor the HDR process. Using this simple method, we successfully generated several transgenic mouse lines harboring loxP insertions or single-nucleotide substitutions with a highly efficiency of 18~38%. Our results demonstrate a simple and efficient method for generating transgenic animals harboring foreign-sequence knock-ins or short-nucleotide substitutions by the use of overlapping sgRNAs.

Biopharmaceuticals produced through bioprocessing are emerging as next generation sources beyond semiconductors in Korea. The large companies in Korea such as Samsung, SK and LG are attracting attention to Bio, and the portion of biopharmaceuticals is rapidly increasing in the global pharmaceutical market, and its proportion is expected to reach 27% of the pharmaceutical market at 2020. What are some of these Bio drugs? I will examine the patentability of the subject after considering the subject from the viewpoint of invention and patent. First, if you classify drugs, you can divide them into chemically synthesized drugs and biopharmaceuticals depending on the molecule type. Biopharmaceuticals can be defined as high-molecular-weight drugs that use raw materials derived from organisms and produce by biological processes such as cell culture. These include recombinant protein drugs such as insulin, antibody drugs used as anticancer drugs, vaccines, cell therapy, gene therapy, tissue culture, tissue isolated from blood, and allergens used for the treatment of allergies. In this seminar, I will look at the necessity of protection of biotechnology patents in general and the direction of protection, especially the recent issues related to biopharmaceuticals. We will examine the patentability of invention, patentability, patent requirements of each invention, and the protection of appropriate rights by understanding these patent systems. We will also look at global patent protection and IP protection strategies for the global business of biopharmaceuticals. I will analyze the patent strategy of a biopharmaceutical company that has infinite competition in the global market based on the published information, compare it with our IP strategy and think about the effective IP strategy of our situation

Although some factors, including season, age, type of estrus (natural estrus vs. induced estrus) and semen type (conventional vs. sexed), affect the conception rate following artificial insemination (AI) in dairy cattle, there is little information about the influence of ovarian characteristics, such as preovulatory follicle (PF) location at estrus, on fertility in dairy cattle. In most breeds of cattle the right ovary appears to function more actively than the left and about sixty percent of pregnancies in dairy cattle occur in the right horn of uterus (Reece and Tuner, 1938). Our study aimed to compare conception rates in dairy cattle between PFs that developed in the left ovary and those that developed in the right ovary at estrus. In this study, we examined the locational effect (left or right ovary) of the preovulatory follicle (PF) on fertility in dairy cattle. In total, 955 artificial inseminations (AI) were analyzed. At AI, PF locations were examined using rectal palpation, and dairy cattle were divided into two groups on their PF locations: (i) the PF located in the left ovary (L-PF); and (ii) the PF located in the right ovary (R-PF). Pregnancy was diagnosed by rectal palpation or ultrasonographic examination 60 days after AI. The conception rate was 38.1% in all dairy cattle. Conception rate was higher in the R-PF (40.8%) than in the L-PF (33.2%). In summary, PF development in the right ovary was associated with increased conception rates in dairy cattle.

한우는 우리나라에서 사육하는 주된 육우로서 그동안 우수한 한우의 보급을 위하여 과배란 처리, 생체난자 흡입술 등을 활용하여 수정란을 생산하여 이식하는 연구가 진행되었다. 또한 농가 현장에서 발정발견 및 수정적기를 놓치는 어려움을 해결하기 위한 발정동기화 방법과 배란동기화 방법 등 한우의 번식률을 향상시키기 위한 많은 연구가 이루어졌으며 이러한 연구의 결과들이 한우사육 농가에 많은 도움을 주었다. 그리고 최근에는 복제와 관련된 연구도 상당한 결과를 나타내고 있으며, 정자 혹은 수정란 시기의 성판별 기술이 개발되어 보급되고 있다. 하지만 한우의 번식과 관련된 많은 연구에도 불구하고 최근에는 한우의 임신기간 송아지 분만 시 체중 등 기본생리에 관련된 연구는 거의 이루어지지 않았다. 따라서 본 연구에서는 대관령에 있는 국립축산과학원 한우연구소 보유축의 2006 년부터 2017 년까지 2,548 두의 번식자료를 수집하여 분석하여 보았다. 이들 자료 중 번식기간이 나오지 않거나 임신기간 250 일 미만, 315 일 초과, 송아지 성별 표시 없는 자료, 산차가 기록되지 않은 자료 등을 제외한 2,452 두의 자료를 분석하였다. 본 연구소에서 지난 12 년 동안의 평균임신기간은 284.4 ± 6.5 일로 나타났으며 2015 년도가 281.0 ± 5.2 일로 가장 짧았으며 2007 년도가 286.7 ± 6.0 일로 가장 길었다. 신생송아지 분만시 평균체중은 23.9 ± 3.7kg 이었으며 2015 년도가 23.1 ± 3.9kg 으로 가장 낮았으며 2014 년도가 25.0 ± 4.0kg 으로 가장 높았다. 송아지의 성별에 따른 평균임신기간과 체중은 암송아지가 283.5 ± 6.5 일과 22.8 ± 3.3kg 이었으며, 수송아지는 285.2 ± 6.4 일과 24.9 ± 3.8kg 이었다. 암소의 산차에 따른 송아지 생시체중을 분석해 본 결과 암송아지의 체중은 1 산차가 22.2 ± 3.2kg 으로 평균체중인 22.87 ± 3.36kg 보다 작은 경향을 나타내었으며 수컷 송아지의 체중 역시 23.7 ± 4.0kg 으로 평균체중인 24.9 ± 3.8kg 보다 작은 경향을 확인할 수 있었다. 임신기간과 신생송아지 체중일 비교해 본 결과 270 일 이전 태어난 송아지의 평균체중이 19.8 ± 4.2kg 으로 전체 평균체중인 23.9 ± 3.7kg 보다 작은 것을 확인할 수 있었다.

OPU 유래 수정란 생산 및 이식은 기존의 체내 및 체외 수정란 생산과 이식의 단점을 해결하고 한우 개량을 촉진 시킬 수 있는 기술이다. 특히 살아있는 공란우를 활용하므로 모계와 부계에 대한 혈통관리가 정확하고 또한 선발된 공란우와 계획 교배를 통하여 가장 적합한 동결정액을 사용하여 개량의 효과를 극대화가 가능하고 또한 현재까지 생산효율이 가장 높은 생산기술이다. 따라서 기존의 수정란 생산방법보다 공란우의 선발강도를 더 높일 수 있어 개량의 효과를 극대화 할 수 있는 장점이 있다. 본 연구실에서는 2009 년도부터 OPU 유래 수정란을 생산하여 이식하여 수정란이식이 산업화로 전개될 수 있도록 진행하고 있다. 특히 한우에 대한 우수성 확보, 개량 및 그 다음 세대 후대의 근친적인 문제가 발생되지 않도록 수정란이식에 의해 탄생된 송아지 혈통관리를 위한 친자검정으로 수정란에 대한 신뢰성을 높여 수정란 이식이 산업화가 될 수 있도록 진행하였다. 본 연구에 대한 조사는 2013 년도 7 개의 시·군을 시작으로 ‘14 년도 11 개, ‘15 년도 15 개, ‘16년도 17 개의 시·군으로 점진적으로 확대됨에 따라 공란우의 두수도 ‘13 년도 35 두를 시작으로 ‘16 년도는 71 두로 증가되었다. 조사된 4 년동안 각 지역에서 선발된 유전능력이 우수한 공란우는 총 211 두에서 수정란을 총 18,839 개, 두당 89.3 개를 생산하였다. 두당 생산 효율도 ‘13 도에는 회당 3.0±4.5 개에서 ‘16 년도에는 4.8±2.2 로 회당 이식 가능한 수정란의 생산량이 년도에 따라 꾸준히 향상되었다. 생산된 수정란은 자연발정 또는 발정동기화를 통하여 매주 2 회 이상 이식으로 ‘13 년 49.7%, ‘14 년 51.9%, ‘15 년 52.9%와 ‘16 년 47.9%의 수태율을 확인하였다. 이는 년도가 증가되면서 수정란의 생산성의 향상과 적정 수준의 수태율의 성과는 시술자와 농가의 적극적인 참여에 의한 결과로 판단되고 또한 유전능력이 우수한 수정란의 대량생산 및 공급으로 개량의 효과가 증가되고 우수한 한우 집단 구축으로 한우산업이 지속적으로 발전 할 것으로 판단된다.

Interferon tau (IFNT), has known as a key signal molecule for a period of pregnancy in ruminants owing to the need on maternal recognition of pregnancy. It is generated in trophectoderm cells of the elongation bovine conceptus at day 13-21 and a peak output is at day 15-17 of pregnancy period. Moreover, other studies indicated that it can be effective in the embryonic development and quality. In previous study, there were 8 bovine IFNT, but only 2 forms of IFNTs, IFNT2 and IFN-tau-c1, were expressed by the conceptuses during the peri-implantation. In this study, we target the one between the two, IFN-tau-c1 and then the effect of IFNT knockout in donor cells to bovine cloned embryonic development by somatic cell nuclear transfer (SCNT) was investigated. In order to proceed this study, the immature oocytes from the ovaries at local slaughterhouse have been matured in vitro for 22 hours. For preparing the donor cell that have a mutation on IFNT gene, somatic cells were transiently transfected with Cas9 protein and single guide RNA targeting IFNT, and various single derived colonies with high proliferation were isolated and confirm the mutation by PCR. Finally, one colony had mono-allelic mutation (4bps deletion) was picked out and applied as the donor cell to SCNT. A donor cell was injected into an oocyte that nucleus was removed. Reconstructed oocytes with the donor cell were fused by electrical shock, activated by chemical stimulation and cultured for 7 days in chemically defined medium. In this study, control (n=199) and IFNT knockout-group (n=219) were compared with four replications. As results, there was no significant difference between control-and IFNT-knockout group not only in cleavage rate, but also blastocyst formation rate (Control: 12.3% ± 9.2, IFNT knockout-group: 20.1 ± 11%). In addition, the number of blastocyst cell was not different between control (91.7 ± 26.2) and IFNT knockout group (83.5 ± 21.3). 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 indicated that the interruption of IFNT did not influence the embryonic development. In future study, we will transfer these mutated embryos toto test the effect of IFNT for pregnancy period. 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.

이종이식 시 사람과 돼지는 면역학 적으로 일치하지 않으므로, 면역세포의 활성화 및 조직의 병변 등을 유발한다. 특히, 영장류에는 존재하지 않고 돼지의 전 조직에서 발현되는 alpha-gal 인자에 의해, 이종이식 시 alpha-gal 항원에 대한 항체의 급격한 증가로 거부반응이 유발되며 영장류를 사망에 이르게 한다. 이에, 본 연구는 alpha-gal 합성에 관여하는 효소인 alpha-galactosyltransferase를 knock out(Gal-/-)한 돼지의 세포를 기반으로, 보체의 활성을 조절하는 membrane cofactor protein(MCP)과 혈액 응고를 저해하는 thrombomodulin(TBM)의 과발현을 유도하고자 하였다. 이는, 이종이식 시 발생하는 염증반응과 혈액응고 현상을 억제하는 복합형질 전환 돼지를 개발함으로 이종이식 후의 생존성 향상에 기여하고자 하였다. 따라서 codon modification 을 통해 염기서열을 변형한 MCP cDNA 는 CAG 프로모터를 이용하여 전 조직에서 발현되도록 하였고, TBM 은 Icam2 프로모터를 통해 혈관 내피세포 특이적인 발현을 유도하였다. 제작된 벡터를 Gal-/- 돼지의 섬유아세포에 도입하고 MCP 발현 수준이 높은 세포를 선별하여 세포주를 구축하였다. 구축된 체세포를 이용하여 체세포 복제란을 생산하고 외과적 방법으로 수란 돈에 이식하였다. 체외성숙(78%) 한 난자를 복제 및 융합(58%)한 후 4 두의 대리모에 각각 약 310 개의 복제란을 이식하였다. 대리모 1 두에서 임신(25%) 및 분만(25%)에 성공하였으며, 9 두의 산자 중 6 두가 생존하고 3 두는 사망하였다. 본 연구에서 생산된 TBM 유전자가 도입된 형질전환 복제돼지는 증식과정을 통해 축군을 조성하고 향후 영장류를 활용한 이종이식 연구에 활용될 예정이다.

Until now, problems related to shortage of organ for transplantation have been continuing. Pigs are the most suitable animal for xenotransplantation. Although primates are most similar to humans, they are not suitable because they have low productivity. Pigs are more productive than primates, and their organ size and physiological characteristics are similar to humans, with the exception of primates. In this study, we breeding the transgenic minipigs using natural mating to produce transgenic pigs. And, transgenic pigs has transmission rate that follow mendel’s rule. There are 20% hDAF gene, 20% US11 gene and 50% both hDAF and US11 gene in transgenic offsprings. Furthermore, transgenic pigs followed normal litter size, and piglets also has normal sex ratio. To suppress the immune function, experiments were performed using porcine ear fibroblast that transfected with hDAF and US11gene. In Cytotoxicity experiment against human complement, hDAF gene and double transgenic cell with both hDAF and US11 gene showed effect to reduce cytotoxicity rate in all of human complement condition. US11 gene and double transgenic cell were significantly reduce the cytotoxicity ratio in human NK cell. Besides, hDAF gene transgenic cell also reduce immune response in 10:1 concentration of human NK cell. In conclusion, natural mating was efficient method for breeding transgenic pigs. And, hDAF and US11 genes has effect for reduce cytotoxicity against human NK cell and human complement conditions.

To preserve the superior genetic resources and restore the endangered species, Somatic cell nuclear transfer (SCNT) has been used widely. In Korea, the research of dog cloning has made outstanding achievements including the production of the world`s first cloned dog. Sapsaree (Sapsalgae), the representative dog of Gyeongsan-si was designated as a Korea natural monument (No. 368). This male dog used in this study has azoospermia due to unknown cause. In this study, the aim was to confirm the cause of infertility in the cell donor dog and to evaluate the reproduction potential of dog cloning using infertile male dog by SCNT. First, to confirm the infertility of the cell donor dog, the reproductive history and the testis were evaluated. The breeding histology was not recorded in individual document. In histopathology, the Sertoli cell tumor was confirmed in biopsy of the cell donor dog after death. But, these tumors are predominantly in older dogs. Second, we produced the cloned dogs with the somatic cells of the infertile dog and the appearance was similar with the cell donor dog. Also, microsatellite analysis confirmed the genetic relationship between the cell donor and clone dogs. Third, the potential breeding capacity of the cloned dog was confirmed. In T4 assay, the normal dog (same age with cloned dogs), cell donor dog, and cloned dogs was investigated. The cell donor dog with azoospermia had very low T4 level, and cloned dogs showed higher level of T4 than normal dogs. In CASA, There was no significant difference in sperm motor ability between normal dogs and cloned dogs. As a result, cloned dogs produced by SCNT had no problem regarding the reproductive function of the testis. In AI experiment, the semen of clone dogs was used to fertilize a natural female bitch and was diagnosed pregnancy by ultrasonography. In total, 7 puppies were born by normal delivery (male: 3, female: 4). In conclusion, this study confirmed that the reproduction problem of non-genetic infertility can generate a normal descendant by SCNT. Also, the first successful research to restore infertile dogs was completed. Furthermore, SCNT would be useful for the restoration of endangered species and application of superior traits.

Rats are an important laboratory animal for biomedical research. Though rats have some physiology and genetic similarities to human, several technical issues such as delicate in vitro culture system and low survival rate after pronuclear microinjection have hindered the development of transgenic rat generation. Accordingly, in this study, to produce transgenic rat, we established transposon-mediated insertional mutagenesis by cytoplasmic microinjection. The sleeping beauty transposon (SB) and SB-transposase recognize the precise genome integration into a TA nucleotide by ‘cut-and-paste’ mechanism. It mediates stable integration and reliable long-term expression. DNA, 0.4ng/ul SB vector (IR/DR-EF1a-eGFP-2A-IL2-pA-IR/DR) and mRNA, 5ng/ul SB-transposase were injected to 1-cell stage embryo and one transgenic rat was generated after full-term gestation. To confirm the genome insertion, GFP was detected by PCR. Further, this method was applied to generate transgenic rats producing Cas9 protein. DNA, 0.4ng/ul SB vector (IR/DR-CAG-Cas9-2A-eGFP-pA-IR/DR) and mRNA, 5ng/ul SB-transposase were injected to 1-cell stage embryo. Some of the in vitro cultured embryos showed GFP positive at blastocyst stage and Cas9 sequence was detected by PCR. One stillbirth pup was born to date and genome PCR on Cas9 was positive. In summary, the SB transposon system could be a highly effective method that contribute to the production of transgenic rats. If the protocols will be optimized, we successfully generated efficiently transgenic rats for human models by SB system. 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.