Earticle

Sperm decondensation must be accomplished before fluorescence in situ hybridization (FISH). However, the lack of studies were conducted to discover the optimal porcine sperm decondensation for improving efficiency of FISH. We therefore sought to investigate the proper conditions of porcine sperm decondensation for FISH. In this trial, semen samples were obtained from eleven boar and immediately washed. After washing, the suspension was smeared and fixed on slides. Air-dried slides were incubated at 37℃ for 0, 15, 30 and 45 min in a 5 mmol/L dithiothreitol (DTT) to cleave protamine disulfide bonds, and thus sperm head decondensation was occurred. Subsequently, slides were counter-stained with hoechst 33258 to measure several parameters of sperm head by using microscope. The area, perimeter, and length of sperm head were significantly increased after 30 min incubation. This result demonstrated that sperm head is effectively expanded, which is essential for sperm decondensation after 30 min incubation. Subsequently, the roundness of sperm head was significantly increased after 15 min incubation. This result showed that the circularity of sperm head, which is important for sperm head decondensation, is ideal after 15 min. Taken together, the incubation time of optimal sperm head decondensation could be 30 min to get better achievement for FISH.

Mammalian spermatozoa must undergo several physiological modifications both prior to in vivo and in vitro fertilization. These functional/physiological modifications of spermatozoa, called capacitation, are prerequisites for fertilization. Therefore, sperm proteins that modify the aforementioned events could regulate their function and fertilization competence. Signal transducer and activator of transcription 1 (STAT1) is a transcriptional protein reported to have a role in sperm capacitation. However, the function of STAT1 in spermatozoa has not yet been clarified. Therefore, Our study was designed to evaluate the role of the STAT1 using the specific inhibitor, fludarabine (Flu) in mouse spermatozoa. In this in vitro study, mouse spermatozoa were incubated for 30, 60, and 90 min accordingly to investigate the role of STAT1 on sperm capacitation. Our results revealed that the expression levels of STAT1 was significantly decreased at 90 min, perhaps as a consequence of sperm capacitation. Simultaneously, we treated mouse spermatozoa with different doses of a specific inhibitor of STAT1, fludarabine (150μM, 1500μM). Although our results showed that motility/motion kinematics, viability, and capacitation status have no significant difference following exposure to fludarabine, capacitation status tends to increase. Taken together, it is tempting to speculate that STAT1 could be associated with sperm capacitation. However, further studies are needed to discover the underlying mechanisms of STAT1 on sperm capacitation.

Peroxiredoxins (PRDXs), which are essential antioxidant enzymes for physiological activity, have reported that influence sperm function and consequently male fertility. Although PRDXs are well known as important factor to affect male fertility, there has been a lack of attempt to discover the accurate role and mechanism of PRDXs in male fertility. Therefore, the principal objective of this study is to elucidate the role of PRDXs in sperm function and fertilization. We treated mouse spermatozoa with different dose of specific inhibitor of PRDXs, conoidin A (1, 10, or 100 μM). Our results revealed that conoidin A significantly decreased oxidized form of PRDXs (PRDXs-SO3) in mouse spermatozoa. Inhibited PRDXs activity are then resulted in a significant decrease in sperm motility/motion kinematics, viability, mitochondrial membrane potential, and intracellular ATP levels. On the other hand, intracellular levels of ROS and DNA fragmentation index were significantly increased following exposure to conodin A. Next, we evaluated capacitation and acrosome reaction status, and subsequently tyrosine phosphorylation and protein kinase-A activity to investigated underlying mechanism of PRDXs on sperm capacitation status. Capacitation and acrosome reaction were significantly decreased perhaps due to reduction of tyrosine phosphorylation and protein kinase- A activity. Finally, we investigated the effect of PRDXs on fertilization and early embryonic development. Our results described that decreased PRDXs activity significantly decreased fertilization and early embryonic development. Consequently, we demonstrated that inhibition of PRDXs activity has a direct impact on male fertility via decreased important physiological sperm function, eventually resulted in poor fertilisation and embryonic development.

Artificial insemination is a commonly used technology in the porcine industry that directly linked to the genetic upgradation of future progeny and profitable farm management. Therefore, accurate prediction of boar fertility is a matter of paramount importance. Although prediction of semen/sperm fertility using conventional analysis provides a preliminary assumption of male fertility, its practical value is limited. Protein content in the cell provides an important and fascinating insight to the dynamics of cell function. Therefore, the study of protein function in spermatozoa and their corresponding relationship with male fertility might be an excellent alternative tool for more accurate prediction of male fertility. Recently we demonstrated that aminopeptidase N (APN) activity is negatively correlated with motility and fertility of mice spermatozoa. Here, we investigated whether APN is also correlated with the fertility of boar. Spermatozoa were collected from high and low fertility boar based on their field fertility data (average litter size 13.2±0.06 and 11.1±0.14, respectively). Simultaneously, APN levels were examined by Western blotting using the corresponding antibody. In addition, spermatozoa from both fertility groups were examined by conventional semen analysis such as computer assisted sperm analysis (CASA) and chlorotetracycline (CTC) staining. Our data showed a significant higher APN expression in low fertility boar spermatozoa compare to the fertile counterpart (p<0.05). Interestingly, no significant difference was noticed in motility, motion kinematics, and capacitation status between both groups. Based on this findings, it is tempting to speculate that APN activity might directly correlate with boar fertility. Therefore, APN activity in spermatozoa is more accurate and sensitive biomarker for the detection of boar fertility then conventional semen analysis. However, further studies and needed to confirm our initial findings

Even human genome has been sequenced completely, we have very limited idea of all characterized genes at the protein level. Therefore, several attempts have been taken to map proteome of human chromosome. Since sex chromosomes determined the sex of individual, it important to study sex chromosome-encoded proteins. Here, we studied human sex chromosome- encoded proteins of the immune system, protein pathways, protein-protein interactions, and diseases association using several bioinformatics tools. We retrieved 30 proteins (X chromosome 28; Y chromosome 7; both 5) from the recent NCBI human genome annotation, based on their association with immune systems (immunological and inflammation pathways) by Pathway studio program. Searching of these proteins in the Human Proteome Project, including neXtProt, PeptideAtlas, and the Human Protein Atlas showed that all proteins were also identified in several cells and tissues of body. Proteins were further investigated using Pathway Studio and STRING programs. Pathway Studio and STRING programs showed 15 and 25 X chromosome-encoded proteins were interacted. All Y chromosome-encoded proteins showed interactions using STRING program, however no interaction was found using Pathway studio. In addition, difference in human sex chromosome-encoded proteins of the immune system showed an indirect relationship with the occurrence of some diseases in a sex specific manner.

Diabetes mellitus, a hyperglycemic condition, in which the patients either fail to secrete insulin because of ß-cells destruction (type I) or shows insulin resistance (type II), affecting more than 300 million people around the globe. Generation of effective ß-cells for the treatment of diabetes is a key area in modern translational medicines. Here we tried to generate porcine induced pancreatic ß-cells (piPan ß-cells) from Gal- TKO+MCP porcine bone marrow mesenchymal stem cell (pBM-MSCs) by overexpressing set of transcription factors included EGFP along with step-wise induction of small molecules. pBM-MSCs were cultured in media with 5-azacytidine for 24 hours and then proceeded with transfection using episomal transfection system. Transfected cells were replaced with basal media containing growth factors cocktail, Activin A and 2 uM of A83-01 followed by retinoic acid treatment. Finally, cells were replaced by maintenance media until maturation. After 3 days of transfection, EGFP expression was observed showing successful transfection which disappeared completely after 21 days. pBM-MSCs attained definitive endoderm morphology after Activin A treatment and formed exact pancreatic clusters after 4 weeks and 8 weeks in maintenance media, respectively. After 4 and 8 weeks, piPan ß-cells were analyzed for the expression of pancreatic markers by immunofluorescence (Insulin, PAX6, Somatostatin) and RT-qPCR (GCG, INS, NKX6.1, PDX1 and NEUROD1), which showed significantly higher expression as compared with pBM-MSCs. Morphological changes and expression of pancreatic markers revealed successfully differentiation of PiPan ß-cells; however, more in depth studies for its functional characteristic and in vivo maintenance are still in progress.

Although there are many studies about pluripotent stem cells, little is known about pluripotent pathways and the difficulties of maintaining the pluripotency of bovine cells in vitro. Here, we investigated differently expressed genes (DEG) in bovine embryo-derived stem-like cells (eSLCs) from various origins to validate their distinct characteristics of pluripotency and differentiation. We identified core pluripotency markers and additional markers which were not determined as pluripotency markers yet in bovine eSLCs. Using the KEGG database, TGFβ, WNT, and LIF signaling were related to the maintenance of pluripotency. In contrast, some DEGs related to the LIF pathway were down-regulated, suggesting that reactivation of the pathway may be required for the establishment of true bovine embryonic stem cells (ESCs). Interestingly, oncogenes were co-down-regulated, while tumor suppressor genes were co-up-regulated in eSLCs, implying that this pattern may induce abnormal teratomas. These data analyses of signaling pathways provide essential information on authentic ESCs in addition to providing evidence for pluripotency in bovine eSLCs.

간 약물 대사에 관여하는 CYP enzymes은 성별 의존적 분비량을 가지고 특히 성호르 몬의 영향을 많이 받는 것으로 알려져 있다. 최근 간 부전이나 노화 등의 원인으로 간 수요가 급증하고 있으나 공급 가능한 간의 수요는 제한적임으로 대안적인 연구로서 체세 포를 간세포로 전환시키는 direct conversion 연구가 시도되고 있고, 이 연구는 마우스 모 델에서 induced Hepatocyte like cells(miHep)의 구축 시 성별의 영향을 평가하였다. 세포 원으로는 동일 부모의 male과 female의 MEF(C57BL/6J)를 사용하고 간 전사인자(hHnf1α, hHnf4α, hFoxa3)들을 Lentiviral vectors로 감염시켜 miHep을 유도하고 특성을 비교하였 다. 성별에 상관없이 miHep은 한달 째 형성이 확인 되었다. Exogenic/endogenic genes의 발현은 모두 3달째 확인 되었고, 특히 male에서 hHnf1α, hHnf4α가 강한 발현을 보였다. 간세포 주요 발현 mRNA와 단백질의 발현을 miHep에서 분석하였다. mRNA발현에서는 Alb은 성별간 차이가 없지만 male에서는 Aat, Cyp3a13이 우세하였고, 반면 female에서는 Afp, Ecad, Cyp1a1, Cyp2a5, Cyp2d22, Cyp3a11가 우세하였다. 면역 형광 염색에 의한 단백질 발현은 성별에 상관없이 Alb, Trf, Aat, Ecad, Cyp3a11이 관찰되었고 Vim의 감소 가 확인되었다. 간의 기능 분석 결과로 지방은 male에서 Lpl, Pparγ가 강하였고, 글리코 겐은 female에서 Glut2의 발현이 강하게 나타났다. LDL의 축적은 male에서 면역 형광 염 색을 통해 강하게 나타났고, 약물의 흡수와 배출 기능을 확인한 ICG test는 male에서 흡 수 능력이 우수함이 밝혀졌다. 3일간의 CYP enzyme induction test에서는 3-Methylcholanthrene의 경우 male에서 Cyp1a1, Cyp3a11, Cyp3a13의 강한 증가가 확인 되었고, Rifampicin(Cyp2a5)과 Dexamethasone(Cyp2d22)의 경우도 역시 male에서 강한 증가를 보였다. 이상의 결과들을 종합하여 볼 때 Lentiviral vectors는 지속적으로 작용함이 규명 되었 고, 간 기능과 약물대사능에서 male이 female보다 우세한 hepatocyte like cells의 특성을 보였다. 이것은 성별보다는 exogenic genes의 발현 정도가 구축된 miHep의 특성에도 강 력한 영향을 미쳤다는 것을 시사한다.

Pluripotent stem cells (PSCs) can be differentiated into all three germ layers in vitro. The pluripotent stem cells are derived from the inner cell mass of blastocyst in vitro, termed embryonic stem cells (ESCs). The epiblast derived pluripotent stem cells (Epi- SCs) represent a more differentiated state than plurioptent ESCs. The pluripotent ESCs and EpiSCs express Oct4 (Pou5f1) gene. Oct4 gene is essential for maintenance of pluripotent ESCs. The pluriopotent ESCs are induced differentiation into trophectoderm by decreasing Oct4 levels below 50%. Oct4 levels regulate cell fate of pluripotent stem cells. Here, we established ZHBTc4-ESCs derived epiblast stem cells like cells (ZHBTc4- EpiLCs). ZHBTc4-EpiLCs could not be maintained in doxycycline (dox) contained EpiSC medium, indicated that Oct4 gene is essential for maintenance of primed EpiLCs. We also tried to differentiate ZHBTc4-EpiLCs into neuroectoderm or mesoendoderm lineages with or without dox. The ZHBTc4-EpiLCs could be efficiently differentiated into neuroectoderm lineage with dox, whereas these cells could not be differentiated into mesoendoderm lineage with dox. This results showed that Oct4 gen

CD133, originally classified as a neural stem cell marker, could be used for enrichment of cancer stem cells (CSCs). In the current study, we analyzed the immunophenotypic characteristics of cancer cell lines derived from brain tumor lesions of 5 meningioma patients. Interestingly, tumor spheres only generated from the specimen of high-grade meningioma cell line (atypical meningioma cell line, WHO grade II), whereas there was no sphere formation in typical meningioma cell line (WHO grade I). The self-renewing capacity of these tumor sphere was assayed by dissociation of primary tumor spheres, and plating of single cells and maintained in the conditional medium. These single cells produced secondary sphere formation after another 7 days, indicating that these meningioma cell lines in spheres were capable of self-renewal and proliferation. Using flow cytometry, we observed the pattern of expression of dual markers CD44/CD133 in atypical meningioma cell line. The cytometry dot plot of CD133 expression in atypical meningioma cells with about 20.84% CD133− (gate Q4) and 79.15% CD133+ cells (gate Q2) before sorting. The CD44+CD133+ subpopulations showed stem cell properties, including extensive proliferation, instead of their CD44+CD- 133- counterparts. A primary culture of brain cancer cells and isolation of CD133+ CSCs might be used as a patient-specific, biology-driven tool to predict the outcome at diagnosis and to study the mechanism of the brain tumor.

The recombinant production of therapeutic proteins from transgenic animal is the major technology of animal biotechnology and is one of the biggest challenges in an efficient bioreactor in the pharmaceutical industry. The useful gene delivery into cells or animals is an important way to express the target gene in mammary glands. Tissue plasminogen activator (tPA) is known to play an important role in the major enzyme responsible for blood clots breakdown. Because it works on the clotting system, tPA is used in clinical medicine to treat thrombolysis, such as pulmonary embolism, myocardial infraction and stroke. The objective of this study is a production of recombinant human tPA protein in the milk of porcine. For this study, we construct knock-in vector that human tPA gene is inserted into the exon 1 locus of porcine whey acid protein (pWAP) gene knock-out somatic cell by homologous recombination. Finally, establishment of tPA knock-in cell lines were confirmed by genomic PCR analysis and identification of karyotype. As a result, we established cell lines of pWAP knock-out expressing human tPA gene from porcine fibroblasts to apply somatic cell nuclear transfer (SCNT). Further study will be performed for the production of transgenic pigs.

Dipeptidyl peptidase IV (DPP-4) is a widely distributed type II transmembrane glycoprotein with known CD26 and its expression is reported in various human cells in its extracellular domain. Recently, DPP-4 inhibitors have been widely used as a second- line therapy for type 2 diabetes because of their glucose-lowering efficacy, weight neutral effects and minimal hypoglycemia. In this study, we introduced a human DPP-4 (hDPP-4) gene into fertilized one cell pig zygotes using microinjection, and produced a piglet that over-expressed human DPP-4 gene. RT-PCR analysis showed that human DPP-4 gene was ove-rexpressed in the various tissues, respectively. Establishment of hDPP-4 transgenic fibroblast cell from ear tissue was verified by mRNA and protein expression. These results suggest that described generation of transgenic pigs that overexpressed human DPP-4. Further study is necessary to confirm whether diabetes occur in these transgenic pigs, especially insulin resistance.

Human Leukocyte Antigen E (HLA-E)는 Natural killer cell의 활성을 억제시키는 리간드 역할을 수행하는 인자로서 모든 세포에서 발현되며, 이러한 기능적 특성에 의하여 Gal 항원이 없음과 동시에 HLA-E를 과발현하는 돼지 장기를 원숭이에 이식할 경우 발생하는 세포성 거부반응을 억제시킬 수 있을 것으로 기대된다. 본 연구는 돼지의 전 조직 및 세 포에서 HLA-E의 과발현을 조절할 수 있는 돼지의 프로모터를 개발하고자 실시하였다. 이 를 위하여 돼지의 모든 세포에서 발현되는 Elongation factor 1 alpha(pEf1a) 유전자의 프 로모터 영역 중 –750 bp에서 엑손 1과 인트론 1, 그리고 엑손 2의 미번역 부위를 포함하 는 1.5 kb의 프로모터를 클로닝한 후 HLA-E cDNA를 연결한 발현 벡터를 제작하였다. HLA-E 유전자의 발현 조절능을 비교하기 위하여 대조군으로 사람 EF1α 프로모터, 그리 고 돼지에서 외래유전자의 발현을 효율적으로 조절한다고 알려진 CMV enhancer chicken β-actin (CAG) 프로모터에 HLA-E cDNA를 각각 연결한 발현 벡터를 제작하였다. 각 각의 발현벡터를 HEK293 세포에 도입후 quantitative real-time RT PCR (qPCR) 방법으 로 분석한 결과, pEf1a 프로모터의 조절에 의한 HLA-E의 발현 수준이 대조군에 비해 유 의적(p<0.05)으로 높은 것을 확인할 수 있었다. 돼지의 귀 조직에서 분리한 섬유아세포 (fibroblasts)와 대동맥 혈관내피세포에 도입한 후 qPCR, western blot 방법으로 HLA-E의 발현 수준을 분석하였다. 그 결과 pEf1a 프로모터는, CAG, EF1a 프로모터와 유의적인 차이 없이 효율적으로 HLA-E의 발현을 조절하는 것을 확인하였다. 이러한 결과로 본 연 구에서 클로닝한 pEf1a 프로모터의 HLA-E의 발현 조절능은 CAG의 것과 유사하다고 판 단되며, 형질전환 돼지에서 HLA-E 효율적인 발현을 조절할 것으로 기대된다.

최근 alpha-Gal knock out (GTKO)/MCP/TBM 형질전환 돼지를 이용한 심장 이식 연구 에서 수여 원숭이가 최대 945일 생존한 것으로 보고되었다. 도입된 유전자에 의해 wild type 돼지의 심장을 이식하였을 때 항체와 보체 활성에 의해 나타나는 초급성 면역거부 반응과 급성 체액성 거부반응이 제어된 것으로 판단된다. 이에 본 연구에서는 Gal KO 기반 위에 일부 염기서열을 변경한 Membrane cofactor protein (MCP) 유전자를 도입함 으로써 발현 효율을 개선하고자 하였다. 동시에 혈액 응고 과정의 억제제로서 Thrombomodulin (TBM)을 발현하는 돼지 체세포주를 구축하는 것을 목적으로 한다. 이를 위하여 CAG 프로모터와 2A sequence를 통하여 MCP와 TBM 유전자가 동시에 전사되도록 한 것과 내피세포 특이적인 Icam2 프로모터를 이용하여 TBM을 따로 발현시킨 두 가지 vector를 제작하였다. 각 vector는 GalT-MCP/-MCP 세포에 도입한 후 MCP에 대한 항체를 이용 하여 두 차례에 걸친 cell sorting을 진행하였다. MCP-positive cell은 single cell로 배양하 였으며 flow cytometry 분석을 통하여 각 clone 별로 유전자의 발현 정도를 비교하였다. TBM 유전자를 CAG 프로모터와 연결한 경우, MCP와 더불어 TBM의 발현도 함께 증가 하였다. 반면, Icam2 프로모터를 통하여 TBM을 따로 발현시킨 clone에서는 TBM의 발현 이 제한됨을 확인할 수 있었다. 또한 선발된 모든 clone에서 MCP 발현이 개선됨에 따라 원숭이 보체에 대한 저항성이 유의적으로 향상되었다. 이 중 염색체 변이가 일어나지 않 은 2개의 clone을 확인하였다. 이를 이용한 개체의 생산은 차후 이종장기 이식 시 발생하 는 거부반응을 억제하여 개체의 생존기간을 연장하는데 기여할 것으로 기대된다.

Porcine skin and preserved cadaver skin are used for temporary wound coverage, but 1 to 2 weeks after grafting, these tissues undergo immune-mediated rejection. To avoid such antigenicity, different types of acellular dermal matrix (ADM) have been studied. The generation of pigs lacking the α-1,3 galactosyl transferase (GT—/—) expressed by α-Gal epitope (Galα1,3Galα 1,4GlcNAc-R) has minimized hyperacute rejection. This study was performed to investigate the expression of α-Gal epitope in the skin derived from GT—/— transgenic pigs. The skin (7/1,000 inches) was obtained by dermatome (Zimmer®Electric Dermatome) from around one month old of wild type (WT), GT—/—,and GT-MCP/-MCP piglets, respectively. The skins treated with decellularization were fixed, dehydrated, cleaned, and embedded. It was confirmed that α-Gal epitope was not detected in the skins from TG pigs stained with BS-IB4 lectin antibody, but in WT skin. To analyze the characteristics of immune response genes in ADM skins, the tissue sections were stained with anti-HLAE and anti-MHC I antibodies. Decellularized skins of GT—/—and GT-MCP/-MCP were not stained in both HLAE and MHC I, but the both immune response genes were strongly expressed in epidermis and dermis, respectively. Taken together, it can be postulated that ADM skin originated from GT KO pigs may be used as a source of xenoskingraft. Further studies are needed to evaluate the functions of the ADM skins of GT—/— and GT-MCP/-MCP pigs after xenograft.

돼지의 장기를 영장류로 이식하기 위해서는 단계적인 면역거부반응을 억제하는 것이 중요하며, 이를 해결하기 위하여 이전 연구에서 MGH 미니돼지로부터 alpha-1,3 Galactocyl transferase(GalT) 유전자 좌위에 membrane cofactor protein(MCP) 유전자가 삽입되 어 이종 이식 시에 생기는 면역거부 반응을 극복한 돼지(GalT-MCP/-MCP)를 생산한 바 있다. 그러나 면역거부반응은 억제되었지만 혈액응고 장애로 인한 문제가 발생하여 이를 억제 할 수 있는 연구가 필요하다. 혈관표면의 CD73(ecto-5’ nucleotidase)과 CD39는 면역억제 및 응고인자를 조절하는 효소로 GalT-MCP/-MCP와 더불어 유전자를 과 발현 시킬 경우 이종 이식 시에 생기는 면역거부, 혈액응고 등의 거부반응을 추가로 억제시킬 수 있을 것으로 판단된다. 따라서 본 실험에서는 GalT-MCP/-MCP MGH 미니돼지의 대동맥에서 혈관 내피세 포를 분리 및 배양하여 형태학적, 면역형광염색법으로 그 특성을 확인하였다. CD73은 다 양한 암조직에서 과 발현되어 암세포의 성장을 촉진한다고 알려져 있어 혈관내피세포에 만 특이적으로 발현하는 돼지의 Icam2 promoter에 2A 시스템을 활용하여 CD39/CD73 유전자를 연결한 동시 발현 벡터를 제작 후 도입하였다. 4주령 MGH 미니돼지로부터 적 출한 대동맥에서 type 1 collagenase 효소를 이용하여 혈관내피세포를 분리하였다. 분리 된 세포는 내피세포의 표지인자인 vWF(von Willebrand factor) 와 CD31 antibody를 사용 하여 면역형광염색법으로 염색하거나 신생혈관생성을 확인하는 tube formation assay를 실시하여 혈관내피세포임을 확인하였다. 그 다음, CD39/CD73 발현 벡터를 nucleofection 방법으로 혈관내피세포 내에 도입한 후 flow cytometry로 발현을 분석하였다. CD39와 CD73의 발현율은 각각 33.4%, 26.8%로 나타났다. 따라서 본 연구에서는 GalT-MCP/-MCP MGH 돼지의 대동맥에서 분리한 혈관내피세포에 혈액응고인자를 조절할 수 있는 유전자 를 도입하였을 경우 이종이식의 부작용을 억제하는 연구에 유용하게 사용될 수 있을 것 으로 판단된다.

The α-Gal epitope (Galα1,3Galα1,4GlcNAc-R), created by alpha-1,3-glycosyltransferase-1 (GT), is recognized by natural anti-α-1,3-galactose antibodies in human serum and responsible for hyperacute rejection (HAR) in pig-to-human/primate xenotransplantation. Although, the generation of GT knockout (KO) pigs has overcome hyperacute rejection, an isoglobotriosylceramide synthase (iGb3s) may produce additional alpha-1,3-terminated galactose residues on glycosphingolipid. This study was performed to investigate effects of iGb3s KO on the glycoproteomic characterization in the transgenic pig cells. We established and used 2 different transgenic cells such as GT-MCP/-MCP (GT/MCP) and GT-MCP/-MCP+iGb3s—/— (GT/MCP/iGb3s) cells. WT pig cells were used as a control. To characterize the expression of α-Gal epitope, we analyzed the cells by FACS using BS-I-B4 lectin antibody. The α-Gal epitope expression in all both TG cells was significantly down regulated as compared with WT (p<0.05). The iGb3s protein level was decreased in GT/MCP/iGb3s cells compared with GT/MCP cells. To analyze changes of glycoproteome level, LC-MS/MS analysis was performed in WT, GT/MCP, and GT/MCP/iGb3s cells. A total amount of glycopeptide in GT/MCP/iGb3s was increased as compared with that of GT/MCP. However, glycopeptide forms of gal-gal were reduced in GT/MCP/ iGb3s compared to both WT and GT/MCP. Our results demonstrated that additional KO of iGb3s gene reduced the expressions of α-Gal epitope and glycopeptide forms of gal-gal in GT/MCP. Further studies are needed to evaluate synergic effects of double gene knock out in order to minimize a HAR response after xenotransplantation.

형질전환 닭을 생산하는데 있어서 필수적인 유전자 전이 방법은 retrovirus 또는 lentivirus vector system을 이용하여 X기의 배반엽 세포에 외래 유전자를 전이시키는 방법이 가장 널리 이용되고 있다. 그러나 최근의 형질전환 닭의 생산에 관한 몇몇의 연구 보고 에서 도입된 virus vector 내의 일부 서열이 결손된 것으로 확인되었으며, 결손된 부위의 유전자 서열은 포유류를 대상으로 한 연구에서 보고된 이소성의 splicing donor 부분과는 상관관계가 없는 다른 염기 서열이 해당되는 것으로 보고되었다. 본 연구에서는 고양이 면역결핍 바이러스(feline immunodeficiency virus, FIV) 유래의 FIV-Ov23p-hEPOW lentivirus vector를 이용하여 난관 조직 특이적으로 hEPO 유전자가 발현되는 형질전환 닭을 생산하였으며, 이로부터 유래한 G1 세대 형질전환 개체에서 닭의 genome에 도입된 lentivirus vector 서열의 결손을 발견하였다. Virus vector 서열의 결손은 Southern blotting, genomic DNA PCR 분석 및 DNA sequencing을 실시하여 확인하였다. 그 결과, 도입된 lentivirus 서열 중 2.3 kb 크기의 ovalbumin promoter 내의 estrogen response element (ERE) 부분에서 443 bp가 결손된 것을 확인할 수 있었으며, 앞서 보고된 여러 연구 결 과와 마찬가지로 특정 서열과는 상관관계가 없는 부분이 소실된 것으로 확인되었다. 그 러나 promoter 부분의 결손에서 불구하고 G1 형질전환 닭이 산란한 계란에는 최대 371 IU/mL (3.1 μg/mL) hEPO 단백질이 생산되었으며, 혈액에서는 22.5 mIU/mL (0.19 ng/ mL)의 hEPO가 포함되어 있었다. 즉, enhancer로 알려진 ERE의 일부 결손이 난관 조직 을 통한 단백질의 발현량을 크게 증가시키진 못하였으나 난관 조직 특이적 발현에는 영 향이 없는 것으로 나타났다. 이상의 연구 결과는 FIV 유래의 lentivirus vector system을 이용하여 생산한 형질전환 닭에서 도입되는 vector 서열의 결손에 대한 실례를 제공하는 것으로, 결손이 일어나지 않는 새로운 형태의 virus vector system의 개발을 모색할 필요 성을 제시하는 데에 의의가 있다.

In the pig industry, foot and mouth disease (FMD) and porcine reproductive and respiratory syndrome (PRRS) cause lots of social and economic losses annually. Many resourses have been used to prevent these viruses, however, vaccinations have obvious limitation on viral diseases. It is difficult to prevent the diseases, because viruses constantly change their genetic information naturally. Therefore, genetic modification of the host has been studied to overcome the limitations of vaccine. In this study, disease resistance was introduced with two genetic modification methods. First the PRRSV viral receptor (CD163) was edited with the CRISPR/Cas9 technique. Porcine fetal fibroblasts (pFF) with the CRISPR/Cas9 vector were single cell cultured to make unique cell lines. As results, CD163 sequences of edited cells showed difference with control cells. Second, shRNAs that target 3D gene (encoding FMDV RNA polymerase) and ORF7 (encoding PRRSV nucleocapsid protein) were introduced into pFF. Those techniques above were combined into single cell line by introducing shRNAs into CD163-edited cells. Vectors expressing the 3D gene and ORF7 gene were lipofected into the cells to confirm shRNA activity in vitro. The cells containg shRNAs showed significantly lower level of transcript than the control cells (about 9-fold in 3D and 160- fold in ORF7). The multi-resistant cells also showed decreasing in transcript level of viral genes (more than 4.8-fold in 3D and 3.8-fold in ORF7). With all these results, we obtained the multi-resistant cell line against FMD and PRRS, and established in vitro shRNA verification system successfully.

As a disease model, rats have similar physiological characteristics to humans and are used in various research methods. However, a protocol to generate genome-edited rats has not been well established. Since its development as a genome editing tool, the CRISPR/ Cas9 system has been widely adapted as an efficient tool to generate knockout (KO) or transgenic rats. In general, microinjection technique, which involves direct injection of CRISPR/ Cas9 into one-cell embryos were utilized for generating KO rodents. However, this technique has several disadvantages: 1. User has to be well-trained to manipulate single zygotes; 2. User can only inject one cell at a time (long and laborious); 3. Low viability after microinjection is observed. To overcome these, we adapted electroporation system which is relatively easy to handle and up to 100 zygotes can be electroporated at once. In this study, female Sprague Dawley (SD) rats at 8-week-old were superovualted and mated with mature male SD rat. Zygotes were obtained the following day and eletroporation was performed (Genome Editor, BEX CO, Japan) to introduce sgRNA and Cas9 protein targeting slc16a2 gene. Zygotes were cultured in a incubator and only two-cell stage embryos were transferred into the recipients. After gestation periods, genome edited founder (F0) offspring were born and confirmed by genomic PCR. Homozygous KO pups (F1) were delivered by mating founder rats. While higher levels of serum triiodothyronine were detected in homozygous KO (1.30±0.26 ng/mL, n=3) than control group (1.13±0.21 ng/mL, n=3), thyroxine levels were lower in the homozygous KO F1 (3.13±0.50 μg/dL, n=3) compared to the control group (6.53±1.19 μg/dL, n=3). In addition, there was a significant difference in body weight between homozygous KO F1 and the control group (homozygous KO F1 : 287.23± 8.26 g vs control group : 339.03+52.26 g, p<0.05, t-test, SPSS). Using this method, KO rats were produced with high efficiency. In the future, electroporation-based genome editing technology can provide simple and efficient methods to produce KO rat models.

The production of pharmaceutical proteins by transgenic animals is one of the major successes of biotechnology. Knock-in system is a more powerful method to produce mammary gland bioreactor. To date, zinc-finger nuclease(ZFNs), transcription activator- like effector nuclease(TALENs), and clustered regularly interspaced short palindromic repeats(CRISPR)/Cas9 systems have been developed for gene targeting. The objective of this study was to develop a knock-in embryo for expression of bovine lactoferrin in the bovine β-casein gene locus by microinjection of knock-in vector with TALEN or CRISPR/Cas9 into bovine zygote. The three kinds of replacement knock-in vectors containing a different length of homologous arm were constructed. These targeting vectors were used enhanced green fluorescent protein(eGFP) as a positive selection marker. These knock-in vectors with TALEN or CRISPR/Cas9 were microinjected into the pronuclear bovine embryo treated cytochalasin B. And the embryos were cultured to blastocyst in the culture medium. These blastocysts were analyzed by PCR to confirm gene targeting by homologous recombination. As a result, when bLF_1kbHR_GFP knock-in vector with TALEN was microinjected into cytoplasm of bovine zygotes, the efficiency of gene targeting was 11.1-14.3%. Also, when bLF_1kbHR and 40HR_GFP knock-in vector with CRISPR/Cas9 was microinjected into cytoplasm of bovine zygotes, the efficiency of gene targeting was 22.2-26.7% but gene targeting by homologous recombination was not detected when bLF_100HR_GFP knock-in vector was microinjected into cytoplasm of bovine zygotes. The precise bovine lactoferrin gene integration of knock-in embryos was confirmed by DNA sequencing analysis. Our knock-in system may help to create transgenic dairy cattle expressing enhanced bovine lactoferrin protein in the mammary gland via the endogenous expression system of the bovine β-casein gene.

The production of transgenic livestock has the opportunity to significantly improve human health, enhance nutrition, protect the environment, increase animal welfare, and decrease livestock disease. In this study, bovine PN embryos were injected in cytoplasm with three groups (Ⅰ: sgRNA+Ca9 protein+KI_hFGF_GFP vector, Ⅱ: sgRNA+Ca9 mRNA+KI_hFGF_GFP vector and Ⅲ: bCNS_EX3_sgRNA+pBSK_sgRNA+Ca9 protein+ KI_hFGF_GFP vector) at 18~20 h after IVF, and treated 7.5 uM RS-1 (RAD51-stimulatory compound 1) for 24 h after microinjection. The survival rate post day 2 was higher in group Ⅰ (84.4%) than other groups. At day 4, embryo development potential (78.1%) and GFP expression was significantly highest in group Ⅲ (54.5%) among treatment groups (p<0.05). At day 8, while the blastocyst formation was significantly high in group Ⅰ(32.1%) among three groups, the GFP expression embryo rate of developed blastocyst was higher in group Ⅱ (34.8%) and Ⅲ (38.5%) than in group Ⅰ (17.1%). The total injection efficiency of GFP embryo production from gene injected embryos was a little higher in group I (5.5%, 6/109) than in group II (4.1%, 8/194) and group III (3.6%, 5/139). However, the GFP expressed healthy embryos were the more presented in group II than other groups. Also, when the effect of 7.5 uM RS-1 treatment to the injected embryos for 24 h were examined, the survival rate post day 2 was higher in RS-1 treatment group (72.5%) than in control group (67.3%). The GFP expression rate was also higher in RS-1 treatment group (57.6% and 58.3%) than in control group (38.8% and 19.2%) at day 4 and 8 development in vitro. This study demonstrates that it can be helpful to the production of hFGF knock-in bovine embryos using combination of bCNS_EX3_sgRNA+pBSK_sgRNA+Ca9 protein+KI_hFGF_ GFP vector and treatment with RS-1.

Xenotransplantation has been getting attention as an attractive strategy to overcome lack of organ-donors in clinical transplantation. However, xenotransplantation show more severe immune responses than organ transplants of the same species. In particular, the hyper acute rejection is caused by the alpha(1,3) galactosyl-transferase expressed on the porcine organ cell membrane and the organ dies within a few minutes. Recently, alpha(1,3)galactosyl-transferase knockout pigs to reduce the problem of hyper acute rejection. However, when the organs survive in the body for a long time, cell- mediated immune rejection occurs due to intrinsic immune response and adaptive immune response, which are left as obstacles for successful xenograft. This immune response is mainly interacted with the MHC class I molecule and various receptors of the immune cell. The US11 gene and the US2 gene are hCMV(human cytomegalovirus)-derived genes that retro-translocate MHC Class I molecules in the endoplasmic reticulum to inhibit expression on the surface of cell membrane, resulting in an immune evasion response. Our study focused on introducing the US11 and US2 genes into the pig fibroblast to evaluate gene function and reduce cell mediated rejection through two genes. The fetal fibroblast was obtained from Korean native pig, and the US2 and US11 genes were inserted into the cells by constructing pCAGGS/US11, pcDNA3.1/ US2 and pcDNA3.1/US2-2A-US11 vectors. G418 (500 ng/mL) and Blasticidin (20 ng/ mL) were treated for 14 days, and gene expression was confirmed by RT-PCR. The MHC Class I expression was confirmed by FACS using SLA-I antibody. NK cells and CD8+T cells were co-cultured with transgene cells and MTT assay was used to confirm results of cytotoxicity. In the US2 and US11 transfected cell lines, MHC class I expression was found to be higher than Wildtype in FACS results, it’s suggested that the expression of this gene was increased as part of the cell defense mechanism due to the insertion of the virus gene. In addition, there were no significant differences in cytotoxicity of NK cell of US11 and US2, it’s presumed that NK cell mainly respond to degranulation of perforin and granzyme or binding of NKG2A, NKG2D receptor than MHC Class I immune response. On the other hand, cytotoxicity of CD8+ T cells was shown lower cell lysis than the control group. Further studies will conduct that the confirm to performance of co-expression of US2 and US11 on immune reject and will apply a novel strategy to attenuate the activity of NK cells.

This study aims to find effect of natural mating and cytotoxicity regulate from US11 and hDAF gene transgenic pig. Piglets that has US11, hDAF or US11&hDAF genes were born from natural mating of female US11 gene transgenic pig and male hDAF gene transgenic pig. Among 10 piglets were born, there are 2 piglets with US11 gene only, 2 piglets with hDAF gene only, 5 piglets with both US11 and hDAF gene, and 1piglets without transgenes. Sex ratio of piglets was 1:1, and transfection rate from natural breeding showed result that only US11 gene was 20%, only hDAF gene was 20%, and both US11 gene and hDAF gene was 50%. In cytotoxicity effect, US11 gene was showed a tendency of reduce human NK cell cytotoxicity in 10:1 ratio. In complement assay, hDAF gene was reduce human complement cytotoxicity in both 1:1 and 10:1 ratio of human complement. hDAF gene was also effected in reduce human NK cell cytotoxicity that showed similar result with US11 gene. Double transgenic piglets fibroblast that has both US11 and hDAF resulted that decrease both human NK cell and complement cytotoxicity compared with wild type cells. In conclusion, Natural mating was effected for US11 and hDAF gene transmitted into offsping piglets, and US11 and hDAF gene were expected to regulate xenograft rejections in transgenic tissues.

In this study, we mutated SH3 domain containing ring finger 2 (SH3RF2) gene to investigate the biological functionality through CRISPR/CRISPR-associated protein 9 (Cas9)-mediated genome editing technology. SH3RF2 has been reported for one of the critical domestication genes in chickens and it expected that SH3RF2 could be closely related to growth improvement in chicken. However, its biofunctionality still remains to be elucidated. Firstly, we cloned the partial genomic structure and identified sequences of the quail SH3RF2 gene. Subsequently, SH3RF2 was knocked out using CRISPR/Cas9 technology and single cell-derived SH3RF2 mutant line was established in quail myoblast (QM7) cells. As a result, we established an SH3RF2 knockout QM7#4 subline which had 61 and 155 nucleotide deletion mutations. After the induction of myotube differentiation, the expression profiles during muscle differentiation were analyzed and compared between regular QM7 and SH3RF2 KO QM7#4 cells by global RNA sequencing and bioinformatics analysis. Additionally, we also mutated SH3RF2 gene in chicken primordial germ cells (PGCs) to generate the SH3RF2 knockout chickens. Thus, to investigate the biofunctional activity of a predicted target gene, in vivo and in vitro functional genomic analyses should be a prerequisite.