The Transgenic livestock can be useful for the production of disease-resistant animals, pigs for xenotranplantation, animal bioreactor for therapeutic recombinant proteins and disease model animals. Previously, conventional methods without using artificial nuclease-dependent DNA cleavage system were used to produce such transgenic livestock, but their efficiency is known to be low. In the last decade, the development of artificial nucleases such as zinc-finger necleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas has led to more efficient production of knock-out and knock-in transgenic livestock. However, production of knock-in livestock is poor. In mouse, genetically modified mice are produced by co-injecting a pair of knock-in vector, which is a donor DNA, with a artificial nuclease in a pronuclear fertilized egg, but not in livestock. Gene targeting efficiency has been increased with the use of artificial nucleases, but the knock-in efficiency is still low in livestock. In many research now, somatic cell nuclear transfer (SCNT) methods used after selection of cell transfected with artificial nuclease for production of transgenic livestock. In particular, it is necessary to develop a system capable of producing transgenic livestock more efficiently by co-injection of artificial nuclease and knock-in vectors into fertilized eggs.

Morphology of cumulus-oocyte-complexes (COCs) at germinal vesicle (GV) stage as one of the evaluation criteria for oocyte maturation quality after in vitro maturation (IVM) plays important roles on the meiotic maturation, fertilization and early embryonic development in pigs. When cumulus cells of COCs are insufficient, which is induced the low oocyte maturation rate by the increasing of reactive oxygen species (ROS) in porcine oocyte during IVM. The ROS are known to generate including superoxide and hydrogen peroxide from electron transport system of mitochondria during oocyte maturation in pigs. To regulate the ROS production, the cumulus cells is secreted the various antioxidant enzymes during IVM of porcine oocyte. Our previous study showed that Mito-TEMPO, superoxide specific scavenger, improves the embryonic developmental competence and blastocyst formation rate by regulating of mitochondria functions in pigs. However, the effects of Mito-TEMPO as a superoxide scavenger to help the anti-oxidant functions from cumulus cells of COCs on meiotic maturation during porcine oocyte IVM has not been reported. Here, we categorized experimental groups into two groups (Grade 1: G1; high cumulus cells and Grade 2: G2; low cumulus cells) by using hemocytometer. The meiotic maturation rate from G2 was significantly (p < 0.05) decreased (G1: 79.9 ± 3.8% vs G2: 57.5 ± 4.6%) compared to G1. To investigate the production of mitochondria derived superoxide, we used the mitochondrial superoxide dye, Mito-SOX. Red fluorescence of Mito-SOX detected superoxide was significantly (p < 0.05) increased in COCs of G2 compared with G1. And, we examined expression levels of genes associated with mitochondrial antioxidant such as SOD1, SOD2 and PRDX3 using a RT-PCR in porcine COCs at 44 h of IVM. The mRNA levels of three antioxidant enzymes expression in COCs from G2 were significantly (p < 0.05) lower than COCs of G1. In addition, we investigated the anti-oxidative effects of Mito-TEMPO on meiotic maturation of porcine oocyte from G1 and G2. Meiotic maturation and mRNA levels of antioxidant enzymes were significantly (p < 0.05) recovered in G2 by Mito-TEMPO (0.1 μM, MT) treatment (G2: 68.4 ± 3.2% vs G2 + MT: 73.9 ± 1.4%). Therefore, our results suggest that reduction of mitochondria derived superoxide by Mito-TEMPO may improves the meiotic maturation in IVM of porcine oocyte.

미토콘드리아는 세포질 칼슘 항상성 및 ATP 생산에 중요한 역할을 하는 세포 소기관으로 이러한 미토콘드리아의 기능은 성숙과 수정 그리고 배 발달에 매우 중요한 역할을 한다. 미토콘드리아 칼슘 축적은 기능장애를 일으킨다. 그러나 돼지 체외성숙란 및 수정란에서 미토콘드리아 칼슘 변화의 관련성에 관한 연구는 보고된 적이 없다. 본 연구의 목적은 미토콘드리아 칼슘 지시자로 알려진 Rhod-2 염색을 이용하여 성숙란 및 수정란에서 미토콘드리아 칼슘 축적의 변화를 확인하였다. 형태학적 모습의 기준을 통해 난구세포의 세포층과 세포질의 균질도를 바탕으로 G1과 G2로 나누어서 체외성숙을 진행하였다. 이후 두 그룹에서 핵 성숙율을 비교하였을 때, G2가 G1에 비해 낮게 나타났다(p<0.001). 돼지 체외성숙란 및 수정란에서 평균적인 Rhod-2 spot 의 수는 G1보다 G2에서 더 많이 나타났다(6시간째 체외수정란: p<0.05). 다음으로 Rhod-2 spot 수에 따른 난모세포의 비율을 확인하기 위해 Rhod-2 spot 의 수를 4개의 군(n<10, 10≤n<20, 20≤n<30, 그리고 30≤n)으로 나누어 해당 난모세포의 비율을 확인하였다. 체외성숙란 및 체외수정란 모두 G1이 G2에 비해 10개 미만(n<10)인 Rhod-2 spot 의 수를 가지는 난모세포가 많았으며, 체외수정란에서는 유의적으로 높았다(p<0.05). 마지막으로 체외성숙란 및 수정란에서 Rhod-2 intensity 값을 측정하여 두 그룹을 비교하였을 때, G2가 G1에 비해 유의적으로 높은 것을 확인 할 수 있었다(성숙란; p<0.001 그리고 수정란; p<0.05). 본 연구의 결과를 토대로 돼지에서 미성숙 난포란의 형태학적인 품질은 체외성숙 및 체외수정 과정 동안 미토콘드리아 내 칼슘 축적과 관련이 있음을 확인하였다.

본 연구는 한우 번식우의 다양한 사료급여 방법에 의한 사양환경에서 체내 수정란의 회수율을 조사하고 혈중 주요 대사성분을 분석하고자 수행되었다. 연구자가 관리하고 있는 강원도 횡성에 위치한 4 농가(A~D)와 경기도 이천에 위치한 1 농가(E)를 대상으로 18 년 7 월~9 월간 실험이 이루어졌으며 5 농가에서의 사료 급여는 각 농후사료 2 kg + 건초 2 kg + 사일리지 1 kg(A 농가), 농후사료 3.5 kg + 볏짚 무제한(B 농가), 농후사료 3 kg + 건초 3 kg 2 회(C 농가), 농후사료 3.6 kg + 건초 4 kg(D 농가), TMR 5 kg(E 농가)의 방법에 따라 이루어졌다. 각 농가별 혈중 주요대사물질 분석을 위해 수정란 채란 당일 경정맥을 통해 혈액을 채취하였고 분리된 혈청은 -20℃에서 냉동보관 후 혈액분석기를 통해 NEFA, Glucose, BUN, Cholesterol, AST, GGT 항목을 일괄적으로 분석하였다. 공란우의 과배란 유도를 위하여 CIDR-plus(InterAg, Hamilton, New Zealand)를 질 내에 삽입 후 7 일째부터 FSH 제제인 Folltropin-V(Vetrepharm, Canada)를 투여하였고 총 FSH 200mg 을 40mg, 30mg, 20mg, 10mg 의 용량으로 오전, 오후 각 2 회씩 점감적으로 주사하여 총 8 회에 걸쳐 12 시간 간격으로 근육에 주사하였다. FSH 투여 3 일째에 PGF2a 제제인 Lutalyse(Zoetis, Belgium)를 25mg 1 회 투여하고 CIDR-plus 를 제거하였으며 FSH 투여 개시 후 5 일째, 오전에 GnRH 제제(Fertagyl; Intervet, USA)를 250ug 투여하고 12시간 간격으로 인공수정을 2 회 실시하였다. 수정란의 채란은 2 차 인공수정 7 일 후에 balloon catheter 를 자궁 내에 주입 및 장착하여 1-way 방법인 DEC(Direct Embryo Collection)에 의해 이루어졌다. 회수된 수정란은 회수된 총 수정란수, 이식가능한 수정란 수를 조사하였다. 실험결과, 회수된 총 수정란수는 A 농가 16±2 개, B 농가 36±3 개, C 농가 18±1 개, D 농가 12±1 개, E 농가 14±4 개로 확인되었으며 이식가능한 수정란수는 각 농가별 14±3 개, 22±3 개, 11±1 개, 6±3 개, 2±2 개로 조사되었다. 총 회수 수정란 대비 이식가능한 수정란의 이용효율은 A 농가가 87.7±3 %로 가장 높게 관찰되었으며 E 농가가 14.5±2 %로 가장 낮게 확인되었다. 공란우의 MPT 분석 결과, NEFA 는 A 농가 125.15 uEq/L, B 농가 185.40 uEq/L, C 농가 177.91 uEq/L, D 농가 103.53 uEq/L, E 농가 232.56 uEq/L 로 조사되었다. Glucose 는 각 농가별 48.00 mg/dl, 66.15 mg/dl, 68.73 mg/dl, 61.42 mg/dl, 39.74 mg/dl 로 분석되었으며 BUN 은 각 13.73 mg/dl, 15.07 mg/dl, 9.01 mg/dl, 16.64 mg/dl, 19.37 mg/dl 로 확인되었다. Cholesterol 은 각 179.85 mg/dl, 162.20 mg/dl, 142.36 mg/dl, 132.68 mg/dl, 152.95 mg/dl 로 분석되었으며 AST & GGT 는 85.00 & 17.60 IU/L, 78.75 & 17.50 IU/L, 75.95 & 22.91 IU/L, 79.26 & 13.42 IU/L, 89.58 & 21.89 IU/L 로 조사되었다. 결론적으로 체내 수정란의 이용효율과 MPT 분석결과를 비교하면 Glucose, BUN, Cholesterol, AST, GGT 의 경우 상관관계를 조사할 수 없었으나, NEFA 의 경우 체내 수정란의 이용효율이 14.3 %로 가장 낮게 관찰된 E 농가의 혈중 NEFA 농도가 232.58 uEq/L 로 가장 높게 관찰된 것이 차이점으로 조사되었다. 본 연구결과는 농촌진흥청 연구 사업(세부과제번호:PJ012695032018)의 지원에 의해 이루어진 것임.

본 연구는 한우 체외 수정란의 동결보존에 hyaluronate(HA), ethylene glycol(EG), glycerol(G), sucrose(S)를 사용한 동결배양액의 효과를 검증하고자 수행되었다. 체외 수정란 생산을 위해 도축장에서 회수된 난소로부터 미성숙 난자를 채취하여 체외성숙, 수정, 발달 배양을 실시하였다. 성숙배양은 0.5 ug/ml FSH, 5 ug/ml LH, 0.125% BSA(v/v)가 첨가된 mTCM199 배양액에서 38.5 ℃, 5 % CO2 의 조건으로 22 h 동안 실시하였으며 체외수정은 mTALP 배양액에서 38.5 ℃, 5 % CO2 의 조건에서 22 h 동안 이루어졌고 발달배양은 mSOFaa 배양액을 사용하여 38.5 ℃, 5 % CO2, 5 % O2 의 조건에서 이루어졌다. 동결에 이용된 수정란은 수정배양 후 7 일차의 배반포를 대상으로 하였다. 실험에 사용된 동결배양액은 대조군으로써 vigro 의 1.8M EG 에 0.1 M S 가 첨가된 제품을 사용하였고 처리군으로써 1.8M EG 배양액, 1.8M EG 에 0.1M S 와 1.7M G 이 첨가된 배양액, 1.8M EG 에 0.1M S, 1.7M G 및 0.0007 g/ml HA 가 첨가된 배양액을 사용하였다. 모든 처리군은 0.01 g/ml Albumax 가 첨가된 CJ buffer 를 동결배양액의 base 로 사용하였다. 동결방법은 0.25 ml 스트로에 장착된 수정란을 CL-8800i(CryoLogic)에 의한 –0.3℃/min 의 완만동결로 –32℃까지 냉각시킨 후 액체질소에 침지하여 실시하였다. 동결과정 중, -6℃에서 스트로의 말단 부분에 식빙을 실시하였다. 동결융해 후 생존율 조사를 위해 동결보존 중인 스트로 32℃의 온수에서 25 초간 융해를 실시하여 발달배양액에서 48 시간 배양하여 총 동결융해 수정란에 대한 재확장율 및 부화율을 통해 생존율을 확인하였다. 실험결과, 동결융해 후 재확장율은 대조군(EG+S)과 EG, EG+S+G, EG+S+G+HA 에서 각 90.1±0.5, 76.0±1.0, ±85.4±2.1, 84.9±0.5 %로 관찰되었으며 부화율은 80.2±1.0, 70.8±4.2, 74.0±1.0, 82.8±1.6 %로 확인되었다. 재확장율은 대조군인 1.8M EG + 0.1M sucrose 배양액에서 90.1±0.5 %로 가장 유의적으로 높게 관찰되었으며 부화율은 대조군과 1.8M EG + 0.1M sucrose + 1.7M glycerol + 0.0007 g/ml HA 처리군에서 각 80.2±1.0 %와 82.8±1.6 %로 가장 유의적으로 높게 관찰되었다(p<0.05). 추가적으로 본 동결방법에 의해 생산된 동결수정란의 수정란이식을 실시하여 수태율을 조사하고 있지만 결과가 부족하여 본 연구결과에서 포함시키지 못하였다. 하지만 수정란 이식과정에서 EG+S+G 처리군의 경우 30 분이 경과된 시점부터 다른 처리군보다 수태율이 감소되는 경향이 뚜렷하게 관찰되었다. 반면, glycerol 과 HA 가 함께 첨가된 다른 동결배양액의 경우에는 이러한 경향이 관찰되지 않았다. 다른 체외수정란의 발달연구에서 HA 의 첨가는 부화율을 증가시킨다는 연구결과가 보고된 바 있는데 이러한 연구결과와 일치된다고 생각된다. 본 연구결과는 농촌진흥청 연구사업(세부과제번호:PJ012695032018)의 지원에 의해 이루어진 것임.

This study aimed to produce high-quality blastocysts and establish appropriate microinjection conditions for the introduction of target gene. First, we identified embryo development to the blastocyst stage after microinjection using the CRISPR/Cas9 system on the Cas9 protein or mRNA. As a result, we confirmed that blastocyst development in the Cas9 mRNA injected group significantly increased when compared to the Cas9 protein injected group (p<0.05). However, the blastocyst gene targeting rate increased in the Cas9 protein injected group when compared to the Cas9 mRNA injected group (p<0.05). Next, we treated the injection medium with 10 μg/ml of cytochalasin B (CB), and the microinjected embryos were cultured in CR1-aa medium supplemented with 0.1 μM of melatonin (Mela). Consequently, the blastocyst formation rate significantly increased in the CB treated group (p<0.05). After microinjecting embryos with the CB treated injection medium, we investigated blastocyst formation and quality via Mela treatment. Consequently, the Mela treated group demonstrated significantly increased blastocyst formation rates when compared to the non-treated group (p<0.05). Furthermore, immunofluorescence assay using RAD51 (DNA repair detection protein) and H2AX139ph (DNA damage detection protein) showed an increase in RAD51 positive cells in Mela treated embryos. Therefore, we verified the improvement in knock-in efficiency in microinjected bovine embryos using Cas9 protein. These results also demonstrated that the positive effect of the CB and Mela treatments improved the embryonic developmental competence and blastocyst qualities in genetically-edited bovine embryos.

The production of therapeutic protein from transgenic domestic animal is the major technology of biotechnology. Insulin-like growth factor-1 (IGF-1) is known to play an important role in the growth of the animal. The objective of this study is construction of knock-in vector that bovine IGF-1 gene is inserted into the exon 7 locus of β-casein gene and expressed using the gene regulatory DNA sequence of bovine β-casein gene. The knock-in vector consists of 5’ arm region (1.02 kb), bIGF-1 cDNA, CMV-EGFP, and 3’ arm region (1.81 kb). To express bIGF-1 gene as transgene, the F2A sequence was fused to the 5’ terminal of bIGF-1 gene and inserted into exon 7 of the β-casein gene. As a result, the knock-in vector is confirmed that the amino acids are synthesized without termination from the β-casein exon 7 region to the bIGF-1 gene by DNA sequence. These knock-in vectors may help to create transgenic dairy cattle expressing bovine bIGF-1 protein in the mammary gland via the expression system of the bovine β-casein gene.

The knock-in efficiency in the fibroblast is very important to produce transgenic domestic animal using nuclear transfer. In this research, we constructed three kinds of different knock-in vectors to study the efficiency of knock-in depending on structure of knock-in vector with different size of homologous arm on the β-casein gene locus in the somatic cells; DT-A_cEndo Knock-in vector, DT-A_tEndo Knock-in vector I, and DT-A_tEndo Knock-in vector II. The knock-in vector consists of 4.8 kb or 1.06 kb of 5’ arm region and 1.8 kb or 0.64 kb of 3’ arm region, and neomycin resistance gene(neor) as a positive selection marker gene. The cEndo Knock-in vector had 4.8 kb and 1.8 kb homologous arm. The tEndo Knock-in vector I had 1.06 kb and 0.64 kb homologous arm and tEndo Knock-in vector II had 1.06 kb and 1.8 kb homologous arm. To express endostatin gene as transgene, the F2A sequence was fused to the 5’ terminal of endostatin gene and inserted into exon 7 of the β-casein gene. The knock-in vector and TALEN were introduced into the bovine fibroblast by electroporation. The knock-in efficiencies of cEndo, tEndo I, and tEndo II vector were 4.6%, 2.2% and 4.8%, respectively. These results indicated that size of 3’ arm in the knock-in vector is important for TALEN-mediated homologous recombination in the fibroblast. In conclusion, our knock-in system may help to create transgenic dairy cattle expressing human endostatin protein via the endogenous expression system of the bovine β-casein gene in the mammary gland.

Humulus japonicus is an ornamental plant in the Cannabaceae family. Although the mode of action of Humulus japonicus is not fully understood, a strong relationship was observed between anti-inflammatory and anticancer in some types of cells. Recent studies also have shown that Humulus japonicus possesses anti-inflammatory activities and may significantly improve antioxidant potential in Raw 264.7 macrophage cells. Thus, the aim of this study was eva-luated the effect of Humulus japonicus extract on sperm motility and subsequent preimplantation developmental com-petence of the bovine embryos. After in vitro maturation, the oocytes with sperms were exposed in in vitro fertilization (IVF) medium supplemented with Humulus japonicus extract (0.01, 0.05, 0.1 μg/mL, respectively) for 1 day. In our results, exposure of IVF medium to Humulus japonicus extract did not affect sperm motility and percentage of pene-trated oocytes but ROS intensity was significantly decreased by 0.01 μg/mL compared with other groups (p< 0.05). Moreover, treatment with 0.01 μg/mL of Humulus japonicus extract was higher the frequency of blastocyst formation than the any other groups (p<0.05). Otherwise, treatment with 0.01 μg/mL of Humulus japonicus extract not increased the total cell number but reduced apoptotic-positive nuclei number. In conclusion, our results indicate that supple-mentation of Humulus japonicus extract in IVF medium may have important implications for improving early embryo-nic development in bovine embryos

Pyracantha is a genus of thorny evergreen large shrubs in the family of Rosaceae, with common names Firethorn or Pyracantha. It's extract has also been used in cosmetics as a skin-whitening agent and functioning through tyrosinase inhibition. Recent studies have shown that pyracantha extract possesses antioxidant activities and may significantly improve lipoprotein metabolism in rats. Although the mode of action of Pyracantha extract is not fully understood, a strong relationship was observed between antioxidant and apoptosis in some types of cells. Thus, the aim of this study was to evaluated the effect of pyracantha extract on blastocysts formation and their quality of the porcine parthenogenetic embryos. After parthenogenetic activation by chemicals, presumptive porcine parthenogenetic embryos were cultured in PZM-3 medium supplemented with extracts of pyracantha leaf, stalk and root for 6 day (1, 5 and 10 μg/ml, respectively). In our results, the frequency of blastocyst formation in pyracantha root extract (5 μg/ml) treated group had increased that of other groups. Furthermore, blastocysts derived from pyracantha root extract (5 μg/ml) treated group had increased the total cell numbers and reduced apoptotic index. Blastocyst development was significantly improved in the pyracantha root extract (5 μg/ml) treated group when compared with the H2O2 treated group (p<0.05). Subsequent evaluation of the intracellular levels of ROS in pyracantha root extract (5 μg/ml) treated groups under H2O2 induced oxidative stress were decreased (p<0.05). In conclusion, our results indicate that treatment of pyracantha root extract may improve in vitro development of porcine parthenogenetic embryos through its antioxidative and antiapoptotic effects.

The key regulators of apoptosis are the interacting protein of the Bcl-2 family. Bcl-2, an important member of this family, blocks cytochrome C release by sequestering pro-apoptotic BH3-only proteins such as Bid, Bad, Bax and Bim. The pro-survival family members (Bcl-2, Bcl-XL, Bcl-W) are critical for cell survival, since loss of any of them causes cell death in certain cell type. However, its role during early porcine embryonic development is not sufficient. In this study, we traced the effects of Bcl-2 inhibitor, ABT-737, on early porcine embryonic development. We also investigated several indicators of developmental potential, including gene expression (apoptosis-related genes) and apoptosis, which are affected by ABT-737. Porcine embryos were cultured in the PZM-3 medium with or without ABT-737 for 6 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without ABT-737 (14.7±3.0 vs 30.3±4.8%, p<0.05). TUNEL assay showed that the number of containing fragmented DNA at the blastocyst stage increased in the ABT-737 treated group compared with control (4.7 vs 3.7, p<0.05). The mRNA expression of the pro-apoptotic gene Bax increased in ABT-737 treated group (p<0.05), whereas expressions of the anti-apoptotic Bcl-2 family members (Bcl-2, Bcl-XL, Bcl-W) decreased (p<0.05). Also, expressions of the ER stress indicator genes (GRP78, XBP-1 and sXBP-1) increased in ABT-737 treated group (p<0.05). In conclusion, Bcl-2 is closely associated with of apoptosis- and ER stress-related genes expressions and developmental potential in pig embryos.

Heat shock protein 90 (Hsp90) is ATPase-directed molecular chaperon and affects survival of several cells. In our previous study, inhibitory effect of Hsp90 by inducing cell cycle arrest and apoptosis in the pig embryonic and primary cells was reported. However, its role during early bovine embryonic development is not sufficient. In this study, we traced the effects of Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), on early bovine embryonic development. We also investigated several indicators of developmental potential, including structural integrity, gene expression (apoptosis-related genes), and apoptosis, which are affected by 17-AAG. Bovine embryos were cultured in the CR1-aa medium with or without 17-AAG for 7 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without 17-AAG (33.1±9.6 vs 21.7± 8.3%). The structural integrity of the blastocysts was examined by differential staining. Blastocysts from the dbcAMP- treated group had higher numbers of ICM, TE, and total cells than those from the untreated group. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 17-AAG treated group compared with control (11.2 vs 3.9, respectively). Blastocysts that developed in the 17-AAG treated group had low structural integrity and high apoptotic nuclei than those of the untreated control, resulting in decrease the embryonic qualities of preimplantation bovine blastocysts. The m-RNA expression of the pro-apoptotic gene (Bax) increased in 17-AAG treated group, whereas expression of the antiapoptotic gene (Bcl-XL) decreased. In conclusion, Hsp90 also appears to play a direct role in bovine early embryo developmental competence including structural integrity of blastocysts. Also, these results indicate that Hsp90 is closely associated with apoptosis-related genes expression in developing bovine embryos.

Embryonic compaction is essential for normal preimplantation development in mammals. The present study was to investigate the effects of compaction patterns on developmental competence of pig embryos. The proportion of blastocyst formation derived from compacted morula was higher than those of compacting and pre-compacting morula (P<0.01). Nuclei numbers of inner cell mass (ICM), trophectoderm (TE), and total of blastocysts derived from compacted group were also superior to those of compacting and pre-compacting groups (P<0.05). Then, compaction patterns, developmental ability and structural integrity were compared between mono- and poly-spermic embryos. The rate of compacted morula in mono-spermic embryos was higher than that of poly-spermic embryos (P<0.05). Especially, the rate of blastocyst formation derived from compacted embryos in mono-spermic embryo group was higher than that of poly-spermic embryo group (P<0.05), although no difference was detected between the two groups in the structural integrity. Finally, we confirmed that beta-catenin was differentially expressed according to compaction patterns in morula and blastocyst stage embryos. In conclusion, our results suggest that the compaction patterns during preimplantation development play a direct role in developmetal competence and quality of pig embryos.

본 연구는 소 배반포의 내부 세포괴로부터 다능성(pluripotency)을 지닌 배아 줄기 세포(embryonic stem cell) 또는 그 유사 세포를 분리 및 배양함으로써 줄기 세포 관련 분야의 기반 기술을 확립하고자 하였다. 소 체외수정란을 10～12일간 체외배양하여 생산된 부화 배반포를 세포분열이 불활성화된 생쥐 태아 섬유아 세포(mouse embryonic fibroblast, MEF) 위에서 배양하여 콜로니 형성을 유도하였으며, 이들로부터 내부 세포괴 유래의 형태를 지닌 것만을 광학현미경 하에서 물리적으로 분리하여 약 5～7일 간격으로 계대배양을 실시하였다. 이러한 방법을 통하여 배아 줄기 유사 세포의 특성을 40계대 이상 유지하는 2개의 세포주를 확립하였다. 각각의 세포주들은 높은 alkaline phosphatase(AP) 활성을 지니고 있었으며, 형광 면역 염색법과 PCR 기법을 사용하여 Oct-4, Nanog, STAT3, SSEA3 및 SSEA4의 발현을 관찰할 수 있었다. 이러한 결과를 종합하여 볼 때 ,본 연구에서는 소 배반포로부터 배아 줄기 세포주를 확립하는 제반 기술이 확립되었다고 판단되며, 향후 관련 분야 연구에 활용될 수 있을 것으로 기대된다.

본 연구는 소의 초기 난할 단계인 2 또는 4세포기 수정란의 특정 분할구가 배반포 단계의 내부 세포괴(Inner Cell Mass) 와 영양막 세포(Trophectoderm cells)로의 발달 운명이 미리 정해지는 지를 확인하기 위해 실시되었다. 먼저 생쥐의 체내 수정란과 소의 체외 수정란에서 배반포의 영양막 세포에서만 특이적으로 발현하는 cdx2 단백질의 발현 양상을 조사하였다. 또한, 소의 경우 2세포기와 4세포기가 내부 세포괴와 영양막 세포로 나눠지는 시점인지를 조사하기 위해 2 또는 4세포기의 특정 분할구에Dextran의 주입 실험과 분할구 제거 실험을 통해 ICM과 TE 형성을 확인하였다. cdx2의 발현 경향은 생쥐와 소의 2세포기일 때 대칭과 비대칭적으로 발현되는 것을 확인하였다. 생쥐의 4, 8세포기 및 상실배기에서는 분할구 전체에서 발현되었으나, 소 수정란의 분할구에서는 전체 또는 부분적으로 발현되었다. 또한, 생쥐와 소의 배반포기에서는 영양막 세포에서만 발현이 되는 것을 확인하였다. 소 수정란의 2세포기와 4세포기 단계에서 특정 분할구에 주입된 Dextran은 배반포의 내부 세포괴와 영양막 세포의 양쪽에 분포된 것을 관찰할 수 있었다. 2세포기 단계에서 하나의 분할구가 제거된 수정란 역시 ICM 및 TE 세포를 지닌 정상 배반포로 발달함을 확인하였다. 따라서 본 연구 결과는 영양막 세포에서만 특이적으로 발현하는 cdx2의 발현이 2 또는 4세포기 단계 소 수정란에서는 특별한 차이를 보이지 않으며, 궁극적으로 난할 초기에는 ICM과 TE 세포로의 운명이 결정되지 않는다는 것을 보여준다.