돼지 태아섬유아세포 Clonal Lines의 배양에 관한 연구

권대진,박춘근,양부근,김정익,정희태 한국동물번식학회 2003 Reproductive & developmental biology Vol.27 No.1

공여세포 처리 조건이 형질전환 복제돼지 생산에 미치는 영향

권대진,곽태욱,오건봉,김동훈,양병철,임기순,김진회,박진기,황성수,Kwon, Dae-Jin,Kwak, Tae-Uk,Oh, Keon-Bong,Kim, Dong-Hoon,Yang, Byoung-Chul,Im, Gi-Sun,Kim, Jin-Hoi,Park, Jin-Ki,Hwang, Seong-Soo 한국동물번식학회 2011 Reproductive & developmental biology Vol.35 No.3

This study was conducted to investigate the effects of donor cell treatments on the production of transgenic cloned piglets. Ear fibroblast cell obtained from NIH MHC Inbred minipig was used as control. The GalT knock-out/CD45 knock-in (GalT/CD46) transgenic cell lines were established and used as donor cells. The reconstructed GalT/CD46 embryos were surgically transferred into oviduct of naturally cycling surrogate sows (Landrace ${\times}$ Yorkshire) on the second day of standing estrus. Unlike control (1.2 kV/cm, 75.4%), the fusion rate of the GalT/CDl6 donor cells was significantly higher in 1.5 kV/cm, (84.5%) than that of 1.25 kV/cm, (20.2%) (p<0.01). When the number of the transferred embryos were more than 129, the pregnancy and delivery rates were increased to 13/20 (65%) and 5/20 (25%) compared to less then 100 group [1/6 (16.7%) and 0/6 (0%)], respectively. To analyze the effect of donor cell culture condition on pregnancy and delivery rates, the GalT/CD46 donor cells were cultured with DMEM or serum reduced medium. In serum reduced medium group, the pregnancy and delivery rates were improved to 8/12 (66.7%) and 5/12 (41.7%) compared to DMEM group [3/7 (42.9%) and 0/7 (0%)], respectively. In conclusion, it can be postulated that an appropriate fusion condition and culture system is essential factors to increase the efficiency of the production of transgenic cloned piglets.

Strategy for the Generation of Porcine Induced Pluripotent Stem Cells by a Non-integrating RNA Sendai Virus Vector

권대진,황인설,곽태욱,양현,오건봉,옥선아,황성수,임기순 한국실험동물학회 2016 한국실험동물학회 학술발표대회 논문집 Vol.2016 No.2

Effect of Porcine Follicular Fluid on Donor Cell Characteristics and Quality of Porcine Cloned Blastocysts

권대진,오건봉,옥선아,이정웅,이승수,박진기,장원경,황성수 사단법인 한국동물생명공학회 2013 Reproductive & developmental biology Vol.36 No.4

This study aimed at investigating whether a porcine follicular fluid (pFF) supplementation positively affects the characteristics of donor cells and the developmental competence of porcine cloned embryos. Ear fibroblast cells (donor cell) from an Massachusetts General Hospital miniature pig were cultured in different culture methods: (1) Dulbecco's modified Eagle's medium (DMEM)+10% FBS (Control); (2) DMEM+0.5% FBS (SS); and (3) DMEM+10% FBS+10% pFF (pFF) for 72 h. In each conditioned medium, the concentrations of 4 amino acids (Thr, Glu, Pro, and Val) in the pFF group were significantly different from those in the control group (p<0.05 or p<0.01). The proliferation of the cells cultured in the SS group was significantly lower than that of the other treatment groups (p<0.01). The population of apoptotic and necrotic cells in the SS group was significantly higher than that of either the control or the pFF group (p<0.01). The number of embryos that cleaved (p<0.05) and developed into blastocysts (p<0.01) in the SS group was significantly lower than that of either the control or the pFF group. Compared to other groups, the blastocysts produced from the donor cells in the pFF group had higher total cells and lower apoptotic cells (p<0.05). It can be concluded that pFF supplementation in the donor cell culture medium positively affects cell death, cell cycle and quality of the cloned blastocyst.

Aberrant methylation of Meg3 in alpha1,3-galactosyltransferase knockout pig induced pluripotent stem cells

권대진,황인설,김혜림,김유라,오건봉,옥선아,임기선,이정웅,황성수 한국통합생물학회 2016 Animal cells and systems Vol.20 No.3

Pigs have been used as a good research model for xenotransplantation. Several groups have generated porcine-induced pluripotent stem cells (piPSCs) from differentiated somatic cells. Transgenic pigs with the alpha1,3-galactosyltransferase gene-knockout (GalT-KO) could successfully govern hyper acute rejection of organ transplants into primates. Thus, GalT-KO piPSCs could be a powerful cell resource for agricultural and biomedical applications. This study was performed to generate iPSCs from GalT-KO pigs and characterize their properties. We successfully generated a GalT-KO iPSC from a genetically modified pig using double alpha1,3- galactosyltransferase knockout alterations. Similar to mouse embryonic stem cells, the GalT-KO piPSCs were positive for classical pluripotency markers: POU5F1, NANOG, SOX2 and SSEA1, and were negative for: SSEA3, TRA-1-60 and TRA-1-81. Furthermore, these cells could form an embryoid body that differentiated into three germ layers in vitro, and were highly proliferative under leukemia inhibitory factor culture conditions. However, the methylation status in DMR2 of the Meg3 gene was higher in GalT-KO piPSCs than in porcine ear fibroblast. In conclusion, GalTKO piPSCs could be successfully generated by six human factors without expression of Galepitopes. Although aberrant methylation observed in GalT-KO piPSCs, this cell line maintained pluripotency and had differentiation properties into all three germ layers. Therefore, GalT-KO piPSCs might be a good cell source for biomedical application and basic research.

소 체외수정란 및 체세포 복제란의 초자화 동결 후 생존성

권대진,박주희,박춘근,양부근,정희태,Kwon, Dae-Jin,Park, Joo-Hee,Park, Choon-Keun,Yang, Boo-Keun,Cheong, Hee-Tae 한국동물번식학회 2007 Reproductive & developmental biology Vol.31 No.1

본 연구는 소 체외수정란과 체세포 복제란의 초자화 동결 및 응해 후 생존능을 검토하였다. 배반포로 발육된 체외수정란 및 체세포 복제란을 초자화 동결법에 의해 동결하였다가 융해하여 생존율 및 배양 후 부화율을 검사하였다. 체외수정란 배반포를 초자화 동결 응해한 결과, 확장배반포가 배반포기 난자에 비하여 생존율(82.1%, 96/117)과 부화율(64.1%, 75/117)에서 모두 유의적으로 높았다(p<0.05). 핵이식 배반포 복제란을 초자화 동결 응해한 경우도 체외수정란과 비슷한 경향을 보여 확장 및 부화배의 생존율과 부화율이 각각 81.1%(30/37)와 78.3%(29/37)로, 배반포(각각 71.8 및 53.8%)에 비하여 다소 높게 나타났다. 본 연구의 결과는 초자화 동결 방법에 의해서 소 체외수정란과 체세포 복제란을 성공적으로 동결할 수 있으며, 특히 후기 배반포기 단계에서 초자화 동결 시 높은 생존율과 부화배 형성율을 얻을 수 있음을 보여준다. This study was conducted to examine the development of in vitro fertilized (IVF) and nuclear transfer (NT) embryos following vitrification IVF and NT embryos developed to the blastocyst stage were equilibrated by 3 steps, vitrified and thawed, and their survival and hatching rates were examined. In IVF embryos, higher survival (82.1%, 96/117) and hatching rates (64.1%, 75/117) were obtained respectively after thawing and culture in expanded blastocysts compared to blastocysts (p<0.05). High survival and hatching rates were also obtained by vitrification of NT blastocysts, especially in expanded and hatching blastocysts (81.1 and 78.3%, respectively). The result of this study shows that IVF and NT blastocysts, especially late stage blastocysts, are successfully cryopreserved by vitrification.

Genome analysis of Yucatan miniature pigs to assess their potential as biomedical model animals

권대진,이영섭,신동현,원경혜,송기덕 아세아·태평양축산학회 2019 Animal Bioscience Vol.32 No.2

Objective: Pigs share many physiological, anatomical and genomic similarities with humans, which make them suitable models for biomedical researches. Understanding the genetic status of Yucatan miniature pigs (YMPs) and their association with human diseases will help to assess their potential as biomedical model animals. This study was performed to identify non-synonymous single nucleotide polymorphisms (nsSNPs) in selective sweep regions of the genome of YMPs and present the genetic nsSNP distributions that are potentially associated with disease occurrence in humans. Methods: nsSNPs in whole genome resequencing data from 12 YMPs were identified and annotated to predict their possible effects on protein function. Sorting intolerant from tolerant (SIFT) and polymorphism phenotyping v2 analyses were used, and gene ontology (GO) network and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses were performed. Results: The results showed that 8,462 genes, encompassing 72,067 nsSNPs were identified, and 118 nsSNPs in 46 genes were predicted as deleterious. GO network analysis classified 13 genes into 5 GO terms (p<0.05) that were associated with kidney development and metabolic processes. Seven genes encompassing nsSNPs were classified into the term associated with Alzheimer’s disease by referencing the genetic association database. The KEGG pathway analysis identified only one significantly enriched pathway (p<0.05), hsa04080: Neuroactive ligand-receptor interaction, among the transcripts. Conclusion: The number of deleterious nsSNPs in YMPs was identified and then these variants-containing genes in YMPs data were adopted as the putative human diseases-related genes. The results revealed that many genes encompassing nsSNPs in YMPs were related to the various human genes which are potentially associated with kidney development and metabolic processes as well as human disease occurrence.

전북지역 한우의 번식 실태 조사 및 평가

권대진,이상민,김도현,오재돈 경상대학교 농업생명과학연구원 2019 농업생명과학연구 Vol.53 No.3

한국에 있어서 한우는 경제 동물일 뿐 만 아니라 중요한 유전자원 중 하나이다. 그러나 현재 한우의 번식 능력 개량을 위한 연구는 매우 미흡한 실정이다. 한우의 번식 능력에 대한 기초 자료를 확보하기 위해 전북지역(정읍시)에서 인공수정 된 암소 총 33,281두에 대한 105,017개의 번식기록(2010-2017)을 이용하여 임신기간(GP), 분만 후 첫 종부기간(CFSI), 분만 후 임신간격(CCI), 수태당 종부회수(NAIPC), 첫 종부 임신율 (FSCR)을 계산하여 분석에 활용하였다. 전체적으로 GP 는 287.602±4.797일, CFSI와 CCI는 각각 77.513±34.417 및 88.433±42.194일 이였다. 또한 FSCR는 62.365%였으며, NAIPC 는 1.411회로 나타났다. 2017년의 FSCR과 NAIPC는 각각 67.497% 및 1.344회로 2013년 62.366% 및 1.402에 비해 유의적으로 증가하였다(p<0.05). 가을과 겨울에는 번식기간이 더 길었으며, NAIPC는 봄과 여름에 비해 유의적으로 증가하는 것으로 나타났다(p<0.05). 3~7산차 암소의 경우 NAIPC가 1.323±0.669~1.339±0.689회로 다른 그룹에의 1.401±0.772~1.446±0.854회 보다 우수한 것으로 나타났다. 반면, 1산차 암소의 경우 FSCR와 NAIPC가 각각 60.217% 및 1.506±0.937회로 가장 저조한 것으로 나타났다. 본 연구 결과 한우의 번식효율은 산차에 따라, NAIPC는 계절에 따라 영향 받는 것으로 나타났으며, 전체적인 번식효율은 과거에 비해 지속적으로 향상되고 있는 것으로 나타났다. Korean Native Cattle (Hanwoo) is an important breed not only as a genetic material but also as an economical livestock resource in Korea. Currently, the information about reproductive performance of Hanwoo is very limited. To investigate reproductive performance of Hanwoo in Jeonbuk province, gestation period (GP), calving to first service interval (CFSI), calving to conception interval (CCI), number of artificial insemination (AI) per conception (NAIPC) and first-service conception rate (FSCR) were analyzed with 33,281 cows and the corresponding 105,017 records for the reproductive traits that were obtained between 2010 and 2017. The average of the GP was 287.602±4.797 days, and 77.513±34.417 and 88.433±42.194 days for CFSI and CCI, respectively. The average of FSCR was 62.365%, and 1.411 for NAIPC. Compared to 2013, FSCR and NAIPC were significantly improved up to 67.497% and 1.344 in 2017, respectively. In autumn and winter, calving interval was longer, and NAIPC were greater than in spring and summer (p<0.05). The cows in parities 3 to 7 had better NAIPC (1.323±0.669 to 1.339±0.689) than the other parities (1.401±0.772 to 1.446±0.854). However, FSCR and NAIPC in parity 1 were shorter and greater, i.e. 60.217% and 1.506±0.937, than other parities, respectively. In conclusion, reproductive efficiency in Hanwoo has been improved, although the efficiency varied depending on parity (1 and ≥9) or season.

돼지 유도만능줄기세포 유래 복제란의 특성 분석

권대진,오재돈,박미령,황인설,박응우,황성수 한국동물생명공학회(구 한국동물번식학회) 2019 Journal of Animal Reproduction and Biotechnology Vol.34 No.3

In general, cloned pigs have been produced using the somatic cell nuclear transfer (SCNT) technique with various types of somatic cells; however, the SCNT technique has disadvantages not only in its low efficiency but also in the development of abnormal clones. This study aimed to compare early embryonic development and quality of SCNT embryos with those of induced pluripotent stem cells (iPSCs) NT embryos (iPSC-NTs). Ear fibroblast cells were used as donor cells and iPSCs were generated from these cells by lentiviral transduction with human six factors (Oct4, Sox2, c-Myc, Nanog, Klf4 and Lin28). Blastocyst formation rate in iPSC-NT (23/258, 8.9%) was significantly lower than that in SCNT (46/175, 26.3%; p < 0.05). Total cell number in blastocysts was similar between two groups, but blastocysts in iPSC-NT had a lower number of apoptotic cells than in SCNT (2.0 ± 0.6 vs. 9.8 ± 2.9, p < 0.05). Quantitative PCR data showed that apoptosis-related genes (bax, caspase-3, and caspase-9) were highly expressed in SCNT than iPSC-NT (p < 0.05). Although an early development rate was low in iPSC-NT, the quality of cloned embryos from porcine iPSC was higher than that of embryos from somatic cells. Therefore, porcine iPSCs could be used as a preferable cell source to create a clone or transgenic animals by using the NT technique.

Catalase와 -Mercaptoethanol이 돼지 태아섬유아세포 Clonal Lines의 배양에 미치는 영향

권대진,박선영,박춘근,양부근,김정익,정희태 韓國受精卵移植學會 2004 한국동물생명공학회지 Vol.19 No.3

본 연구에서는 clonal cell lines을 효율적으로 확립할 수 있는 방법을 제시하기 위하여 배양액 내에 catalase와 ME 첨가가 clonal cell line 확립 효율에 미치는 영향을 검토하였다. 임신 50일령의 암퇘지에서 얻은 태아섬유아세포를 2회 passage한 후 동결 보관하였다가 실험에 이용하였다. 단일세포를 catalase나 ME가 첨가된 배양액이 들어 있는 96-well dish로 옮겨 배양하였다. 단층이 형성된 세포는 4-we This study was performed to examine the effects of catalase and -mercaptoethanol (ME) on the establishment of clonal lines from porcine fetal fibroblast cells. Fibroblasts derived from a pig fetus (Day 50) were passaged two times before use. A single cell was seeded in 96-well plates and cultured in medium supplemented with or without catalase or ME. Cell colonies were passaged two times into 4-well dish. Cell lines with proliferating potential were classified as an established clonal cell line. In experience 1, the establishment efficiencies were examined by addition of catalase (100ng/) or ME (100 uM) in culture medium. The establishment efficiency of ME-added group (8.3%) was significantly higher than that of control group (3.2%, P<0.05). However, catalase did not have a positive efffct on the establishment efficiency. In experience 2, the establishment efficiencies were examined by addition of different concentrations of catalase (0-1,000 ng/) in culture medium. However, establishment efficiencies were not different among the different concentrations of catalase (0-2.6%). In experience 3. the establishment efficiencies were examined by addition of different concentrations of ME(0-1,000 uM) in culture medium. The establishment efficiency was significantly higher in 100 uM ME-added group (9.4%) compare to others (0-1.6%). The result of present study shows that the establishment efficiency of clonal cell lines can be enhanced by the culture in media supplemented with 100uM ME. However, catalase did not have a positive effect on the establishment efficiency.