Genetic characteristics of Korean Jeju Black cattle with high density single nucleotide polymorphisms

Alam, M. Zahangir,Lee, Yun-Mi,Son, Hyo-Jung,Hanna, Lauren H.,Riley, David G.,Mannen, Hideyuki,Sasazaki, Shinji,Park, Se Pill,Kim, Jong-Joo Asian Australasian Association of Animal Productio 2021 Animal Bioscience Vol.34 No.5

Objective: Conservation and genetic improvement of cattle breeds require information about genetic diversity and population structure of the cattle. In this study, we investigated the genetic diversity and population structure of the three cattle breeds in the Korean peninsula. Methods: Jeju Black, Hanwoo, Holstein cattle in Korea, together with six foreign breeds were examined. Genetic diversity within the cattle breeds was analyzed with minor allele frequency (MAF), observed and expected heterozygosity (HO and HE), inbreeding coefficient (FIS) and past effective population size. Molecular variance and population structure between the nine breeds were analyzed using a model-based clustering method. Genetic distances between breeds were evaluated with Nei's genetic distance and Weir and Cockerham's FST. Results: Our results revealed that Jeju Black cattle had lowest level of heterozygosity (HE = 0.21) among the studied taurine breeds, and an average MAF of 0.16. The level of inbreeding was -0.076 for Jeju Black, while -0.018 to -0.118 for the other breeds. Principle component analysis and neighbor-joining tree showed a clear separation of Jeju Black cattle from other local (Hanwoo and Japanese cattle) and taurine/indicine cattle breeds in evolutionary process, and a distinct pattern of admixture of Jeju Black cattle having no clustering with other studied populations. The FST value between Jeju Black cattle and Hanwoo was 0.106, which was lowest across the pair of breeds ranging from 0.161 to 0.274, indicating some degree of genetic closeness of Jeju Black cattle with Hanwoo. The past effective population size of Jeju Black cattle was very small, i.e. 38 in 13 generation ago, whereas 209 for Hanwoo. Conclusion: This study indicates genetic uniqueness of Jeju Black cattle. However, a small effective population size of Jeju Black cattle indicates the requirement for an implementation of a sustainable breeding policy to increase the population for genetic improvement and future conservation.

Comparisons of Beef Fatty Acid and Amino Acid Characteristics between Jeju Black Cattle, Hanwoo, and Wagyu Breeds

Seung-Hoon Lee,Chung-Nam Kim,Kyoung-Bo Ko,Se-Pill Park,Ho-Kyoung Kim,Jun-Mo Kim,Youn-Chul Ryu 한국축산식품학회 2019 한국축산식품학회지 Vol.39 No.3

Jeju black cattle are known as one of Korea’s traditional cattle. However, Hanwoo is more well-known to Korean meat consumers as representative beef cattle. Despite the popularity of these two breeds, comparison of the nutritional characteristics between Jeju black cattle and Hanwoo have not been studied. Here, we compared the fatty acid and amino acid characteristics between two Korean traditional cattle and Wagyu breeds. A total of 62 cattle were used in this study. The Jeju black cattle beef had significantly higher unsaturated fatty acids than Hanwoo (p<0.05). Savory fatty acids, including oleic acid were also higher than in Hanwoo cattle (p<0.05). The negative flavor fatty acids, such as palmitic acid were significantly lower than in Hanwoo (p<0.001). On the other hand, linoleic acid which imparts a negative flavor was higher than Hanwoo (p<0.05). Amino acids, including alanine and glutamine, usually representative of the umami taste were present in significantly higher proportions in Jeju black cattle (p<0.05). In addition, bitter tasting amino acids, including valine, leucine, isoleucine, and methionine were lower in Jeju black cattle beef than in Hanwoo (p<0.05, p<0.001, p<0.001, and p<0.001 each). Taken together, our results suggest that Jeju black cattle beef had higher savory flavor and umami taste which affected consumers preference for the meat.

Spermatozoa characteristics after L-methionine ingestion during 6 weeks in Jeju black cattle over 10 years of age

Sang-Min Shin,Jun-Gyu Son,Byung-Chul Yang,Nam-Young Kim,Jai-Hoon Woo,Moon-Cheol Shin,Ji-Hyun Yoo 한국수정란이식학회 2018 한국수정란이식학회 학술대회 Vol.2018 No.11

Purpose The Jeju black cattle are a type of traditional Korean native cattle with a characteristic black fur that covers the entire body. The Jeju black cattle are rare breed and designated as national natural monuments in 2013. It is necessary to improve reproductive techniques for the preservation and proliferation of Jeju black cattle. Methionine acts as a precursor amino acid for glutathione in protection of cells from oxidative damage, and plays a vital role in detoxification. Low sperm motility causes infertility because when sperm do not have progressive motility, it was unable to reach the ovum. The purpose on this study was to investigate whether intake of L-methionine improves sperm motility and fertility. Materials and Methods 6 Jeju black cattles over 10 years of age are raised in Subtropical Livestock Research Institute, National Institute of Animal Science, RDA. 6 Jeju black cattles was fed with L-methionine with 10g/day during 6 weeks. Semen of Jeju black cattle were collected by artificial vaginal technique. Collected sperm was diluted with AndroMed® extender and cryopreserved in 0.5 ml French straws. Fresh and freeze-thawed sperm viability and motility were evaluated by CASA. Results The viability and progressive motility of fresh spermatozoa(live spermatozoa means: from 74.41% to 80.22%, progressive motility means: from 80.60% to 95.40%) and freeze-thawed spermatozoa(live spermatozoa means: from 51.26% to 62.05%, progressive motility: from 29.68% to 45.44%) incresed after L-methionine ingestion. Conclusion The intake of L-methionine in genetically valuable cattle over 10 years of age seems to be useful for improving sperm function.

흑한우와 한우 및 수입우를 판별하기 위한 multiplex PCR 기술

김찬수(Chan-Su Kim),고정문(Jung-Moon Ko),차현철(Hyeon-Cheol Cha),박중국(Joong Kook Park),정준(Joon Jeong) 한국생명과학회 2014 생명과학회지 Vol.24 No.8

본 연구는 현행 한우확인시험법에 이용되는 Microsatellite (MS)와 소의 유전형질 중 품종 판별에 주로 이용되는 Melanocortin Receptor 1 (MC1R) 유전자상의 Single Nucleotide Polymorphism (SNP)를 분석하여 설계한 Primer를 Multiplex PCR을 활용하여 소의 품종을 판별하였다. MC1R 유전자형은 E<SUP>D</SUP>, E<SUP>+</SUP>, e형의 3Type으로 나뉘며 E<SUP>D</SUP>/E<SUP>D</SUP>, E<SUP>D</SUP>/E<SUP>+</SUP>, E<SUP>D</SUP>/e, E<SUP>+</SUP>/E<SUP>+</SUP>, E<SUP>+</SUP>/e, e/e의 6가지 유전자형을 가진다. E<SUP>D</SUP>유전자형은 외래종이 지닌 유전자형으로 E<SUP>D</SUP>/E<SUP>D</SUP>, E<SUP>D</SUP>/E<SUP>+</SUP>, E<SUP>D</SUP>/e이 이에 속하며, e유전자형은 한우가 지닌 유전자형으로 e/e의 유전자형을 가진다. 흑한우의 경우 E<SUP>D</SUP>, E<SUP>+</SUP>, e의 모든 유전자형을 가지고 있으나 이는 교잡에 의해 나타난 것으로 보이며 E<SUP>+</SUP>유전자형이 흑한우 고유의 유전자형으로 추정되어 본 연구에서는 이에 따라 E<SUP>+</SUP>/E<SUP>+</SUP>, E<SUP>+</SUP>/e의 유전자형을 흑한우로 분류하였다. 그러나 E<SUP>D</SUP>, E<SUP>+</SUP>의 경우 단순 PCR기법만으로는 그 판별에 어려움이 있어, MS Maker를 활용한 다형성 분석을 통해 흑한우와 수입우를 판별할 수 있는 새로운 Primer를 설계하였으며, 이를 통해 소의 품종을 판별하였다. For the identification of the Jeju black cattle, Hanwoo and imported beef, we performed a multiplex polymerase chain reaction (PCR) associated with microsatellite (MS) and melanocortin 1 receptor (MC1R) gene. The MC1R gene plays an important role in regulation of the melanin synthesis within mammalian melanocytes. MC1R encoded by extension (E) locus was almost fixed with recessive red e allele in the Hanwoo. We estimated that the specific genotypes (E<SUP>+</SUP>/E<SUP>+</SUP>, E<SUP>+</SUP>/e) of MC1R gene were characteristic genotypes of Jeju black cattle. But the PCR products resulted from using the MC1R gene derived primers only are not sufficient to identify Jeju black cattle from other relatives. We performed two times of successive multiplex PCR to provide a more accurate result for the identification of Jeju black cattle. The results suggest that two types of successive multiplex PCR methods using MC1R gene and Microsatellite derived primer set will be more useful to identification of Jeju black cattle, Hanwoo and imported beef.

제주도산 한우와 제주흑한우 집단의 도체형질에 대한 Lysophospholipase I (LYPLA1) 유전자형의 효과

한상현(Sang-Hyun Han),성필남(Pil-Nam Seong),조인철(In-Cheol Cho) 한국생명과학회 2017 생명과학회지 Vol.27 No.7

본 연구는 Lysophospholipase I (LYPLA1) 유전자의 SNP 마커인 rs385360448 g.23655332G>C의 유전자형이 제주도 한우와 제주흑한우(한우×제주흑우, JCC) 집단의 도체형질에 미치는 영향을 시험하였다. 한우 거세우 집단에서 LYPLA1 rs385360448 G/-를 보유한 도체는 C/C 동형접합에 비해 유의적으로 높은 수준의 근내지방도(MARB)와 조직감지수(TI)를 나타내었다(p<0.05). 반면, LYPLA1 유전자형은 도체중, 등지방두께, 등심단면적, 육색, 지방색에서는 유의적인 차이를 보이지 않았다(p>0.05). JCC 거세우에서는 LYPLA1 G/- 도체에서 C/C 동형접합에 비해 등 심단면적(EMA)의 수준이 유의적으로 더 높았다(p<0.05). 이번 연구의 결과들은 LYPLA1 유전자형이 소의 등심에서 인지질 대사를 통해 근내지방도와 조직감의 수준과 등심단면적의 수준을 변화시킬 수 있음을 보여주고 있다. 이러한 결과들은 LYPLA1 유전자형이 한우와 제주흑우-유래의 산업화축군에서 도체형질을 개선할 수 있는 유전자 마커로써의 기능을 수행할 수 있을 것으로 기대된다. Genotypes of the nucleotide substitution g.23655332G>C of SNP marker rs385360448 at Lysophospholipase I (LYPLA1) gene intron 7, were tested for their effect on the carcass traits of Hanwoo and Jeju Crossbred cattle (F1 progeny produced between Hanwoo × Jeju Black cattle) populations on Jeju Island. In the Hanwoo steer population, the meats containing LYPLA1 rs385360448 G/- genotypes showed significantly higher marbling scores and greater texture indices, compared to those of rs385360448 C/C homozygous animals (p<0.05). However, the LYPLA1 genotypes were not associated with the levels of carcass weight, backfat thickness, eye muscle area (EMA), meat color, and fat color (p>0.05). On the other hand, in the JCC steer population, the LYPLA1 G/- harboring meats showed significantly greater EMA levels, compared to those of C/C homozygotes (p<0.05). The results of the present study indicate that the LYPLA1 genotypes could alter the levels of intramuscular fat deposition, texture index, and eye muscle area via phospholipid metabolism in the Longissimus dorsi muscle of the cattle. These findings suggested that LYPLA1 genotypes may effect molecular genetic markers in the improvement of carcass traits of Hanwoo and Jeju Black industrial cattle populations on Jeju Island.

Coat Color Patterns and Genotypes of Extension and Agouti in Hanwoo and Jeju Black Cattle

Sang-Hyun Han(한상현),In-Cheol Cho(조인철),Jae-Hwan Kim(김재환),Moon-Suck Ko(고문석),Young-Hoon Kim(김영훈),Eun-Young Kim(김은영),Se-Pill Park(박세필),Sung-Soo Lee(이성수) 한국생명과학회 2011 생명과학회지 Vol.21 No.4

소의 Extension(E), Agouti(A) 유전자형과 모색유전양상의 연관관계를 이해하기 위해, 한우와 제주흑우(JBC), 이들 사이에서 생산된 교배후손에서 melanocortin-1 receptor( MC1R)과 agouti signaling protein( ASIP)의 유전자형을 분석하였다. 흑모색의 JBC에서는 세 가지 MC1R대립인자들(E<SUP>D</SUP>, E?, e)이 모두 발견되었다. JBC 집단은 한우에서 우점적으로 나타나는 열성동형접합자(e/e)가 발견되지 않았다. 반면, MC1R E?/e한우는 흑모색이 아닌 갈색이나 적색의 모색을 나타내었다. ASIP유전자에서, 한우에서는 L1-BT 전위인자의 삽입/결실에 의해 세 가지 유전자형(A/A, A/A<SUP>Br</SUP>, A<SUP>Br</SUP>/A<SUP>Br</SUP>)이 모두 관찰되었다. JBC 집단에서는 ASIP A<SUP>Br</SUP> 대립인자가 드물게 관찰되고, ASIP A<SUP>Br</SUP>/A<SUP>Br</SUP> 동형접합인 개체들은 발견되지 않았다. ASIP A<SUP>Br</SUP> 대립인자를 보유하더라도 한우와 JBC, 이들의 교배후손에서도 agouti-유사 얼룩무늬 모색은 발견되지 않았다. 교배후손의 모색은 Agouti 유전자형과는 무관하고, Extension유전자형과 직접적으로 연관되어 황-갈색(e/e)과 암-갈/흑색(E?/e)으로만 구분되었다. ASIP유전자형은 아마도 한우 또는 교배 후손에서 모색의 발달에 영향이 없는 것으로 추정된다. 본 연구 결과는 한우, 제주흑우, 이들의 교배후손에서 MC1R유전자형이 모색 표현형을 결정하며, ASIP유전자형은 모색의 변이에는 핵심적인 역할을 수행하지 않는다는 것을 시사한다. To understand the relationship between coat color inheritance patterns and genotypes of Extension( E) and Agouti( A) loci in cattle, the genotypes for melanocortin-1 receptor( MC1R) and agouti signaling protein ( ASIP) were analyzed in Hanwoo, Jeju black cattle (JBC), and their crossbred progeny. Three MC1R alleles ( E<SUP>D</SUP>, E?, and e) were found in the black-colored JBC population. JBC had no recessive homoygotes (e/e), but this genotype was predominant in the Hanwoo breed. However, MC1R E?/eHanwoo did not produce a black coat color as they appeared either as brown or solid red. For ASIP, three genotypes ( A/A, A/A<SUP>Br</SUP>, and A<SUP>Br</SUP>/A<SUP>Br</SUP>) were determined by insertion/deletion of an L1-BT element in Hanwoo. The ASIP A<SUP>Br</SUP> allele was rarely observed, and no ASIP A<SUP>Br</SUP>/A<SUP>Br</SUP> homozygotes were detected in the JBC population. Cattle carrying ASIP ABr did not show any agouti-like brindle pigmentation patterns in either breed or their progeny. The coat colors of the crossbred progeny were discriminated by two colors, yellowish-brown versus dark-brown or black, and their coat colors were directly related to the genotypes of the Extensionlocus, yellowish-brown (e/e) and dark-brown or black (E?/e), but not to the Agouti locus. ASIP genotypes probably did not affect coat color development in the Hanwoo or crossbred progeny. Our results suggest that the ASIP genotypes do not play key roles in coat color variation, but the MC1R genotypes do direct the phenotypes of Hanwoo, JBC, and their progeny.

제주도 한우와 제주흑우실용화축군 집단에서 ADD1 유전자의 다형성이 경제형질에 미치는 영향

한상현(Sang-Hyun Han),오홍식(Hong-Shik Oh),이재봉(Jae-Bong Lee),좌은숙(Eun-Sook Jwa),강용준(Yong-Jun Kang),김상금(Sang-Geum Kim),양성년(Sung-Nyun Yang),김유경(Yoo-Kyung Kim),조인철(In-Cheol Cho),조원모(Won-Mo Cho),고문석(Moon-Suck Ko 한국생명과학회 2015 생명과학회지 Vol.25 No.1

한우와 제주흑우실용화축군(JBC-DC) 집단에서 ADD1 유전자의 유전적 다형성을 조사하였다. ADD1 유전자형은 intron 7 영역에서 84-bp 절편의 유무에 따라 결정하고, 두 집단에서 ADD1 유전자형과 경제형질의 상관관계를 시험하였다. 한우 거세우집단에서는 ADD1 D/- 도체들이 WW 개체들보다 유의적으로 더 두꺼운 등지방두께를 보여주었다(p<0.05). 하지만, JBC-DC 집단에서는 이형접합인 WD에서 가장 두꺼운 등지방두께를 나타냄을 볼 수 있었고(p<0.05), 이는 W와 D 대립인자의 상승효과에 의해 등지방 축적이 증가하는 것으로 추정된다. 반면 육질등급지수나 근내지방도으로 측정된 근육조직에서 근내지방의 축적과 ADD1 유전자형과은 상관없는 것으로 조사되었다. 이상의 결과들로부터, ADD1 유전자는 근육조직의 근내지방보다는 피하조직에서 등지방에 영향을 주는 것으로 보인다. 또한 DD 유전자형에서 W/-인 동물들에 비해 더 높은 수준의 육색을 나타내었다(p<0.05). 흥미로운 것은 JBC-DC 집단에서만 도체중과 유전자형 사이에 고도의 유의적인 차이를 나타내었는데, D/-인 동물들이 WW인 동물들에 비해 38 kg 이상 더 무거웠다(p<0.001). 연구결과는 ADD1 유전자형에 따라 빠른 성장률과 거세우의 생산성에 차이들을 나타내는 것을 보여주고 있다. 이러한 발견들은 ADD1 유전자형이 유전적 분자 마커로써 한우와 제주흑우-유래 축군의 개량을 위한 교배육종에서 효과적인 역할을 수행할 수 있음을 보여주는 결과이다. Genetic polymorphisms of adipocyte determination and differentiation factor 1 (ADD1) gene were screened in Hanwoo and Jeju Black cattle-derived commercial (JBC-DC) populations. The ADD1 genotypes were determined using the presence/absence of 84-bp fragment at intron 7 region. The association of ADD1 genotypes for economic traits was examined in both populations. In the Hanwoo steers, ADD1 D/-carcasses showed significantly thicker backfat levels than those from WW (p<0.05). However, the thickest level of backfat appeared in WD heterozygotes, whereas thicker backfat did not appear in DD homozygotes in the JBC-DC population (p<0.05), leading to the supposition that synergic effects of alleles W and D increase backfat deposition. On the other hand, there was no association between the ADD1 genotypes and intramuscular fat deposition measured as meat quality index and marbling score. From these results, we concluded that the bovine ADD1 affected the backfat in subcutaneous tissue, rather than intramuscular fat in muscle tissue. In addition, the DD animals showed higher levels of meat color than those from W/- (p<0.05). Interestingly, a highly significant difference was found between the genotypes and carcass weights only in the JBC-DC population, and D/- animals were heavier by more than 38 kg than those from WW (p<0.001). The results of this study reveal faster growth rate and differences in steer productivity according to genotypes of the ADD1 gene. These findings demonstrate that ADD1 genotypes may effectively function as molecular genetic markers for the improvement of Hanwoo and Jeju Black cattle-related crossbreeding systems.

Evaluation of Jeju Black Cattle Bull Sperm

Jae-Wook Yoon,Seung-Eun Lee,Won-Jae Kim,Sang-Gi Jeong,Chan-Oh Park,Hyo-Jin Park,Dong-Yong Hong,Min-Jee Park,Eun-Young Kim,Se-Pill Park 한국동물생명공학회(구 한국동물번식학회) 2018 발생공학 국제심포지엄 및 학술대회 Vol.2018 No.06

The Jeju Black Cattle (JBC) are a type of traditional Korean native cattle with a characteristic black fur that covers the entire body. Jeju black cattle are smaller than other breeds and their disposition is very calm. The breed’s qualities and genetic uniqueness helped it to be named as a protected, state-designated national monument in 2013. However, they are endangered crisis. Therefore, in vitro fertilization (IVF) technology is needed to preserve these endangered animals and to further improve their traits. In this context, semen analysis is the most commonly used procedure to evaluate male fertility potential in humans and animals. This study was performed on 3 Jeju Black Cattle (JBC) Bulls belonging to Jeju Special Self-Governing Province Promotion Institute. [JBC A, B, C grade]. The freezing medium (20% egg yolk plus 20% triladyl) to a final concentration of 100×106 sperm cells/mL. For sperm cooling, the 0.5 mL plastic straws filled with diluted samples were kept in refrigerator at 4°C for 2 h. They were placed 3 or 7 cm over liquid nitrogen (LN2) vapor for 10 or 30 min and then directly plunged into LN2 for storage. Thawing was done by transferring the frozen straws into water bath at 37°C for 30 sec for analysis. The sperm motility in each group was assessed using the Sperm Analysis Imaging System (SAIS Plus; Medical Supply Co, Ltd., Korea). The sperm vitality in each group was assessed using the eosin-nigrosin stain. The sperm morphology in each group was assessed using the diff-quik kit. The motility was significantly lower in JBC-C frozen-thawing sperm group (35∼65%) than in JBC-C fresh sperm group (98.5%) (p<0.05). The vitality of fresh sperm group (45∼61%) was similar to that of frozen-thawed sperm group (30∼65%). The morphology of fresh sperm group (72∼89%) was similar to that of frozen-thawed sperm group (83∼93%). These results demonstrate that there were similar in sperm quality (vitality and motility) between fresh sperm and frozen-thawed sperm.

Quality Analysis of Fresh or Frozen-Thawed Jeju Black Cattle Bull Sperm

Jae-Wook Yoon,Min-Jee Park,Chan-Oh Park,Seung-Eun Lee,Eun-Young Kim,Won-Jae Kim,Hyo-Jin Park,Se-Pill Park 한국수정란이식학회 2018 한국수정란이식학회 학술대회 Vol.2018 No.11

The Jeju Black Cattle (JBC) are a type of traditional Korean native cattle with a characteristic black fur that covers the entire body. Semen analysis is the most commonly used procedure to evaluate male fertility potential. This study was to evaluate the quality of 10 JBC bulls belonging to Jeju Special Self-Governing Province Promotion Institute. [JBC A∼J grade]. The freezing medium (20% egg yolk plus 20% triladyl) was added in semen sample to a final concentration of 100×106 sperm/ml. For sperm cooling, diluted semen was filled in 0.5 ml plastic straws and then kept in refrigerator at 4°C for 2 h. They were placed in 7 cm over liquid nitrogen (LN2) vapor for 10 min and then directly plunged into LN2 for storage. Thawing was done by transferring the frozen straws into water bath at 37°C for 30 sec for analysis. The sperm motility, vitality and morphology in each group was assessed using the Sperm Analysis Imaging System (SAIS Plus; Medical Supply Co, Ltd., Korea), eosin-nigrosin stain and diff-quik kit. There was no difference in the motility of the fresh groups (87.4 ~ 100%), while it was difference in the frozen-thawed groups (42.8 ~ 98.6%) (p<0.05). The best motility was shown in JBC-B (100/fresh and 98.6%/frozen-thawed). There was significant difference in the vitality of the fresh group (19.8 ~ 59.2%) and frozen-thawed group (21.2 ~ 49.8%)(p<0.05). The highest vitality was also shown in JBC-B (59.2/fresh and 49.8%/frozen-thawed). Morphologically, in fresh semen the highest normal ratio was indicated in JBC-E (90.9%) and in frozen-thawed group the highest was in JBC-C (90.2%). These results demonstrated that the analysis including motility, vitality and morphology of fresh or frozen-thawed semen is valuable to select the high quality sperm using for reproduction.

Derivation of Induced Pluripotent Stem Cell–Like Cells from Jeju Black Cattle Cells

Sang-Gi Jeong,Seung-Eun Lee,Yun-Gwi Park,Yeo-Jin Son,Eun-Young Kim,Se-Pill Park 한국동물생명공학회(구 한국동물번식학회) 2017 Reproductive & Developmental Biology(Supplement) Vol.41 No.2

Jeju Black Cattle (JBC, endangered native Korean cattle), a species characterized by their pitch-black coat color, live in Jeju Special Self-Governing Province. Pluripotent stem cells (PSCs) derived from farm animals have potential for various applications in transgenic breeding, basic research and agriculture. However, there were a few reports for establishment of bovine induced PSC (iPSC) and their culture conditions were not optimized yet. In this study, JBC bull #5293 (date of birth, October 31, 2013) ear cells were transfected by retroviral vectors that include 5 pluripotent reprogramming factors, hOCT4, hSOX2, hKLF4, hc-MYC and hNANOG. The JBC cells were exposed to retrovirus in suspended state in a fibroblast culture medium (10% FBS+DMEM) for 3 h and then plated on culture dish (3×104 cells/60mm-dish). After three days, infected cells were dissociated and replated to a new feeder-free culture dish or mouse embryonic fibroblast (MEF) feeder cell dish (3×104 cells/60 mm-dish). And these cells were cultured in two different ES media (15% FBS+DMEM; 15% FBS or 20% SR+DMEM/F12; 20% SR) commonly supplemented with 5 ng/mL bFGF and 1,000 U LIF. Produced iPSC-like colony was separated by mechanical dissociation and transferred to a new MEF cell dish. In the result, cell aggregation before colony formation was predominantly appeared in 20% SR group compared with 15% FBS group, while the JBC cell growth was faster in 15% FBS group. In comparison of feeder-dependent or -free culture, differentiation was more frequently appeared in feeder-free condition. However, a iPSC-like colony was recovered in 15% FBS group under feeder-free culture. These iPSC-like colonies were maintained up to 3 passages. When we checked the pluripotent cell marker alkaline phosphatase (AP), the activity was strongly detected in 2nd passage JBC iPS-like cells. Further experiments were needed to find the better stable culture condition able to more passages culture (>15) for establishment of JBC-iPSCs.