Earticle

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.

Silencing of Dicer1 by siRNA did not inhibit development up to the blastocyst stage, but decreased expression of selected transcription factors, including Oct‐4, Sox2 and Nanog, suggesting that Dicer1 gene expression is associated with differentiation processes at the blastocyst stage (Cui et al., 2007). In order to get insights into genes which may be linked with microRNA system, we compared gene expression profiles in Gapdh and Dicer1 siRNA‐microinjected blastocysts using the Applied Biosystem microarray technology. Our data showed that 397 and 737 out of 16354 genes were up‐ and down‐regulated, respectively, following siRNA microinjection (p<0.05), including 24 up‐ and 28 downregulated transcription factors. Identification of genes that are preferentially expressed at particular Dicer1 knock down embryos provides insights into the complex gene regulatory networks that drive differentiation processes in embryos at blastocyst stage.

이 연구는 HPV 16 E6/E7 도입 불멸화 구강상피세포의 배양 미세환경과 세포 분화간의 관계를 분석하였다. 배양환 경을 변화시켜서 IHOK-EF 세포와 IHOK-EFKGM 세포를 얻었고, 이들 세포의 특성변화를 세포증식분석, 면역형광분석 및 마이크로어레이와 실시간 정량 PCR분석으로 알아보았다. IHOK-EF 세포는 상피세포의 특성을 상실하고 간엽세포의 특성을 획득하였고, 마이크로어레이 분석결과, 분화억제 유전자인 ID2, IL6, TWIST1이 과발현 되었다. 이러한 변화는 초기 의 배양환경으로 회복되었을 때, 특별히, ID2와 IL6에서 유전자발현의 복귀를 나타내면서 세포의 특성이 부분적으로 회복되 었다. 이 연구는 세포의 특성을 결정하는 연구에서 배양 미세환경의 변화에 따른 세포의 생존을 위한 적응양상을 이해하는 데 공헌할 것이며, 향후, 암세포의 미세환경변화에 따른 생존연구에 적용하여 질병에 대한 치료적 접근을 가능하게 할 것이다.

This study was analyzed about the relationship between culture microenvironment and cell differentiation of HPV 16 E6/E7-transfected immortalized oral keratinocyte(IHOK). By the alteration of culture environment, IHOK-EF and IHOK-EFKGM were obtained, and the modulation of cell properties was observed by cell proliferation assay, immunofluorescence, microarray, and quantitative real-time PCR analysis. IHOK-EF losed the properties of epithelial cells and obtained the properties of mesenchymal cells, and in the result of microarray analysis, genes related to the inhibition of differentiation such as IL6, TWIST1, and ID2 were highly expressed in IHOK-EF. When the culture environment was recovered to initial environment, these changes were recovered partially, presenting the return of genes involved in the inhibition of differentiation such as IL6, and ID2, particularly. This study will contribute to understand adjustment aspect for cell surviving according to the change of culture microenvironment in the study for determining the cell characteristic, and facilitate therapeutic approach for human disease by applying surviving study according to the change of cancer microenvironment.

Osteoarthritis is one of the commonest causes associated with age-related damage of articular cartilage. Non-steroidal anti-inflammatory drugs are commonly used in osteoarthritic patient. However, long term administration of these drugs results gastrointestinal disorders. Though, most studies have demonstrated in the past that bee venom has therapeutic effect on diseases related to inflammation and pains, but its anti-inflammatory properties have not been so far studied on inflamed chondrocytes (LPS induced) invitro. For the purpose, the study was carried out to determine the effect of bee venom on porcine articular chondrocyte cell using microarray. In this study, we found that 2,235 significantly associated gene (1,404 up-regulated genes and 831 down-regulated genes) that were expressed on inflamed and non inflamed chondrocytes during proliferation. Among the 1,404 up-regulated genes and 831 down-regulated genes, known genes were 372 and 237, respectively. On the other hand, bee venom significantly reduced expression of fetuin involved in acute inflammatory reaction. Our results suggest that this study could be useful database in gene expression profiling of chondrocyte cell treated with bee venom.

Muscle satellite cell (SC) is responsible for postnatal muscle growth, repair, and regeneration. Satellite cell is an important source of multi-potent stem cell process and differentiation into adipogenic, myogenic, and osteoblastogenic. The objective of this study was to identify alter of transcriptome during differentiation in porcine satellite cell and to elevated transcriptome at different stages of postnatal development to gain insight into the differences in differentiated PSC. We used RNA-seq technique to investigate the transcriptomes during differentiation in pig muscle. Sequence reads were obtained from Illumina HiSeq2000. Differentially expressed genes (DEG) were detected by EdgeR. Gene ontology (GO) terms are powerful tool for unification among representation genes or products. In study of GO biological terms, functional annotation clustering involved in cell cycle, apoptosis, extracellular matrix, phosphorylation, proteolysis, and cell signaling in differences stage. Taken together, these results would be contributed to a better understanding of muscle biology and processes underlying differentiation. Our results suggest that the source of DEGs could be better understanding of the mechanism of muscle differentiation and transdifferentiation.

In order to investigate genetic stability and gene expression profile after cloning procedure, two groups of cloned pigs were used for swine leukocyte antigen (SLA) gene nucleotide alteration and microarray analyses. Each group was consist of cloned pigs derived from same cell line (n=3 and 4, respectively). Six SLA loci were analyzed for cDNA sequences and protein translations. In total, 16 SLA alleles were identified and there were no evidence of SLA nucleotide alteration. All SLA sequences and protein translations were identical among the each pig in the same group. On the other hand, microarray assay was performed for profiling gene expression of the cloned pigs. In total, 43,603 genes were analyzed and 2,150～4,300 reliably hybridized spots on the each chip were selected for further analysis. Even though the cloned pigs in the same group had identical genetic background, 18.6～47.3% of analyzed genes were differentially expressed in between each cloned pigs. Furthermore, on gene clustering analysis, some cloned pigs showed abnormal physiological phenotypes such as inflammation, cancer or cardiomyopathy. We assumed that individual environmental adaption, sociality and rank in the pen might have induced these different phenotypes. In conclusion, the results of the present study indicate that SLA locus genes appear to be stable following SCNT. However, gene expressions and phenotypes between cloned pigs derived from the same cell line were not identical even under the same rearing conditions.

본 연구는 한우의 자궁내막염에서 발현 변화를 보이는 유전자를 마이크로어레이를 이용하여 조사하였다. 정상적인 자궁내막과 자궁내막염이 있는 자궁내막을 비교한 결과, 전체 확인된 4,560개의 유전자 중 2,026개의 유전자가 자궁내막염에서 증가하였고, 2,534개의 유전자가 감소하였다. 본 연구에서는 상위 조절되는 유전자 10개씩을 정리하였다. 자궁내막염에서 filamin A, pancreatic anionic trypsinogen, Rho GDP dissociation inhibitor alpha, collagen type VI alpha 1, butyrate response factor 2, aggrecanses-2, annexin 14, aminopeptidease A, orphan transporter v7-3 및 epithelial stromal interaction 1의 발현율이 26배 이상 증가하였다. MHC class II antigen, integrin-binding sialoprotein, uterine milk protein precursor, down-regulated in colon cancer 1, glycoprotein 330, dickkopf-1, cfh protein, Ca2+-dependent secretion activator, UL16 binding protein 3 및 proenkephalin은 25.5배 이상 감소하였다. 본 연구에서 얻어진 유전자 정보는 한우의 자궁내막염 진단에 필요한 유용한 생물지표로 사용되어질 수 있을 것으로 사료된다.

This study was carried out to examine gene expression altered in endometrium of Korean cattle (Hanwoo) with endometritis using microarray. In this study, 4,560 differentially expressed genes (DEGs) were identified in the endometrium of Hanwoo. Of 4,560 DEGs, 2,026 genes were up-regulated, while 2,536 genes were down-regulated in endometritis. Of them, top 10 regulated genes were listed. Filamin A, pancreatic anionic trypsinogen, Rho GDP dissociation inhibitor alpha, collagen type VI alpha 1, butyrate response factor 2, aggrecanses-2, annexin 14, aminopeptidease A, orphan transporter v7-3, and epithelial stromal interaction 1 were up-regulated, while MHC class II antigen, integrin-binding sialoprotein, uterine milk protein precursor, down-regulated in colon cancer 1, glycoprotein 330, dickkopf-1, cfh protein, Ca2+-dependent secretion activator, UL16 binding protein 3, and proenkephalin were down- regulated in the endometritis. Our results suggest that these genes could be useful biomarkers for diagnosis Hanwoo’s endometritis.

본 연구는 소의 부위별 근육에 특이하게 발현하는 유전자 마커를 발굴하여 소고기의 부위를 과학적으로 판명할 수 있는 기술을 개발하고자 실시하였다. 이러한 연구 목표 아래 먼저 사태(Beef shank), 등심(Longissimus dorsi), 양지(Deep pectoral), 홍두깨 (Semitendinosus) 부위의 근육조직에서 MSC(myogenic satellite cell, 근육줄기세포)를 순수 분리하고 이를 MFC(myotube-formed cell; 근관이 형성된 세포)로 분화시키거나 ALC(adipocyte-like cell; 지방세포와 유사한 세포)로 이형분화 시킨 후 3가지의 세포로부터 각각의 RNA를 추출하였다. 이렇게 추출한 RNA는 24,000개의 bovine oligo-nucelotide(70 mer)가 집적된 microarray를 이용해 4개의 조직 중 1개의 조직에서만 MSC의 분화(MFC) 또는 이형분화 과정에서 mRNA의 발현이 증감을 보이는 유전자 135개를 먼저 발굴하였다. 135개의 유전자에 대해 microarray 분석에 사용한 동일한 RNA를 이용하여 real-time PCR 기술로 검증한 결과 총 29개의 유전자가 microarray 분석 결과와 유사함을 보였다. 29개의 유전자를 다시 4개 부위의 생체 조직에서 추출한 RNA를 이용해 real-time PCR 방법으로 분석한 결과 TS(thymi- dlyate synthase), TE(tropoelastin), RAD52(similar RAD52 motif- containing protein 1), unknown gene), MLC2(myosin light 2, regulatory cardiac, slow), TXNIP(thioredoxin-interating protein) 6개의 유전자만이 다른 부위에 비해 사태 부위에서 현저한 발현의 차이를 나타냈다. 결론적으로 본 연구를 통해 소 부위별 근육을 구분할 수 있는 과학적 기술의 토대를 확립하였다.

Myogenic satellite cells(MSCs) are mononuclear, multipotent progenitors of adult skeletal muscle possessing a capacity of forming adipocyte-like cells(ALC). To identify the skeletal muscle type-specific myogenic and adipogenic genes during MSCs differentiation, total RNA was extracted from bovine MSCs, myotube-formed cell(MFC), and ALC from each of Beef shank, Longissimus dorsi, Deep pectoral, and Semitendinosus. DNA microarray analysis(24,000 oligo chip) comparing MSCs with MFC and ALC, respectively, revealed 135 differentially expressed genes(>4 fold) among four cuts. Real-time PCR confirmed expression of 29 genes. Furthermore, the whole tissue sample RNAs analysis showed 6 differentially expressed genes in Beef shank. Among which, 1 gene in MSCs, 4 in MFC, and 1 in ALCs were highly expressed. This study will provide an insight for better understanding the molecular mechanism of differentiation of skeletal muscle type-specific MSCs. The identified genes may be used as marker to distinguish skeletal muscle types.

The present study was undertaken to investigate specific immune response of Rubus coreanus Miquel (raspberry) in pig spleen lymphocytes and gene expression induced by the extracts of raspberry using gene chip technology. The 70% ethyl alcohol extracts of raspberry were treated to pig spleen lymphocytes. The extracts of raspberry stimulated the proliferation of splenocytes and increased the population of CD3 & CD4 T-cells and B-cells in pig spleen lymphocytes. The extracts of raspberry improved immune response by increasing the viability of splenocytes. In microarray study we found eight genes were significantly up- regulated by the extracts of raspberry in pig splenocytes, including genes known to be involved in cell structure and immune response, particularly microtubule-associated protein 4, cytoplasmic dynein heavy chain, tumor necrosis factor alpha, lymphotoxin-beta receptor precursor. However, ten genes were down- regulated by the extracts of raspberry treatment.

최근 가축에 있어서 DNA marker를 이용하여 경제적으로 유용한 유전자를 찾아내는 연구가 활발히 진행되고 있다. 소의 경우 생산성을 향상시키기 위하여 경제형질관련 양적 형질좌위에 존재하는 후보 유전자를 선발한 후 형질변이의 원인이 되는 염기서열을 찾아 표지인자로 이용을 하고 있다. 본 연구는 소의 중요 경제형질인 육질 및 육량과 관련된 분자 유전학적 연구와 더불어 경제형질관련 후보유전자를 찾아내기 위하여 최근에 많이 이용되고 있는 microarray에 대하여 고찰하였다. 특히 microarray의 경우 cDNA microarray에서 oligoarray를 제작하여 이용함으로서 실험의 오차를 최대한 줄이는 방향으로 연구가 진행되고 있다. 소의 형질 관련 유전자에 관한 연구는 bovine genome sequencing이 끝난 현 시점에서 연구의 속도가 가속화될 것으로 생각되며 경제형질 원인 유전자의 분석 뿐 아니라 질병 저항성과 환경에 영향을 많이 받는 유전자를 확인하여 선발에 이용하기 위한 연구가 계속될 것으로 생각된다.

Researches in livestock are currently actively progressing to improve economically important traits using DNA markers. In cattle, the candidate genes have been selected based on their known functions in the target QTL (quantitative trait locus) region in order to identify QTN (quantitative trait nucleotide) for improving productivities. In this review, molecular genetic studies for the meat related traits, one of the major determinant of market prices, have been fully described. Also recent emerging microarray technique for identifying candidate genes in cattle has been discussed. In case of microarray, cDNA microarrays have been replaced to oligoarrays in order to minimize the experimental errors in cattle. Since the first draft of bovine genome sequences was appeared in the public domain, more markers in relation to the quantitative traits will be discovered in a short period of time and genes affecting difficult-to-measure traits, such as disease resistance, can also be selected for marker assisted selection in near future.

The effects of angiogenesis inhibitor from the extract libraries of Korean and Chinese medicinal plants were investigated using a protein microarray chip. Protein chip was constructed by immobilization of integrin α5β1 on protein chip base plates and employed for screening active extracts that inhibit the integrin-fibronectin interaction from the extract libraries. The 100 extracts of medicinal plants were obtained from extract bank of National Institute of Crop Science, RDA. The 14 extracts among 100 extract libraries were shown efficient inhibition activity for the interaction between integrin-fibronectin. The medicinal plants of 14 extracts were Vitex negundo var. incisa (Lam.) C.B. Clarke, Epimedium koreanum Nakai, Cedrela sinensis A. Juss, Ipomea aquatica Forsk, Schisandra chinensis Baill, Pulsatilla koreana Nakai, Paeonia lactiflora Pall. var.hortensis Makino, Oenothera odorata, Allium chinense, Allium victorialis var. platyphyllum MAKINO, Polygonatum odoratum Druce var. pluriflorum Ohwi, Hosta lancifolia, Agrimonia pilosa L. var. japonica Nakai and Potentilla chinensis SER. The Paeonia lactiflora, Oenothera odorata, and Agrimonia pilosa from these 14 extracts libraries were shown strong inhibition activity of integrin α5β1.