Earticle

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.

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.