• Clinical and Experimental Reproductive Medicine
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• 제23권3호
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• pp.269-275
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• 1996

Biosynthesis and secretion of anterior pituitary hormones are under the control of specific hypothalamic stimulatory and inhibitory factors. Among them, Growth Hormone Releasing Hormone (GHRH) is the major stimulator of pituitary somatotrophs activating GH gene expression and secretion. Human GHRH is a polypeptide of 44 amino acids initially isolated from pancreatic tumors, and the gene for the hypothalamic form of GHRH is organized into 5 exons spanning over 10 kilobases (kb) on genomic DNA and encodes a messenger RNA of 700-750 nucleotides. Several neuropeptides classically associated with the hypothalamus have been found in the extrahypothalamic regions, suggesting the existence of novel sources, targets and functions. GHRH-like immunoreactivity has been found in several peripheral sites, including placenta, testis, and ovary, indicating that GHRH may also have regulatory roles in peripheral reproductive organs. Furthermore, higher molecular weight forms of the GHRH transcripts were identified from these organs (1.75 kb in testis; 1.75 and >3 kb in ovary). These tissue-specific expression of GHRH gene suggest the existence of unique regulatory mechanism of GHRH expression and function in these organs. In fact, placenta-specific and testis-specific promoters for GHRH transcripts which are located in about 10 kb upstream region of hypothalamic promoter were reported. The use of unique promoters in extrahypothalamic sites could be refered in a different control of GHRH gene and different functions of the translated products in these tissues. Somatotrophs and lactotrophs have been thought to be derived from a common bipotential progenitor, the somatolactotrophs, which give origins to either phenotypes. Although the precise mechanism responsible for the lactotroph differentiation in the anterior pituitary gland has not been yet clalified, there are several candidators for the generation of lactotrophs. In human, the presence of GHRH peptides with different size from authentic hypothalamic form in the normal anterior pituitary and several types of adenoma were demonstrated. Recently our group found the existence of immunoreactive GHRH and its transcript from the normal rat anterior pituitary (gonadotroph> somatotroph> lactotroph), and the GHRH treatment evoked the increased proliferation rate of anterior pituitary cells in vitro. The transgenic mouse models clearly shown that GHRH or NGF overexpression by anterior pituitary cells induced development of pituitary hyperplasia and adenomas particularly GH-oma and prolactinoma. Taken together, we hypothesize that the pituitary GHRH could serve not only as a modulator of hormone secretion but as a paracrine or autocrine regulator of anterior pituitary cell proliferation and differentiation. Interestingly enough, the expression of Pit-1 homeobox gene (the POU class transcription factor) was confined to somatotrophs, lactotrophs and somatolactotrophs in which GHRH receptors are expressed commonly. Concerning the mechanism of somatolactotroph and lactotroph differentiation in the anterior pituitary, we have focused following two possibilities; (1) changes in the relative levels or interactions of both hypothalamic and intrapituitary factors such as dopamine, VIP, somatostatin, NGF and GHRH; (2) alterations of GHRH-GHRH receptor signaling and Pit-1 activity may be the cause of lactotroph differentiation or pituitary hyperplasia and adenoma formation. Extensive further studies will be necessary to solve these complicated questions.

• Journal of Animal Science and Technology
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• 제49권5호
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• pp.577-584
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• 2007

본 연구는 후대검정 한우 295두의 혈액으로부터 genomic DNA를 추출하여 PCR 방법에 의한 증폭과 염기서열 분석을 통하여 ADSF/resistin 유전자의 단일염기다형을 발굴하고 이들과 한우 육질관련형질과의 상관관계를 분석하기 위하여 실시하였다. 확보된 DNA로부터 염기서열을 결정한 결과 promoter와 4개의 exon영역에서는 SNP를 찾지 못하였으나 intron 영역에서 7개의 SNP를 발굴하였다. 발굴된 SNP의 출현 빈도는 0.027에서 0.16까지 그 차이가 많았다. 육질형질과의 상관분석에서 이들 SNP 중 intron 2에서 발굴된 764A ins 만이 근내지방도와 상관관계가 발견되었다(P<0.05). 근내지방도는 유전력이 매우 높기 때문에 이번에 발굴된 ADSF/resistin 유전자의 SNP 764A ins와 같이 유전표지인자를 이용하는 것이 근내 지방도의 개량을 위해 우수한 결과를 가지는 것으로 사료된다. 가축의 경제형질의 경우 다수의 유전자가 관여하기 때문에 한 개의 유전자를 이용한 가축의 선발 또는 개량에 이용한다는 것은 제한적일 수 있다. 따라서 다수의 관련 유전자를 이용한 다형현상과 경제형질과의 연관성 연구가 동반될 때 육질개선 및 가축개량에 실질적인 증대효과가 있을 것으로 사료된다.

• 한국수정란이식학회지
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• 제33권3호
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• pp.129-137
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• 2018

Acute vascular rejection has been known as a main barrier occurring in a xenograted tissue of alpha 1,3-galactosyltransferase knock-out (GalT KO) pig into a non-human primate (NHP). Adenosine which is a final metabolite following sequential hydrolysis of nucleotide by ecto-nucleotidases such as CD39 and CD73, act as a regulator of coagulation, and inflammation. Thus xenotransplantation of CD39 and CD73 expressing pig under the GalT KO background could lead to enhanced survival of recipient NHP. We constructed a human CD39 and CD73 expression cassette designed for endothelial cell-specific expression using porcine Icam2 promoter (pIcam2-hCD39/hCD73). We performed isolation of endothelial cells (pAEC) from aorta of 4 week-old GalT KO and membrane cofactor protein expressing pig ($GalT^{-MCP/-MCP}$). We were able to verify that isolated cells were endothelial-like cells using immunofluorescence staining analysis with von Willebrand factor antibody, which is well known as an endothelial maker, and tubal formation assay. To find optimal condition for efficient transfection into pAEC, we performed transfection with GFP expression vector using four programs of nucleofection, M-003, U-023, W-023 and Y-022. We were able find that the program W-023 was optimal for pAEC with regard to viability and transfection efficiency by flow cytometry and fluorescent microscopy analyses. Finally, we were able to obtain $GalT^{-MCP/-MCP}/CD39/CD73$ pAEC expressing CD39 and CD73 at levels of 33.3% and 26.8%, respectively. We suggested that pACE isolated from $GalT^{-MCP/-MCP}$ pig might be provided as a basic resource to understand biochemical and molecular mechanisms of the rejections and as an alternative donor cells to generate $GalT^{-MCP/-MCP}/CD39/CD73$ pig expressing CD39 and CD73 at endothelial cells.

• Journal of Plant Biotechnology
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• 제2권2호
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• pp.61-71
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• 2000

By screening a cDNA library of auxin-treated mung bean (Vigna radiata L.) hypocotyls, we have isolated two full-length cDNA clones, pVR-ACS6 and pVR-ACS7, for 1-aminocyclopropane-1-carboxylate (ACC) synthase, the rate-limiting enzyme in the ethylene biosynthetic pathway. While PVR-ACS6 corresponds to the previously identified PCR fragment pMBA1, pVR-ACS7 is a new cDNA clone. A comparison of deduced amino acid sequences among auxin-induced ACC synthases reveal that these enzymes share a high degree of homology (65-75%) to VR-ACS6 and VR-ACS7 polypeptides, but only about 50% to VR-ACS1 polypeptide. ACS6 and ACS7 are specifically induced by auxin, while ACS1 is induced by cycloheximide, and to lesser extent by excision and auxin treatment. Results from nuclear run-on transcription assay and RNA gel blot studies revealed that all three genes were transcriptionally active displaying unique patterns of induction by IAA and various hormones in etiolated hypocotyls. Particularly, 24-epibrassinolide (BR), an active brassinosteroid, specifically enhanced the expression of VR-ACS7 by distinct temporal induction mechanism compared to that of IAA. In addition, BR synergistically increased the IAA-induced VR-ACS6 and VR-ACS7 transcript levels, while it effectively abolished both the IAA- and kinetin-induced accumulation of VR-ACS1 mRNA. In light-grown plants, VR-ACS1 was induced by IAA in roots, whereas W-ACS6 in epicotyls. IAA- and BR-treatments were not able to increase the VR-ACS7 transcript in the light-grown tissues. These results indicate that the expression of ACC synthase multigene family is regulated by complex hormonal and developmental networks in a gene- and tissue-specific manner in mung bean plants. The VR-ACS7 gene was isolated, and chimeric fusion between the 2.4 kb 5'-upstream region and the $\beta$-glucuronidase (GUS) reporter gene was constructed and introduced into Nicotiana tobacum. Analysis of transgenic tobacco plants revealed the VR-ACS7 promoter-driven GUS activity at a highly localized region of the hypocotyl-root junction of control seedlings, while a marked induction of GUS activity was detected only in the hypocotyl region of the IAA-treated transgenic seedlings where rapid cell elongation occurs. Although there was a modest synergistic effect of BR on the IAA-induced GUS activity, BR alone failed to increase the GUS activity, suggesting that induction of VR-ACS7 occurs via separate signaling pathways in response to IAA and BR.

• 식물조직배양학회지
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• 제22권2호
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• pp.95-99
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• 1995

본 연구는 이미 확립되어 있는 고려인삼의 체세포배발생을 통한 식물체 재분화와 Agrobacterium을 매개로 한 형질전환 시스템을 이용하여 항곰팡이성 인삼을 개발하고자 염기성인 강낭콩 키틴가수분해효소 유전자를 인삼으로 도입하였다. CaMV 35S promoter-강낭콩 키틴가수분해효소 유전자와 선발표지로서의 neomycin phosphotransferase II (NPT II) 유전자를 가진 pChi/748 binary 벡터를 pGA748로부터 제조하여 이를 도입한 A. tumefacience LBA4404와 인삼 접합배의 자엽절편을 1 mg/L 24-D, 0.1 mg/L kinetin이 첨가된 MS 액체배지에서 48시간 동안 공동배양한 후 동일배지에 100 mg/L kanamycine 500 mg/L carbenicillin을 첨가한 고체 배지에 옮겨 배양하였다. 배양 한달 후부터 절편의 절단면 부근으로부터 캘러스가 유도되기 시작하였으며 이어서 수많은 체세포배가 형성되었다. 이들 체세포배를 BA와 GA3가 각각 1 mg/L 첨가된 배지로 옮겨서 5주 경과되었을 때 식물체로 전환되었다. 재분화된 개체 중 선발된 8개의 식물체로부터 PCR과 이 산물의 Southern분석 결과 6개의 재분화 개체에서 강낭콩 키틴가수분해효소 유전자가 도입되었음을 확인하였다.

• 한국가축번식학회지
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• 제26권2호
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• pp.87-94
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• 2002

본 연구는 섬유소분해효소 유전자(CelD)가 도입된 형질전환 돼지를 생산하기 위해 사계절동안 수행하였다. 약 8∼15개월령의 순종의 랜드레이스경산돈 및 미경산돈 126두는 유전자 미세주입을 위한 1세포기 단계의 수정란 채란 및 이식을 위해 사용하였으며, 발정동기화 및 과배란 방법은 PG600 주입 후 9일간 매일 20mg의 altrenogest를 사료에 첨가하여 급여하였다. Altrenogest를 9일간 급여 후 1000IU의 PMSG와 750IU의 hCG를 주입하므로서 과배란을 유도하였다. 미세주입을 위한 유전자는 Rat elasterase promoter에 CelD유전자를 연결하여 준비하였으며, 호르몬 처리후 91두의 공란돈으로부터 1,422개의 난자를 회수하였으며 이중 95.6% (1,359/1,422)는 DNA미세주입을 위해 전핵을 관찰 할 수 있는 1세포기의 수정란이었다. 이중 유전자가 미세주입된 725개의 난자를 35두의 수란돈에 이식하였으며 13두의 임신돈으로부터 65두의 자돈을 생산하였다. 한편 수란돈당 수정란 이식수가 임신율에 미치는 영향을 조사한 결과 약 21∼24개의 수정란을 이식한 구에서 임신율이 50%로 타구의 20.0%(20개 이하)와 33.3%(25개 이상)보다 높았으며, 꼬리조직으로부터 분리된 DNA의 PCR검정결과 65두중 5두가 형질전환 양성 반응을 나타내어 7.69%의 형질전환율을 나타내었다 따라서 본 연구는 생체반응기를 통한 형질전환 돼지생산을 위한 유용한 정보를 제공하게 될 것이다.

• BMB Reports
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• 제40권2호
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• pp.212-217
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• 2007

The chicken oviduct is a dynamic organ that produces secretory proteins such as ovalbumin during the laying period. In this study, we identified oviduct-specific proteins in hens during the egg-laying period by proteomic analysis. Proteins extracted from the magnum of hens of different ages (5, 35, and 65 weeks) were analyzed by two-dimensional gel electrophoresis to compare the intensity of proteins among samples. Approximately 300 spots were detected on each gel. Based on the comparison of image gels, we found that the intensity of eight spots in 35-week magnums was increased at least by 2-fold compared with the others. Five of the eight spots were identified as calumenin, acidic ribosomal phosphoproteins (ARP), prohibitin, heart fatty acid-binding protein, and anterior gradient-2 (AGR-2). In particular, ARP and AGR-2 were highly expressed in 35- week magnums compared with 5- and 65-week magnums. In addition, the level of these proteins was consistent with their RNA levels. Expression of AGR-2 mRNA was detected in the mature magnum, whereas no signal was observed in premature tissue. Among various tissues, expression of AGR-2 mRNA was highest in the magnum, high in the isthmus, and five fold lower in muscle. It was undetectable in the liver and in other tissues (heart and kidney). However, the mRNA levels of other proteins were ubiquitous among tissues. In transcriptional activity of AGR-2, a 3.0 kb fragment of promoter region containing potential estrogen receptor binding sites had enhanced its activity strongly. In conclusion, these results suggest that AGR-2 has functional regulatory roles in the chicken oviduct during the egglaying period.

• Asian-Australasian Journal of Animal Sciences
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• 제23권7호
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• pp.855-862
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• 2010

Among the known interferon-induced antiviral mechanisms, the Mx pathway is one of the most powerful pathways. The Mx protein has direct antiviral activity and inhibits a wide range of viruses by blocking an early stage of the viral replication cycle. Cloning, characterization, and expression of Mx in vivo and in vitro have been conducted. The chicken Mx gene spans 21 kb and is made up of 14 exons and 13 introns, of which the promoter region was analyzed. The real-time PCR results showed that Mx expression was increased in chicken embryo fibroblasts (CEF) after 12- and 24-h induction with polyI: C. Induction of Mx expression by poly I: C in vivo revealed tissue-specific patterns among the chicken tissues tested. A trace expression of Mx was detected in healthy chicken liver tissues from adult chickens without inducement; the expression levels in the liver, heart, and gizzard were higher than in the muscle and kidney. This is the first report to demonstrate the expression of a glutathione-S-transferase-tagged-Mx fusion protein of 75 KDa, as well as the biological activity tested by SDS-PAGE and western blotting.

• Reproductive and Developmental Biology
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• 제35권1호
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• pp.23-31
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• 2011

Two piglets and one juvenile pig were used to investigate closely what types of cells express green fluorescent protein (GFP) and if any, whether the GFP-tagged cells could be used for stem cell transplantation research as a middle-sized animal model in bone marrow cells of recloned GFP pigs. Bone marrow cells were recovered from the tibia, and further analyzed with various cell lineage markers to determine which cell lineage is concurrently expressing visible GFP in each individual animal. In the three animals, visible GFP were observed only in proportions of the plated cells immediately after collection, showing 41, 2 and 91% of bone marrow cells in clones #1, 2 and 3, respectively. The intensity of the visible GFP expression was variable even in an individual clone depending on cell sizes and types. The overall intensities of GFP expression were also different among the individual clones from very weak, weak to strong. Upon culture for 14 days in vitro (14DIV), some cell types showed intensive GFP expression throughout the cells; in particular, in cytoskeletons and the nucleus, on the other hand. Others are shown to be diffused GFP expression patterns only in the cytoplasm. Finally, characterization of stem cell lineage markers was carried out only in the clone #3 who showed intensive GFP expression. SSEA-1, SSEA-3, CD34, nestin and GFAP were expressed in proportions of the GFP expressing cells, but not all of them, suggesting that GFP expression occur in various cell lineages. These results indicate that targeted insertion of GFP gene should be pursued as in mouse approach to be useful for stem cell research. Furthermore, cell- or tissue-specific promoter should also be used if GFP pig is going to be meaningful for a model for stem cell transplantation.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 2001년도 추계학술대회 및 정기총회
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• pp.108-108
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• 2001

Axin2/Conductin/Axil and its ortholog Axin are negative regulators of the Wnt signaling pathway, which promote the phosphorylation and degradation of ${\beta}$-catenin. While Axin is expressed ubiquitously, Axin2 mRNA was seen in a restricted pattern during mouse embryogenesis and organogenesis. Because many sites of Axin2 expression overlapped with those of several Wnt genes, we tested whether Axin2 was induced by Wnt signaling. Endogenous Axin2 mRNA and protein expression could be rapidly induced by activation of the Wnt pathway, and Axin2 reporter constructs, containing a 5.6 kb DNA fragment including the promoter and first intron, were also induced. This genomic region contains eight Tcf/LEF consensus binding sites, five of which are located within longer, highly conserved non-coding sequences. The mutation or deletion of these Tcf/LEF sites greatly diminished induction by ${\beta}$-catenin, and mutation of the Tcf/LEF site T2 abolished protein binding in an electrophoretic mobility-shift assay. These results strongly suggest that Axin2 is a direct target of the Wnt pathway, mediated through Tcf/LEF factors. The 5.6 kb genomic sequence was sufficient to direct the tissue specific expression of d2EGFP in transgenic embryos, consistent with a role for the Tcf/LEF sites and surrounding conserved sequences in the in vivo expression pattern of Axin2. Our results suggest that Axin2 participates in a negative feedback loop, which could serve to limit the duration or intensity of a Wnt-initiated signal.