To test the muscle cell specific gene expression, we examined the ability of human α-skeletal muscle actin (ACTA) promoter or human myoglobin (hMb) promoter to direct the expression of the GFP gene in both muscle and non-muscle cells, respectively. C2C12 cells, a mouse myoblast cell line, provide a powerful model to study skeletal muscle differentiation in vitro. We intended to use this cell line as a model for skeletal muscle-specific gene expression during myogenic differentiation from myoblast to myotubes. We compared marker gene expression profiles of proliferating and differentiated C2C12 cells using RT-PCR and fluorescent microscopy analysis. Also, we found that the expression of PCK1 gene under the control of ACTA promoter was proportionally increased as C2C12 differentiated into myotube form. PCK1 is involved in the regulation of gluconeogenesis. In previous research, transgenic mice with overexpressing PCK1 in skeletal muscle showed a greatly enhanced level of physical activity, which extends well into old age. This is due, in part, to an increased number of mitochondria and a high concentration of triglyceride in their skeletal muscles. These mice also had very little body fat, despite eating 60% more than controls. We also constructed a mesenchymal stem cell line and fetal fibroblast cell line for the experiments aiming to make transgenic animals in which the PCK1 gene is specifically expressed in muscle tissue. Accumulated knowledge of this approach could be applicable to a variety of related biological areas including transgenic animal research, gene function study, anti-aging study, etc. This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through Export Promotion Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (316002-5).

Variance of conceptus interferon tau (IFNT), produced by the embryonic trophectoderm, is known as a major conceptus protein that signals the process of maternal recognition of pregnancy in ruminants, essential for the maintenance of early pregnancy. Similar to other IFN genes such as IFNA and IFNB, multiple IFNT genes are present. However, some kinds of IFNT genes actively transcribed and regulated in bovine conceptuses have not been well characterized. In this study, during the course of bovine IFNT gene transcription through the use of next generation sequencer SOLiD3, revealed that among 38 IFN genes registered, only two transcripts, IFNT1 and IFNTc1, were found in conceptuses during early pregnancy. Also, to identify a transcription factor(s) involved in the regulation of IFNT genes, mRNAs for various known transcription factors were investigated by real-time PCR in conceptus tissues, respectively. Furthermore, compared to the IFNT genes, IFNT1 and IFNTc1 had same active levels, which were previously shown to correlate with the appearance of effective antiviral activity. However, the expression levels of these Luc activities differed. Bovine ear fibroblast (EF) cells were cotransfected with luciferase reporter constructs carrying upstream (–631 to -51) promoter regions of IFNT1 or IFNTc1 and various transcription factor expression plasmids, CDX2, AP1(JUN), ETS2 and/or cAMP-response element binding protein (CREB)-binding protein (CREBBP). CDX2, either alone with the other 2 transcription factors, was found to increase luciferase activity approximately 14- and 11-folds, respectively. The degree of transcriptional activation of the IFNTc1 gene was not similar to that IFNT1 gene by AP1, ETS2 or/and CREBBP, expression plasmid. These results suggest that two isoforms of bovine conceptus IFNT genes are regulated differently in conceptuses during early pregnancy.

The present study was undertaken to evaluate the effect of trisaccharides supplementation in glycerol-free tris (GFT) for the cryopreservation of dog spermatozoa. In the first experiment (E1), dog spermatozoa were resuspended with 50, 75, 100 or 125 mM of raffinose, melezitose or maltotriose and cooled at 4 ℃ for 10 min. To determine the effect of different cooling time, the spermatozoa resuspended with 100 mM of raffinose, melezitose or maltotriose were cooled during 10, 20, 30 or 40 min at 4 ℃ (second experiment; E2). The straws were then aligned horizontally for 10 min on the rack and then plunged into LN2. In the third experiment (E3), to determine the effect of different vapor freezing time, the spermatozoa resuspended with 100 mM raffinose were cooled at 4 ℃ for 20 min and frozen in LN2 for 5, 10, 15 or 20 min and then plunged into LN2. In the fourth experiment (E4), to compare different freezing methods [cooling plus vapor freezing (CV), cooling plus step-down freezing (CS) and direct step-down freezing (SD)], the spermatozoa resuspended with 100 mM raffinose were cooled for 20 min and frozen in LN2 vapor for 5 min in case of CV method. In case of CS method, spermatozoa were cooled for 20 min at 4℃ and then frozen by the step-down freezing method. The straws were then aligned horizontally at 18, 15, 5, and 2 cm respectively from the surface of LN2 for 1, 1, 1.4, and 5 min, respectively in an L shaped straw holder and then plunged into LN2. For SD method, the straws were directly aligned horizontally at the same levels as CS from the surface of LN2 for 1, 1, 1.9, and 5 min, respectively and then plunged into LN2. After thawing at 37℃ for 25 sec, the spermatozoa were then incubated for 30 min in the freezing extender (E1) or in the 50 mM sucrose supplemented GFT (E2, E3, and E4) at 24℃. Following post-thaw incubation, sperm progressive motility and viability were assessed in E1, E2, E3, and E4. In addition, acrosome integrity, and gene expression related to apoptosis (BAX, BCL2, and Caspase10) and sperm motility (SMCP) were evaluated in E4. The results demonstrated that, in E1, using 75 mM trisaccharides resulted in significantly (p<0.05) higher sperm motility in all sugar groups. Using 100 mM melezitose significantly (p<0.05) improved the post-thaw viability than the 100 mM raffinose. The viability in 100 mM maltotriose was similar with 100 mM raffinose and melezitose group. In E2, the different cooling time has no significant effect on post-thaw sperm progressive motility in all the sugar types. In addition, the viability was variable among the different groups. In E3, liquid nitrogen vapor freezing for 5 min resulted in improved motility and viability. The sperm progressive motility was significantly (p<0.05) higher in CV and SD group compared to CS group and the sperm viability was significantly (p<0.05) higher in CV group compared to the other groups in E4. However, the acrosomal integrity of spermatozoa in the group CV was significantly (p<0.05) higher than the group CS and SD. In addition, the expression of SMCP gene was significantly (p<0.05) higher in the CV group than the CS group. In contrast, the expression of Caspase10 significantly (p<0.05) lower in the group CV and SD than the group CS. Furthermore, the ratio of gene expression of BAX and BCL2 was significantly (p<0.05) lower in the group CV than the group CS. Therefore, cryopreservation of dog spermatozoa in 100 mM of raffinose supplemented GFT cooled for 20 min and vapor freezing for 5 min provides better progressive sperm motility, viability, and acrosome integrity with higher expression of SMCP gene and lower expression of caspase10 and BAX/BCL2 ratio following post-thaw incubation in 50 mM sucrose supplemented GFT for 30 min at 24℃.

Honey bee, Apis mellifera L., have been widely used as a model organism for biological science because of its highly developed sociality, specialized labor division and passive population management. In order to examine the expression patterns of genes putatively involved in social development in honey bee, quantitative real-time PCR (qRT-PCR) that has been widely used to investigate the expression level of target gene can be used in honey bee study. However, the selection and validation of optimal reference genes is a crucial step prior to running qRT-PCR. In the present study, therefore, the seasonal expression stability of five candidate reference genes in the abdomen of forager and nurse was investigated using three programs (geNorm, NormFinder and BestKeeper), and selected reference genes were validated by the normalization of expression level of vg encoding vitellogenin. Although three programs revealed slightly different gene stability values, overall the combination of two genes (rpS18 and gapdh encoding ribosomal protein S18 and glyceraldehyde-3-phosphate dehydrogenase, respectively) was resulted in the most suitable use for normalization of the target gene in forager. However, a single gene, either rpL32 or rpS18 in the nurse or either rpL32, rpS18, or gapdh in the comparison between foragers and nurses, were suggested to be applied for normalization of seasonal and labor-specific gene expression by qRT-PCR.

The pea aphid, Acyrthosiphon pisum, requires the nutritional endosymbiont, Buchnera, for the production of essential amino acids. However, it is unclear if host plant diet that varies in essential amino acids influences aphid regulation of its nutritional symbioses. We hypothesized that aphid genes respond to host plant diet when aphids feed on their specialized (alfalfa) compared to universal host plant diet (fava), which vary in essential amino acid concentrations. Using RNA-Seq and whole genome bisulfite sequencing, we compared the gene expression profiles and DNA methylation distributions of specialized aphid cells that harbor Buchnera (bacteriocytes) when aphids feed on their specialized compared to their universal host plant diets. Our results show that bacteriocyte transcription and methylation patterns differ between host plant diets. When aphids feed on their specialized host plant, they significantly up-regulate and/or hypo-methylate key aphid genes in bacteriocytes related to the amino acid metabolism, including glutamine synthetase (GS) and the glutamine transporter ApGLNT1. Moreover, regardless of which host plant aphids feed on, we observed significant up-regulation and differential methylation of the key genes in the amino acid metabolism and the glycine/serine metabolism in aphid bacteriocytes. We suggest that the regulatory response of key symbiosis genes in bacteriocytes allows aphids to feed on a specialized host plant diet with suboptimal nitrogen concentrations.

곤충은 넓은 범위의 온도영역에 사는 것으로 알려져 있으나, 40℃가 넘는 고온이나 빙결온도 이하의 저온에서는 생존이 어렵다. 본 연구는 사육온도 조건이 다른 환경에서 대사중심 조직인 지방체의 유전자 발현을 분석하기 위해, 온도조건을 달리하여 담배나방을 저온 사육충 (3~10℃), 고온 사육충 (35℃)로 나누고 상온 사육충 (25℃)을 대조구로 사용하여 전사체 분석을 수행하였다. 저온에서 특이적으로 높은 발현을 보인 유전자는 표피단백질, △9 불포화효소, 글리세롤 3-인산 탈수소효소이며, 저온에서 발현이 낮아진 유전자는 키틴 합성효소, catalase, UDP-당전이 효소이다. 고온에서 특이적으로 높은 발현을 보인 유전자는 과산화물제거효소, metallothionein 2, phosphenolpyruvate carboxykinase, 트레할로스 운반단백질이었다. 고온에서 높고 저온에서 낮은 대조적 발현을 보인 유전자는 열충격단백질, glutathione peroxidase이었다. 이들 온도 특이적이거나 대조적 발현을 보이는 유전자는 기후변화에 관련한 특이마커로 활용이 가능할 것으로 사료된다.

Adhesive capsulitis of the shoulder is a common cause of pain that occurs during shoulder movement, thereby restricting shoulder rotation in clinical practice. Although most patients respond to pain relief treatment (NSAID or corticosteroids) by improving their range of motion, it remains poorly understood without any definitive treatment algorithm. In addition to immune cells, synoviocytes, chondrocytes and osteoblasts in the joint are known to produce pro-inflammatory mediators such as reactive oxygen species (ROS), inflammatory cytokines and lipid mediators, presumably contributing to the pathogenesis of osteoarthritis (OA) and adhesive capsulitis. Although inflammation and also fibrosis are proposed to be the basic pathological changes of a frozen shoulder, there is a lack of information regarding the downstream targets of the pro-inflammatory ROS signaling pathway in the synoviocytes and also how these ROS targets are modulated at the transcription level by a corticosteroid - dexamethasone. In this study, we used human fibroblast like synoviocytes (HFLS) to characterize the signaling targets of ROS by employing a human DNA microarray tool and studied the role of dexamethasone in this process. Our data suggest that several genes such as FOS, FOSB and NFkBIZ, which are known to be involved in pro- or anti- inflammation response, are modulated at the transcription level by ROS and dexamethasone.

화색변형 화훼 형질전환체 개발을 위한 유전자를 개발하고자 클레마티스로부터 청색소 발현 유전자를 동정하여 ClF3 ′5 ′H로 명명하였다. 아그로박테리움 공동배양 경유 형질전환기술을 이용 하여 페튜니아 Dream’s Red 품종에 ClF3 ′5 ′H 유전자를 도입하였 고, 선발배지에서 재분화한 식물체 51개체를 PCR분석하여 49개체로 ClF3 ′5 ′H 유전자의 도입을 확인하였다. 또한 22개체의 서던분석 을 통해 ClF3 ′5 ′H 유전자가 1~3개 copy가 도입되었음을 확인하였 고, 노던분석을 통해 형질전환체에서의 ClF3 ′5 ′H 유전자의 정상 적인 발현을 확인하였다. ClF3 ′5 ′H 유전자가 도입된 페튜니아 형질전환 식물체 7개체가 개화할 때 ClF3 ′5 ′H 유전자가 도입된 페튜니아아의 꽃잎 주요색이 적색에서 진적색으로, 화관 내부색이 적보라색에서 진적보라색 또는 자주색 계역로, 암술머리색이 연두색에서 자주색 계열로, 꽃잎 가장자리 모양이 물결모양으로 변형되는 것을 확인하였다. 또한 실시간 정량 유전자 분석을 통해 형질전환 7개체에서의 표현형적 변화가 ClF3 ′5 ′H 유전자의 발현에 의한 것임을 입증하였다.