Enamel knot (EK)—a signaling center—refers to a transient morphological structure comprising epithelial tissue. EK is believed to regulate tooth development in early organogenesis without its own cellular alterations, including proliferation and differentiation. EKs show a very simple but conserved structure and share functions with teeth of recently evolved vertebrates, suggesting conserved signaling in certain organs, such as functional teeth, through the course of evolution. In this study, we examined the expression patterns of key EK-specific genes including Dusp26 , Fat4, Meis2, Sln , and Zpld1 during mice embryogenesis. Expression patterns of these genes may reveal putative differentiation mechanisms underlying tooth morphogenesis.

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.

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.

Effectiveness of transgene transfer into genome is crucially concerned in mass production of the bio-pharmaceuticals using genetically modified transgenic animals as a bioreactor. Recently, the mammary gland has been considered as a potential bioreactor for the mass production of the bio-pharmaceuticals, which appears to be capable of appropriate post-translational modifications of recombinant proteins. The mammary gland tissue specific vector system may be helpful in solving serious physiological disturbance problems which have been a major obstacle in successful production of transgenic animals. In this study, to minimize physiological disturbance caused by constitutive over-expression of the exogenous gene, we constructed new retrovirus vector system designed for mammary gland-specific expression of the hEPO gene. Using piggyBac vector system, we designed to express hEPO gene under the control of mammary gland tissue specific and lactogenic hormonal inducible goat β-casein or mouse Whey Acidic Protein (mWAP) promoter. Inducible expression of the hEPO gene was confirmed using RT-PCR and ELISA in the mouse mammary gland cells treated with lactogenic hormone. We expect the vector system may optimize production efficiency of transgenic animal and reduce the risk of global expression of transgene.

Norovirus (NoV) is an etiologic agent of human and animal acute gastroenteritis and is a member of the family Caliciviridae. NoV is classified based on nucleotide sequences of the VP1 gene into at least six genogroups (GI-GVI), among which GI, GII, and GIV are known to infect humans and GII is the most prevalent genogroup. In this study, VP1, the full gene of GII human NoV, was cloned from a human fecal sample and expressed using a baculovirus expression system. Human NoV VP1-specific monoclonal antibodies (MAbs) were produced using expressed recombinant VP1. Expressed VP1 in the recombinant virus was confirmed by polymerase chain reaction (PCR), indirect fluorescence antibody (IFA) test, and Western blot analysis. Eight hybridomas secreting VP1-specific MAbs against human GII NoV were generated and characterized. All of the MAbs produced in this study reacted with human GII NoV VP1-recombinant baculoviruses but not with other non-human calicivirus recombinant baculoviruses. These MAbs reacted specifically with human NoV GII.4-2009 virus-like particles (VLPs), and some MAbs showed cross-reactivity with other GII.4 variant VLPs. Expressed human GII NoV VP1-recombinant protein and MAbs specific to this protein can be used as useful reagents for detecting and characterizing human NoV.

Temperature and insecticide are two important factors that affect survival, reproduction and other physiological processes of insects. To determine interactions of temperature and insecticide treatment on susceptible (S) and imidacloprid-resistant strains (IMI-R) of Aphis gossypii, adults were exposed to three temperatures (17, 22, and 28℃) or combinations of three temperatures (17, 22 and 28℃) and imidacloprid (LC20), and the expression of several genes (heat shock protein 70, cuticle protein, cytochrome P450, and elongation factor) were analyzed. The expression level at 17℃ of heat shock protein 70, cuticle protein, and elongation factor in S strain were up-regulated with increased time and higher than those of IMI-R strain. However, expression of cytochrome P450 was not affected by elevated temperature both S and IMI-R strain. Combined treatment of elevated temperature and imidacloprid were significantly up-regulated only cuticle protein in S strain and higher than those of IMI-R strain.

Transgenic chickens have been spotlighted as an highly potent bioreactor for their fecundity, short generation time, and eggs associated with mass production of protein. In this study, we generated transgenic chickens exhibiting oviduct specific expression of human growth hormone fused to human transferrin for oral administration. Gene of the modified growth hormone located at downstream ovalbumin promoter (∼3.6 kb) was introduced to stage X blastodermal cell employing retrovirus vector system. Several transgenic chickens were successfully generated. However, genomic analyses showed unexpected deletion within the transgene. The modification of the transgene seemed to occur during germ cell formation because the deletion was detected only from the sperm DNA of the G0 founder animal. There was no evidence of deletion in the somatic cell DNA samples of the same chicken. Consequently, same pattern of the deletion was confirmed in both somatic and germ cells of the G1 progeny.

The miniature pig is considered to be a better organ donor breed for xenotransplantation than other pig breeds because the size of the organs of the miniature pig is similar to that of humans. In this study, we aimed at identifying differentially expressed genes in the miniature pig ovary during pregnancy. For this, we used the miniature pig ovary model, annealing control primer‐based reverse transcription polymerase chain reaction (PCR), quantitative real‐time PCR (qRT‐PCR), and northern blotting analysis. We identified 13 genes showing differential expression on the based of pregnancy status and validated 8 genes using qRT‐PCR. We also sequenced the full‐length cDNA of ephrin receptor A4 (EphA4), which had a significant difference in expression level, and validated it by northern blotting. These genes may provide a better understanding of the cellular and molecular mechanisms during pregnancy in miniature pig ovary.

This study was conducted to investigate the specific expression genes in the cloned bovine tissues. Donor cells, cloned tissues were analysed by RAPD-RFLP method. The results were detected three genes (CH-U7B, CH-U7M and CH-U7P) in the cloned fetus. It was found a single copy genes by southern hybridization. Sequence analysis of CH-U7M gene was shown 99% homology to a previously reported EST from a cloned bovine fetus. The putative ORF was encode a protein of hydrophobicity index 0.03. Semi-quantitative RT-PCR by using the CH-LS001 specific primer was remarkably detected in the lung tissue of cloned fetus. Further investigation of these genes may provide one of the key information to explain the early death, abnormal fetus, large off-spring and the low pregnancy rate in the production of cloned bovine.

Ionizing radiation is a well- known therapy factor for human carcinoma cells. Genotoxic stress mediates cell cycle control, transcription and cellular signaling. In this work, we have used a microarray hybridization approach to characterize the cell type-

Periodontalligament (PDL) fibroblasts have an ectomesenchymal origin and are known to participate not only in formation of PDL but also in the repair and regeneration of the a이acent alveolar bone and cementum. However, little is known about the molecular mechanism which is related to the development and differentiation of PDL cells. Recendy, we reported the PDLs (a periodontalligament-specific) 22 as a PDL fibroblast-specific mRNA which is not expressed in gingival fibroblasts. In this study, to examine the expression and functional characterization of PDμ22 mRNA and prαein in development and differentiation of periodontal 따sue ， we carried out northem analysis, insitu hybridization, immunofluorescence and immunohistochemistry. The expression of PDLs22 mRNA was increased with PDL cell differentiation from the confluent to multilayer stage but decreased slighdy with mineralized nodule formation in vitro. πle PDLs22 protein was localized on the nuclear membrane and expressed throughout the differentiation of PDL fibroblasts in vitro. The PDLs22 mRNA and protein were expressed in the differentiating cementoblasts, PDL fibroblasts and osteoblasts along the r∞t surface and alveolar bone of the developing rat teeth. These results indicate that the PDLs22 plays an irnportant role in the differentiation of cementoblasts and osteoblasts and thus homeostasis of cementum, PDL and alveolar bone.