• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.60-60
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• 2001

Human thrombopoietin (hTPO) is a cytokine that plays a central role in megakaryopoiesis by influencing on the development and maturation of megakaryocyte and platelet production. To induce hTPO production in the mammary gland, expression vector was constructed by combining the promoter of bovine beta-casein gene, cDNA of hTPO and neomycine resistance gene for transfection into fibroblasts. Bovine fibroblast cells derived from female ear skin were transfercted with the expression vector using Lipofectamine (Life Technology, NY). Transected cells resistant to G4l8 treatment (600 $\mu\textrm{g}$/$m\ell$) were recovered and colony formation was initiated at 13 days. The colonies with about 1 cm diameter were picked and analysed by PCR. Single transfected cells were individually transferred to enucleated oocytes. After electrofusion, the reconstructed embryos were exposed to calcium ionophore (5uM) for 5 min followed by treatment with 6-DMAP (2.5 mM) for 4h. The nuclear transfer embryos were cultured in CRlaa medium at 38.5C, 5% $CO_2$ for 7 days. Twenty three of 29 (79.3%) colonies were proved to be hTPO transfectants by PCR. The colonies were further passaged and used to produce transgenic embryos using nuclear transfer. Cleavage and developmental rates of reconstructed embryos to the blastocyst stage were 65.1% and 39.4%, respectively Of 22 blastocysts that developed from reconstructed embryos with the transfected cell, 20 embryos (90.9%) were positive for hTPO by using PCR analysis. The results suggest that somatic cell nuclear transfer is efficient for production of transgenic embryos.

• 한국수정란이식학회지
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• 제23권3호
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• pp.133-139
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• 2008

DNA methylation is one of the reasons for poor survival of clone animals. The OCT-4 gene is essential for maintaining pluripotency of embryonic stem (ES) cells and early embryos. We previously reported that the 5'-promoter region of Oct-4 gene was a target of DNA methylation and the methylation status was changed variously during embryonic development in bovine. The study conducted to examine the expression and methylation pattern of tissue-dependent differentially methylated region (T-DMR) of Oct-4 gene in bovine somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF) blastocysts. The Oct-4 gene expression was evaluated by RT-PCR and fluorescence immunocytochemistry. The methylation pattern of T-DMR was analyzed using restriction mapping and bisulfite sequencing methods. The Oct-4 transcripts were highly expressed in IVF, while they were not expressed in SCNT. The Oct-4 protein was not detected or expressed at very low level in SCNT, the intensity of Oct-4 protein, however, was strong in IVF. On the other hand, the T-DMR of Oct-4 gene was hypermethylated in SCNT compared to that of IVF. These results suggested that expression and the failure of demethylation of Oct-4 gene was closely associated with incomplete development of SCNT embryos.

• 한국잠사곤충학회지
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• 제37권1호
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• pp.46-51
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• 1995

국내 분리주의 BmNPV를 이용하여 유용 단백질을 생산할 수 있는 새로운 전이벡터의 제작과 외내 유전자의 발현에 대한 연구결과는 다음과 같다. 1. PCR 기법에 의하여 다각체 단백질 유전자의 ＋1 - －194에 해당하는 promoter 부위를 증폭하고 클로닝 하였다. 2. 다각체 단백질 유전子의 5' 및 3'의 leader 부위를 promoter 부위와 함께 단계적으로 클로닝하여 전이벡터를 완성하였다. 3. 완성된 전이벡터는 pBmKSK1으로 명명하였으며, 염기서열 결정을 통하여 다각체 단백질 유전자의 ＋2 부터 ＋597까지 제거되고 외내 유전자의 클로닝 sites로 EcoRI, KpnI과 SacI을 가짐을 확인하였다. 4. 외래 유전자로서 E. coli의 $\beta$-galactosidase 유전자를 pBmKSKl에 클로닝하고 누에 세포주에 전이시킨 후, X-gal 염색 및 SDS-PAGE에 의하여 pB- mKSKl이 제 기능을 수행함을 확인하였다.

• International Journal of Industrial Entomology and Biomaterials
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• 제2권1호
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• pp.19-26
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• 2001

Transformed Spodoptera frugiperda (Sf9) cells expressing baculovirus very late factor (VLF-1) were constructed by using Autograha nuclear polyhedrosis virus (AcNPV) immediate earthy gene (ie1). Neomycin-resistance gene as a selectable marker was introduced under the control of AcNPV ie1 promoter, and Bombyx mori nuclear polyhedrosis (BmNPV-K1) vlf-1 gene was introduced under the control of the Drosophila heat shock protein gene (hspr70) promoter to yield dual expression plasmid with two independent transcription units. It was transfected into Sf9 cells and cell clones expressing vlf-1 were selected by G4l8 treatment. Genomic DNA from transformed cells was isolated and integration of AcNPV iel harboring vlf-1 was confirmed by PCR using AcNPV iel-specific primers and Southern blot analysis. The transformed cells expressing VLF-1 in an infection-independent manner expressed foreign gene product of recombinant baculovirus in the earlier stage of infection compared with control Sf9 cells. These results suggest the possible to develop highly efficient transformed insect cells for baculovirus expression vector system.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 2003년도 Annual Meeting of KSAP : International Symposium on Pharmaceutical and Biomedical Sciences on Obesity
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• pp.89-89
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• 2003

Exposure of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes a variety of biological and toxicology effects, most of which are mediated by aryl hydrocarbon receptor (AhR). The ligand-bound AhR as a heterodimer with AhR nuclear translocator (ARNT) binds to its specific DNA recognition site, the dioxin-responsive element (DRE), and it results in increased transcription of CYP1A1 gene. Retinoic acid (RA) regulates the transcription of various genes for several essential functions through binding to two classes of nuclear receptors, the retinoic acid receptor (RAR) and retinoid X receptor (RXR). Constitutive androstane receptor (CAR) also regulates the transcription of gene. In this study, we have examined how RAR, RXR and CAR regulated CYP1A1 in rainbow trout hepatoma cell (RTH 149) using luciferase reporter gene assay system. We did transient transfection with CYP1A1 luciferase reporter gene and treated with TCDD, all-trans RA, 9-cis RA and phenobarbital. Treatment of all-trans RA, 9-cis RA or phenobarbital decreased the TCDD induced transcription of CYP1Al. When we did transient cotransfection with CYP1A1 luciferase reporter gene and RXR, as increase of RXR concentration, the TCDD induced transcription of CYP1A1 was decreased. Transfection with CAR also decreased the TCDD induced transcription of CYP1A1 in RTH 149 cells.

• 한국환경독성학회:학술대회논문집
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• 한국환경독성학회 2003년도 추계국제학술대회
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• pp.179-179
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• 2003

Exposure of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes a variety of biological and toxicology effects, most of which are mediated by aryl hydrocarbon receptor (AhR). The ligand-bound AhR as a heterodimer with AhR nuclear translocator (ARNT) binds to its specific DNA recognition site, the dioxin-responsive element (DRE), and it results in increased transcription of CYP1A1 gene. Retinoic acid (RA) regulates the transcription of various genes for several essential functions through binding to two classes of nuclear receptors, the retinoic acid receptor (RAR) and retinoid X receptor (RXR). Constitutive androstane receptor (CAR) also regulates the transcription of gene. In this study, we have examined how RAR, RXR and CAR regulated CYP1A1 in rainbow trout hepatoma cell (RTH 149) using luciferase reporter gene assay system. We did transient transfection with CYP1A1 luciferase reporter gene and treated with TCDD, all-trans RA, 9-cis RA and phenobarbital. Treatment of all-trans RA, 9-cis RA or phenobarbital decreased the TCDD induced transcription of CYP1A1. When we did transient cotransfection with CYP1A1 luciferase reporter gene and RXR, as increase of RXR concentration, the TCDD induced transcription of CYP1A1 was decreased. Transfection with CAR also decreased the TCDD induced transcription of CYP1A1 in RTH 149 cells.

• Journal of Microbiology and Biotechnology
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• 제11권3호
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• pp.515-523
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• 2001

The dinoflagellates have some primitive nuclear features and are evolutionarily intermediate between prokaryotes and eukaryotes. The small subunit ribosomal RAN gene, the 5.8S ribosomal RNA gene, and the internal transcribed spacer (ITS) of Gonyaulax polyedra were cloned, and their sequences were analyzed to better understand their evolutionary position. The small subunit ribosomal RNA gene was 1,794 nt long, the large subunit ribosomal RNA gene was approximately 3,500 nt long, and the 5.8S ribosomal RNA gene was 159 nt long. The first internal transcribed spacer (ITS1) was 191 nt long, and the second internal transcribed spacer (ITS2) was 185 nt long. The intergenic spacer of the ribosomal RNA gene (IGS) was about 2,200 nt long, indicating that 5,800 nt of transcribed sequences were separated by roughly 2,200 nt of intergenic spacer. The ribosomal RNA genes were repeated many times and arranged in a head-to-tail, tandemly repeated manner. The repeating unit of ribosomal RNA gene of G. polyedra was proposed to be 8,000 nt long. Based on the lengths of ribosomal RNA, sequence alignments with representative organisms, and phylogenetic analysis on ribosomal RNA, G. polyedra appears to be one of the alveolates branched from the eukaryotic crown and, among dinoflagellates, it seems to not have emerged early.

• Journal of Ginseng Research
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• 제21권3호
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• pp.147-152
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• 1997

Ginsenosides $Rh_1$ and $Rh_2$ Induced the differentiation of F9 teratocarcinoma stem cells. These agents are structurally similar to the steroid hormones, therefore, we speculated that the steroid receptor (s) or novel nuclear receptor (s) could be involved in the differentiation process induces by them. Based on this speculation, we tried to alone new nuclear receptors with reverse transcription-polymerase chain reaction (RT-PCR) method by isolating RNA from F9 teratocarcinoma cells induced by ginsenosides. By using RT-PCR with degenerated primers from highly conserved DNA binding domain of nuclear receptors, we identified several nuclear receptors. In northern blot analysis we found that these clones are transcriptionally regulated by ginsenoside Rhl or Rh2 treatment. Further characterizations of these clones are needed to identify the mechanism of gene expression, which has an important role in the differentiation of F9 cells induced by ginsenosides.

• 대한핵의학회:학술대회논문집
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• 대한핵의학회 1997년도 제36차 춘계학술대회 초록집
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• pp.172-172
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• 1997

• BMB Reports
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• 제51권7호
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• pp.327-337
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• 2018

Lamin A and its alternative splicing product Lamin C are the key intermediate filaments (IFs) of the inner nuclear membrane intermediate filament. Lamin A/C forms the inner nuclear mesh with Lamin B and works as a frame with a nuclear shape. In addition to supporting the function of nucleus, nuclear lamins perform important roles such as holding the nuclear pore complex and chromatin. However, mutations on the Lamin A or Lamin B related proteins induce various types of human genetic disorders and diseases including premature aging syndromes, muscular dystrophy, lipodystrophy and neuropathy. In this review, we briefly overview the relevance of genetic mutations of Lamin A, human disorders and laminopathies. We also discuss a mouse model for genetic diseases. Finally, we describe the current treatment for laminopathies.