• Clinical and Experimental Reproductive Medicine
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• v.36 no.4
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• pp.275-281
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• 2009

Objective: This study was performed to evaluate the effect of oxytocin antagonist on the outcome of IVF/ICSI cycles in infertile patients with repeated failure of IVF/ICSI treatment. Method: Forty patients who had experienced two or more failures of IVF/ICSI treatment without low ovarian reserve, were recruited for this prospective randomized study. All patients received controlled ovarian stimulation (COS) using GnRH antagonist multidose protocol (MDP). For the intervention group, intravenous administration of atosiban (mixed vasopressin $V_{1A}$/oxytocin antagonist) started with a bolus dose 6.75 mg one hour before embryo transfer (ET) and continued at an infusion rate of 18 mg/hour. After ET, administered atosiban was reduced to 6 mg/hour and continued for 2 hours. The main efficacy endpoints were clinical pregnancy rate and implantation rate. Results: Patients' characteristics were comparable in the intervention and control groups. COS parameters and IVF results were also similar. The number of uterine contractions for 3 minutes measured just before ET was significantly lower in the intervention group than control group ($3.5{\pm}1.4$ vs $8.7{\pm}2.2$, p<0.001). While there was no statistically significant difference in the clinical pregnancy rate between control group and intervention group (20.0% and 40.0%, p=0.168), the implantation rate was significantly higher in the intervention group, with 16.9% (11/65) compared with 6.0% (4/67) in the control group (p=0.047). There were no differences in ectopic pregnancy rate and miscarriage rate between the two groups. Conclusion: This study demonstrates that administration of oxytocin antagonist during ET can improve the implantation rate probably by decreasing the frequency of uterine contractions in infertile patients undergoing IVF/ICSI treatment.

• Korean Journal of Poultry Science
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• v.40 no.4
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• pp.299-304
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• 2013

Tracheal epithelial cells (TECs) are an important tool for studies of viral respiratory diseases. Primary TECs have been cultured from human, mouse and hamster. It is also necessary to diagnose viral respiratory disease and reveal infection mechanisms in chicken. In this study, we isolated tracheal epithelial layers from tracheal of 20-day-old chicks and cultured primary TECs from the isolated layers. Ciliated cells which were a typical morphology of TECs were observed in cultured primary TECs and maintained until cell passage 5 (15 to 20 days). When we analyzed expression patterns of epithelial marker genes (retinoic acid responder, FGF-binding protein, virus activating protease (VAP) in TECs compared to immortalized chicken embryonic fibroblast cell line (DF-1), all the marker genes are highly expressed in TECs than in DF-1. When TECs were cultured with 0.1 and 1 MOI of ND virus (rNDV-GFP strain) to test the susceptibility of TECs for ND virus, 12.6% and 48.2% of the incubated TECs were infected respectively. In addition, when DF-1 was incubated with 1 MOI of ND virus, the virus infection rate of DF-1 was three times lower than the virus infection rate of TECs. These data could contribute to study infection mechanisms of viral respiratory diseases and control them in chicken.

• Korean Journal of Microbiology
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• v.43 no.4
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• pp.243-249
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• 2007

The centromere is a highly differentiated structure of the chromosome that fulfills a multitude of essential mitotic and meiotic functions. Alphoid DNA (${\alpha}$-satellite) is the most abundant family of repeated DNA found at the centromere of all human chromosomes, and chromosomes of primates in general. The most important parts in the development of Human Artificial Chromosomes (HACs), are the isolation and maintenance of stability of centromeric region. For isolation of this region, we could use the targeting hook with alphoid DNA repeat and cloned by Transformation-Associated Recombination (TAR) cloning technique in yeast Saccharomyces cerevisiae. The method includes rolling-circle amplification (RCA) of repeats in vitro to 5 kb-length and elongation of the RCA products by homologous recombination in yeast. Four types of $35\;kb{\sim}50\;kb$ of centromeric DNA repeat arrays (2, 4, 5, 6 mer) are used to examine the stability of repeats in homologous recombination mutant strains (rad51, rad52, and rad54). Following the transformation into wild type, rad51 and rad54 mutant strains, there were frequent changes in inserted size. A rad52 mutant strain showed extremely low transformation frequency, but increased stability of centromeric DNA repeat arrays at least 3 times higher than other strains. Based on these results, the incidence of large mutations could be reduced using a rad52 mutant strain in maintenance of centromeric DNA repeat arrays. This genetic method may use more general application in the maintenance of tandem repeats in construction of HAC.

• The Korean Journal of Nuclear Medicine
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• v.38 no.1
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• pp.99-108
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• 2004

Purpose: The ability to noninvasively track the migration of neural progenitor cells would have significant clinical and research implications. We generated stably transfected F3 human neural progenitor cells with human sodium/iodide symporter (hNIS) for noninvasively tracking F3. In this study, the expression patterns of hNIS gene in F3-NIS were examined according to the cultured time and the epigenetic modulation. Materials and Methods: F3 human neural stem cells had been obtained from Dr. Seung U. Kim (Ajou University, Suwon, Korea). hNIS and hygromycin resistance gene were linked with IRES (Internal Ribosome Entry Site) under control of CMV promoter. This construct was transfected to F3 with Liposome. To investigate the restoration of hNIS gene expression in F3-NIS, cells were treated with demethylating agent (5-Azacytidine) and Histone deacetylase inhibitor (Trichostatin A: TSA). The expression of hNIS was measured by I-125 uptake assay and RT-PCR analysis. Results: The iodide uptake of the F3-NIS was higher 12.86 times than F3 cell line. According to the cell passage number, hNIS expression in F3-NIS gradually diminished. After treatment of 5-Azacytidine and TSA with serial doses (up to $20{\mu}M$, up to 62.5nM, respectively) for 24 hours, I-125 uptake and mRNA of hNIS in F3-NIS were increased. Conclusion: These results suggest that hNIS transfected F3 might undergo a change in its biological characters by cell passage. Therefore, the gene ex[ressopm of exogenous gene transferred human stem cell might be affected to the epigenetic modulation such as promoter methylation and Histone deacetylation and to the cell culture conditions.

• Nuclear Medicine and Molecular Imaging
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• v.42 no.5
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• pp.394-400
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• 2008

Purpose: Quantitative comparison of transgene expression within stem cells between lentivirus and adenovirus-mediated delivery systems has not been reported. Here, we evaluated the human sodium iodide symporter (hNIS) gene expression in rat mesenchymal stem cell (rMSC) transduced by lentivirus or adenovirus, and compared the hNIS expression quantitatively between the two delivery systems. Materials and Methods: Lentiviral-mediated hNIS expressing rMSC (lenti-hNIS-rMSC) was constructed by cloning hNIS gene into pLenti6/UbC/V5-DEST (Invitrogen) to obtain pLenti-hNIS, transducing rMSC with the pLenti-hNIS, and selecting with blasticidin for 3 weeks. Recombinant adenovirus expressing hNIS gene (Rad-hNIS) was produced by homologous recombination and transduction efficiency of Rad-hNIS into rMSC evaluated by Rad-GFP was $19.1{\pm}4.7%$, $54.0{\pm}6.4%$, $85.7{\pm}8.7%$, and $98.4{\pm}1.3%$ at MOI 1, 5, 20, and 100, respectively. The hNIS expressions in lenti-hNIS-rMSC or adeno-hNIS-rMSC were assessed by immunocytochemistry, western blot, and 1-125 uptake. Results: Immunocytochemistry and western blot analyses revealed that hNIS expressions in lenti-hNIS-rMSC were greater than those in adeno-hNIS-rMSC at MOI 20 but lower than at MOI 50. However in vitro 1-125 uptake test demonstrated that iodide uptake in lenti-hNIS-rMSC ($29,704{\pm}6,659\; picomole/10^6\;cells$) was greater than that in adeno-hNIS-rMSC at MOI 100 ($6,168{\pm}2,134\;picomole/10^6\;cells$). Conclusion: Despite lower amount of expressed protein, hNIS function in rMSC was greater by lentivirus than by adenovirus mediated expression. Stem cell tracking using hNIS as a reporter gene should be conducted in consideration of relative vector efficiency for transgene expression.

• Reproductive and Developmental Biology
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• v.31 no.3
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• pp.161-167
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• 2007

In this study, we constructed and tested retrovirus vectors designed to express the human thrombopoietin gene under the control of the tetracycline-inducible promoters. To increase the hTPO gene expression at him-on state, WPRE sequence was also introduced into retrovirus vector at downstream region of either the hTPO gene or the sequence encoding reverse tetracycline-controlled transactivator (rtTA). Primary culture cells (PFF, porcine fetal fibroblast; CEF, chicken embryonic fibroblast) infected with the recombinant retrovirus were cultured in the medium supplemented with or without doxycycline for 48hr, and induction efficiency was measured by comparing the hTPO gene expression level using RT-PCR, western blot and ELISA. Higher hPTO expression and tighter expression control were observed from the vector in which the WPRE sequence was placed at downstream of the hTPO (in CEF) or rtTA(in PFF) gene. This resulting tetracycline inducible vector system may be helpful in solving serious physiological disturbance problems which have been a major obstacle in successful production of transgenic animals.

• Korean Journal of Veterinary Research
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• v.33 no.4
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• pp.657-664
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• 1993

To reveal the immunogenicity of ${\gamma}-irradiated$ E tenella and its progeny, a series of experiments on the effects of Cobalt-to ${\gamma}-irradiation$ was performed. The SPF chickens inoculated with diffenrt doses of inoculum were challenged with $1{\times}10^5$ oocysts of virulent E tenella. The levels of 100 Gy ${\gamma}-irradiation$ from $^{60}Co$ and of inoculum with $1{\times}10^4$ oocysts were recognized as proper as immunogen by comparison of survival rates, body weight gains, blood in feces and lesion scores in the chickens. In these trials of challenge with virulent E tenella after inoculation with $1{\times}10^4$ oocysts of the ${\gamma}-irradiated$ E tenella and its progeny, the survival rates of the chickens challenged with the virulent E tenella after immunization with the 1st and the 3rd progeny groups of ${\gamma}-irradiated$ E tenella oocysts were higher(l00%) than that(87.0%) of the challenged control group. The signs of blood in feces and the lesion scores were seen markedly lower with the ourput of the smaller number of oocysts, i.e. OPG 103,900 and 25,800 in the groups of the 1st and the 3rd progeny, respectively, than those(OPG 1,658,900) of the challenged control group. The body weight gains of the 1st and the 3rd progeny groups, the 1st week and the 2nd week after challenge, were higher (2.6g and 155.4g, 11.6g and 168.9g respectively) than those(-85.8g and 63.6g, respectively) of the challenged control group, and the feed conversion ratios(FCR 3.28 and 2.96) of the 1st and the 3rd progeny groups were lower than that(FCR 5.60) of the groups challenged control group. The anticoccidial indices(70.5 and 93.9) of the groups challenged with the virulent oocysts of E tenella after immunization with the 1st and the 3rd progeny of the ${\gamma}-irradiated$ E tenella were significantly higher than that (ACI -81.9) of the challenged control group. It was thought that the immunogenicity of ${\gamma}-irradiated$ E tenella would be increase according to increase the number of generation passaged in chicken. That might be because of increasing the pathogenicity of ${\gamma}-irradiated$ E tenella according to increase the number of generation passaged in chicken.

• Korean Journal of Poultry Science
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• v.36 no.2
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• pp.177-186
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• 2009

It is well-known that unregulated over-expression of foreign gene may have unwanted physiological or toxic effects in transgenic animals. To circumvent these problems, we constructed retrovirus vector designed to express the foreign gene under the control of the tetracycline-inducible promoter. However, gene expressions in the tetracycline-inducible expression system (Tet system) are not completely regulated but a little leaky due to the inherent defects in conventional Tet-based systems. A more tightly controllable regulatory system can be achieved when the advanced versions ($rtTA2^SM2$) of rtTA and a minimal promoter in responsive components (pTRE-tight) are used in combination therein. In this study, we tried to produce human thrombopoietin (hTPO) from various target cells and transgenic chickens using the retrovirus vector combined with Tet system. hTPO is the primary regulator of platelet production and has an important role in the survival and expansion of hematopoietic stem cells. In a preliminary experiment in vitro, higher hTPO expression and tighter expression control were observed in chicken embryonic fibroblast (CEF) cells. We also measured the biological activity of the hTPO using Mo7e cells whose proliferation is dependant on hTPO. The biological activity of the recombinant hTPO from CEF was higher than both its commercial counterpart and hTPO from other target cells. The recombinant retrovirus was injected beneath the blastoderm of non-incubated chicken embryos (stage X). Out of 138 injected eggs, 15 chicks hatched after 21 days of incubation. Among them, 8 hatched chicks were hTPO positive. When the Go transgenic chicken was fed doxycycline (0.5 mg per 1 gram of feed), a tetracycline derivative, hTPO concentration of the transgenic chicken blood was 200 ng/mL. Germline transmission of the transgene was confirmed in sperm of the Go transgenic roosters. These results are informative to establish transgenic chickens as bioreactors for the mass production of commercially valuable and biological active human cytokine proteins.

• Microbiology and Biotechnology Letters
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• v.37 no.4
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• pp.389-398
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• 2009

Development of expression vectors is important for the basic and applied researches on kimchi LAB (lactic acid bacteria). An expression vector, pSJE6c was constructed by inserting P6C promoter sequence from Lactococcus lactis into pSJE, a shuttle vector for E. coli and Leuconostoc species. To test the efficiency of pSJE6c, aga ($\alpha$-galactosidase) and lacZ ($\beta$-galactosidase) genes were expressed in Lactobacillus brevis 2.14. Compared to the pSJE, expression levels of both genes were increased, indicating P6C promoter was better than indigenous promoters. Enzyme activities of L. brevis cells harboring pSJE6caga (pSJE6c with aga) or pSJE6Z (pSJE6c with lacZ) were 1.5-2 fold higher than those with pSJEaga (pSJE with aga) or pSJEZ (pSJE with lacZ). More RNA transcripts were detected in cells harboring pSJE6c based recombinant plasmid. The results indicated that heterologous gene expressions in kimchi LAB could be improved significantly by use of efficient expression vectors.

• Development and Reproduction
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• v.3 no.1
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• pp.75-85
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• 1999

To investigate the origin and action mechanism of cytoplasmic factors as regulators of morphogenesis, the embryonic development, RNA synthesis and protein phosphorylation were examined in reconstituted embryos. A half of 1-cell mouse embryo with both pronuclei was electrofused with the enucleated cytoplasm of 1- or 2-cell embryos which were cultured for 24 hrs from post 20 hrs hCG in CZB with or without cycloheximide (CHX, an inhibitor of protein synthesis; P+P-CHX group), genistein (Gen, an inhibitor of tyrosine protein kinase; P+2-Gen group) and 6-dimethylaminopurine (6-DMAP, an inhibitor of serine-threonine protein kinase; P＋2-DMAP group), and co-cultured with Vero cells for 5 days. And their development, cell numbers at compaction, [5, 6-$^3$H]-uridine incorporation into RNA and the pattern of protein phosphorylation after labeling of [$^{32}$ P] orthophosphate were compared with that of the reconstituted embryos such as P+2 or P+P (control group). Embryonic development and the time of RNA synthesis in P+P-CHX were similar to those in P+P. But the time and the cell stages of embryonic compaction in P+P-CHX were similar to those in P+2. The compaction was initiated at 4-cell in P+2 and P+2-Gen, but at 8-cell in P+P and P+2-DMAP. On a two-dimensional gel electrophoresis, phosphorylation of 80KD and 110KD proteins were inhibited after 3 hrs of reconstruction in the embryo of P+2-DMAP when compared with that of P+2 and P+2-Gen. These results suggest that protein synthesis between 1- and 2-cell stage affects the timing of embryonic genome activation, and that cytoplasmic factors derived from oocyte or their modification regulates the time schedule of embryonic compaction in mouse. Also, serine-threonine protein kinase has an important role on the regulation of compaction.