• 한국습지학회지
• /
• 제16권2호
• /
• pp.269-274
• /
• 2014

본 연구의 목적은 항생제 내성 균과 유전자 및, 항생제 내성 전달을 제어하는데 필요한 살균능 (CT, 농도 * 접촉 시간)을 서로 비교하는데 있다. 이를 위하여, 이를 위해 각기 다른 염소 주입농도(C)와 접촉시간(T)에 따라 각각의 항생제 내성 제거를 산정하였다. 그 결과 항생제 내성균 90%(1 log)이상 제어를 위해서는 CT 값(176~353 mg min/L)이 필요하였으며, 항생제 내성 유전자의 제거를 위한 CT 값은 195~372 mg min/L 이었다. 또한 항생제 내성 유전자의 전이 90% 이상 제거를 위한 CT 값은 187~489 mg min/L이었다. 따라서, 본 연구조건에서는 항생제 내성 유전자 및 유전자의 전이에 대한 제어를 위해서는 항생제 내성균 제어보다 더 높은 소독능이 필요함을 알 수 있었다.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
• /
• 제32권4호
• /
• pp.308-316
• /
• 2006

Objectives Despite considerable advances in technique, experience and skill, the precise place of surgery in the treatment of facial nerve injury remains uncertain. We designed a facial nerve crush injury model in rats and evaluated the recovery of crushed nerve which is the most common injury type of facial nerve using adenovirus vector mediated in vivo gene transfer of Brain derived neurotrophic factor(BDNF). Materials and methods In 48 Sprague Dawley rats, we made a facial nerve crush injury model to main trunk before the furcation, and injected a $10^{11}$pfu adenoviral BDNF in experimental group(BDNF adenoviral injection group; ad-BDNF) and $3{\mu}l$ saline in control group(Saline injection group; saline). After a period of regeneration from 10 to 40 days, nerve regeneration was evaluated with functioinal test (vibrissae and ocular movement), electrophysiologic study(threshold, peak voltage, conduction velocity) and histomorphometric study of axon density. Results Vibrissae and ocular movement, threshold and conduction velocity improved as time elapse in both group, however axon density was increased significantly only in experimental group. Functional test in 10 days and 20 days showed no difference between experimental group and control group. Vibrissae movement, threshold, conduction velocity and axon density in 30 days revealed that the regeneration in quality of experimental group was significantly superior to that of control group. Conclusion In general, there is tendency for nerve regeneration in experimental group (BDNF-adenovirus injection group) during 40 days, functional recovery was detected successfully after facial nerve crush in 30 days postoperatively.

• 한국수정란이식학회지
• /
• 제22권4호
• /
• pp.257-263
• /
• 2007

This study was performed to investigate the relationship between PSS-HSP70 gene polymorphism and artificial insemination (AI) reproductivity in the pigs. The RFLP polymorphism of PSS and the SSCP polymorphisms of HSP70 K1, K3 and K4 PCR product were detected different patterns. In the experiment for AI of fresh semen, spring and fall season showed higher litter size born of 10.89 head than 10.47 head of summer season. Landrace was showed higher litter size of 9.96 head than that of Duroc and Yorkshire (p<0.05). Stress relating PSS and HSP70 polymorphism of PSS-Normal, HSP70 K1-BB, K3-AB, K4-AA showd a highest litter size born of 10.97 head and litter size born alive of 10.69 head than that of the other polymorphisms(p<0.05). In the experiment for AI of frozen semen, effects of season and pig breeds were not showed for litter size born. The stress relating polymorphism of PSS-Carrier, HSP70 K1-BB, K3-BB, K4-AB showed highest litter size born of 11.29 head and litter size born alive of 10.82 head and PSS-Normal, HSP70 K1-BB, K3-AB, K4-AA showed the lowest litter size born of 8.48 head and litter size born alive of 7.33 head than that of the other polymorphisms(p<0.05). These results suggest that AI litter size born for the stress of forzen thawed semen may be affected by PSS and HSP70 polymorphism in pigs.

• 한국수정란이식학회지
• /
• 제31권1호
• /
• pp.61-64
• /
• 2016

We developed a polymerase chain reaction (PCR)-based molecular method for sexing and identification using sexual dimorphism between the Zinc Finger-X and -Y (ZFX-ZFY) gene and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for mitochondrial DNA (mtDNA) cytochrome B (CYTB) gene in meat pieces and commercial sausages from animals of different origins. Sexual dimorphism based on the presence or absence of SINE-like sequence between ZFX and ZFY genes showed distinguishable band patterns between male and female DNA samples and were easily detected by PCR analyses. Male DNA had two PCR products appearing as distinct two bands (ZFX and ZFY), and female DNA had a single band (ZFX). Molecular identification was carried out using PCR-RFLP of CYTB gene, and showed clear species classification results. The results yielded identical information on the sexes and the species of the meat samples collected from providers without any records. The analyses for DNA isolated from commercial sausage showed that pig was the major source but several sausages originated from chicken and Atlantic cod. Applying this PCR-based molecular method was useful and yielded clear sex information and identified the species of various tissue samples originating from livestock.

• Molecules and Cells
• /
• 제26권4호
• /
• pp.409-414
• /
• 2008

The cholesteryl ester transfer protein (CETP), a key player in cholesterol metabolism, has been shown to promote the transfer of triglycerides from very low density lipoprotein (VLDL) and low density lipoprotein (LDL) to high density lipoprotein (HDL) in exchange for cholesterol ester. Here we demonstrate that farnesoid X receptor ${\alpha}$ ($FXR{\alpha}$; NR1H4) down-regulates CETP expression in HepG2 cells. A $FXR{\alpha}$ ligand, chenodeoxycholic acid (CDCA), suppressed basal mRNA levels of the CETP gene in HepG2 cells in a dose-dependent manner. Using gel shift and chromatin immunoprecipitation (ChIP) assays, we found that $FXR{\alpha}$ could bind to the liver X receptor ${\alpha}$ ( $LXR{\alpha}$; NR1H3) binding site (LXRE; DR4RE) located within the CETP 5' promoter region. $FXR{\alpha}$ suppressed $LXR{\alpha}$-induced DR4RE-luciferase activity and this effect was mediated by a binding competition between $FXR{\alpha}$ and $LXR{\alpha}$ for DR4RE. Furthermore, the addition of CDCA together with a $LXR{\alpha}$ ligand, GW3965, to HepG2 cells was shown to substantially decrease mRNA levels of hepatic CETP gene, which is typically induced by GW3965. Together, our data demonstrate that $FXR{\alpha}$ down-regulates CETP gene expression via binding to the DR4RE sequence within the CETP 5' promoter and this $FXR{\alpha}$ binding is essential for $FXR{\alpha}$ inhibition of $LXR{\alpha}$-induced CETP expression.

• Asian-Australasian Journal of Animal Sciences
• /
• 제27권11호
• /
• pp.1644-1651
• /
• 2014

Transgenic animals have become important tools for the production of therapeutic proteins in the domestic animal. Production efficiencies of transgenic animals by conventional methods as microinjection and retrovirus vector methods are low, and the foreign gene expression levels are also low because of their random integration in the host genome. In this study, we investigated the homologous recombination on the porcine ${\beta}$-casein gene locus using a knock-in vector for the ${\beta}$-casein gene locus. We developed the knock-in vector on the porcine ${\beta}$-casein gene locus and isolated knock-in fibroblast for nuclear transfer. The knock-in vector consisted of the neomycin resistance gene (neo) as a positive selectable marker gene, diphtheria toxin-A gene as negative selection marker, and 5' arm and 3' arm from the porcine ${\beta}$-casein gene. The secretion of enhanced green fluorescent protein (EGFP) was more easily detected in the cell culture media than it was by western blot analysis of cell extract of the HC11 mouse mammary epithelial cells transfected with EGFP knock-in vector. These results indicated that a knock-in system using ${\beta}$-casein gene induced high expression of transgene by the gene regulatory sequence of endogenous ${\beta}$-casein gene. These fibroblasts may be used to produce transgenic pigs for the production of therapeutic proteins via the mammary glands.

• 한국발생생물학회지:발생과생식
• /
• 제22권1호
• /
• pp.73-83
• /
• 2018

This study investigates the endoplasmic reticulum (ER) stress and subsequent apoptosis in duced during somatic cell nuclear transfer (SCNT) process of porcine SCNT embryos. Porcine SCNT and in vitro fertilization (IVF) embryos were sampled at 3 h and 20 h after SCNT or IVF and at the blastocyst stage for mRNA extraction. The x-box binding protein 1 (Xbp1) mRNA and the expressions of ER stress-associated genes were confirmed by RT-PCR or RT-qPCR. Apoptotic gene expression was analyzed by RT-PCR. Before commencing SCNT, somatic cells treated with tunicamycin (TM), an ER stress inducer, confirmed the splicing of Xbp1 mRNA and increased expressions of ER stress-associated genes. In all the embryonic stages, the SCNT embryos, when compared with the IVF embryos, showed slightly increased expression of spliced Xbp1 (Xbp1s) mRNA and significantly increased expression of ER stress-associated genes (p<0.05). In all stages, apoptotic gene expression was slightly higher in the SCNT embryos, but not significantly different from that of the IVF embryos except for the Bax/Bcl2L1 ratio in the 1-cell stage (p<0.05). The result of this study indicates that excessive ER stress can be induced by the SCNT process, which induce apoptosis of SCNT embryos.

• Reproductive and Developmental Biology
• /
• 제30권3호
• /
• pp.189-194
• /
• 2006

Sperm-mediated gene transfer(SMGT) can be used to transfer exogenous DNA into the oocyte at fertilization. The main objective of this study was to assess efficiency of transferring mitochondrial DNA(mtDNA) fragment into boar spermatozoa in either presence or absence of liposome and quality of transfected spermatozoa. The mtDNA of chicken liver was isolated and purified by phenol and alkaline lysis extraction, and it was inserted to plasmid. The genome of transfected spermatozoa treated with DNase I was purified by alkaline lysis, and then amplified by the PCR analysis. After electrophoresis, DNA quantitation of each well was calculated by comparison of the band intensity with standard. As a result, exogenous DNA was composed of mtDNA fragment(1.2 kb) and plasmid(2.7 kb). On the other hand, efficiency of transfection by liposome($9.0{\pm}0.34ng/{\mu}l$) in SMGT was higher than that by DNA solution($6.9{\pm}0.53ng/{\mu}l$). However, there was no significant difference. Transfering exogenous DNA into spermatozoa was completed within 90 min of incubation. In another experiment, there were significant (p<0.05) differences between transfected spermatozoa using both DNA solution and DNA/liposome completes with unheated spermatozoa for viability ($70.8{\pm}1.80$ and $68.0{\pm}2.16%$ vs. $83.3{\pm}1.69%$, respectively) and motility($78.7{\pm}1.59$ and $79.3{\pm}2.14%$ vs. $86.7{\pm}1.59%$, respectively). This study indicates that exogenous mtDNA can be efficiently transferred into boar spermatozoa regardless of the presence of liposome, and transfected spermatozoa can also use insemination and in vitro fertilization to generate transgenic pig.

• Journal of Animal Science and Technology
• /
• 제66권4호
• /
• pp.726-739
• /
• 2024

This study was conducted to investigate whether lysophosphatidic acid (LPA) could improve the development of porcine somatic cell nuclear transfer (SCNT) embryos. Porcine SCNT-derived embryos were cultured in chemically defined polyvinyl alcohol (PVA)-based porcine zygote medium (PZM)-4 without or with LPA, and the development, cell proliferation potential, apoptosis, and expression levels of pluripotent markers were evaluated. LPA significantly increased the rates of cleavage and blastocyst formation compared to those seen in the LPA un-treatment (control) group. The expression levels of embryonic development-related genes (IGF2R, PCNA and CDH1) were higher (p < 0.05) in the LPA treatment group than in the control group. LPA significantly increased the numbers of total, inner cell mass and EdU (5-ethynyl-2'-deoxyuridine)-positive cells in porcine SCNT blastocysts compared to those seen in the control group. TUNEL assay showed that LPA significantly reduced the apoptosis rate in porcine SCNT-derived embryos; this was confirmed by decreases (p < 0.05) in the expression levels of pro-apoptotic genes, BAX and CASP3, and an increase (p < 0.05) in the expression level of the anti-apoptotic gene, BCL2L1. In addition, LPA significantly increased Oct4 expression at the gene and protein levels. Together, our data suggest that LPA improves the quality and development of porcine SCNT-derived embryos by reducing apoptosis and enhancing cell proliferation and pluripotency.

• KSBB Journal
• /
• 제14권5호
• /
• pp.593-599
• /
• 1999

본 연구는 제대혈과 골수로부터 얻은 조혈모세포를 재조합 retrovirus로 감염시킬 때의 최적조건을 human growth hormone (hGH)과 $\beta$-galactosidase를 발현하는 두 가지의 다른 retroviral vector를 이용하여 찾았다. Retrovirus는 자라는 세포에만 감염하는 것으로 알려져 있어 이에 대한 최적조건을 구하기 위해 세포 배양을 통해 조혈모세포의 성장곡선을 얻었으며, 또한 감염된 세포를 환자에게 다시 넣는 유전자요법에서는 이 세포가 체내에서 가능하면 조혈모세포의 기능을 가지는 것이 요구되어 이 때 얻어진 세포의 분열능을 나타내는 집락형성 세포분율을 구하였다. 우선, 세포성장에 대해 조사한 결과 초기에 넣은 세포농도가 5$\times$$10^4$세포/mL일 때 세포성장속도가 가장 빠른 것으로 나타났다. 그러나, 배양시간이 지남에 따라 집락을 형성할 수 있는 능력은 급격하게 감소하여 유전자요법을 위한 최적조건을 구하기 위해서는 이를 고려한 최적화가 필요하였다. 이를 위한 예비실험으로 감염이 잘 된다고 알려진 NIH3T3 세포에 retrovirus 상층액으로 감염시킨 결과 성공적으로 유전자가 전달된 것을 배지에 분비되는 hGH을 측정하여 확인하였다. 이러한 결과로부터 hGH을 발현하는 재조합 retrovirus는 정상적으로 작동하는 것을 확인하였다. 그러나, 조혈모세포와 retrovirus를 분비하는 packaging cell을 동시 배양하는 방법을 채택하였다. 제대혈로부터 얻은 조혈모세포와 대장균 lacZ 유전자로부터 $\beta$-galactosidase를 분비하는 packaging cell을 이용한 경우 동시배양의 경우 조혈모세포를 3일 동안 세포배양을 한 후 이 증식된 세포를 48시간 동안 동시배양하면서 감염시켰을 때 최대의 감염율을 나타내었다. 한편, 골수로부터 얻은 조혈모세포와 hGH을 분비하는 packaging cell과 동시배양시켰을 때 세포농도가 다름에도 불구하고 제대혈에서와 마찬가지로 조혈모세포를 3일 동안 세포배양한 후 48시간 동안 동시배양하는 경우에 hGH이 최대로 분비되었다. 이러한 결과로부터 세포의 source나 세포농도와 관계없이 유전자전달을 통한 단백질의 발현에 있어서 최적조건이 존재하였다. 그러나, 이러한 경우에 유전자전달이 완료되는 시점이 배양을 시작한지 5일이 되므로 집락을 형성할 수 있는 세포의 분율이 약 1/3로 감소하였다. 따라서, 이러한 결과를 유전자요법에 적용하는 경우에는 그 목적에 따라 적절한 실험조건을 선정하는 것이 필요하리라 사료된다.