• Journal of Aquaculture
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• v.10 no.4
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• pp.409-415
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• 1997

The carp $\beta$-actin regulatory sequences and RSV/LTR promoter were tested whether they are functinal to express linked structure gene (chloramphenicol acetyltransferas, CAT) in olive flounder (Paralichthys olivaceus) by determining the patterns of gene expression following intramuscular in vivo direct injection. The injection experiments with various concentrations of both pRSVCAT and pFV4CAT clearly revealed the effectiveness of DNA dosage on expression of CAT. The increase of CAT activity was linear in both plasmids, and maximal CAT activity was obtained with 100 ug of pFV4CAT injection. The amounts of CAT expression with pFV4CAT-injected fist were higher than those with pRSVCAT-injected fish. CAT activity was readily detectable as early as one day after injection, slightly increased at day 2, and declined over time. Most amount of DNA intramuscularly injected into olive flounder muscles persisted extrachromosomally without showing any integrated or replicated form in vivo.

• Korean Journal of Ichthyology
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• v.28 no.4
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• pp.215-222
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• 2016

Triploidy was induced in the marine medaka, Oryzias dancena by cold shock treatment ($0^{\circ}C$) of fertilized eggs for 30, 45, or 60 min, applied two minutes after fertilization. The triploid genotype was induced by each of the thermal shock regimes tested. The best result was obtained when the eggs were treated for 45 min, which induced triploidy in all the resulting fish. Triploidy was confirmed using chromosomal and flow cytometer analyses, and erythrocyte measurements. The surface areas and volumes of the erythrocytes of triploid fish were significantly larger than those of diploid fish, and their chromosome number (3N=72) was 1.5 times greater that for the diploids (2N=48). Based on a flow cytometer analysis, the triploid fish had approximately 1.5 times the cellular DNA content (2.40 pg/cell) of the diploid specimens (1.61 pg/cell). Data from this study provide the basis for the development of unique models for studying reproductive confinement in transgenic fish.

• BMB Reports
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• v.36 no.1
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• pp.35-42
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• 2003

Heterocyclic amines (HCAs) are potent mutagens generated during the cooking of meat and fish, and several of these compounds produce tumors in conventional experimental animals. During the past 5 years or so, HCAs have been tested in a number of novel in vivo murine models, including the following: lacZ, lacI, cII, c-myc/lacZ, rpsL, and $gpt{\Delta}$ transgenics, $XPA^{-/-}$, $XPC^{-/-}$, $Msh2^{+/-}$, $Msh2^{-/-}$ and $p53^{+/-}$ knock-outs, Apc mutant mice ($Apc^{{\Delta}716}$, $Apc^{1638N}$, $Apc^{min}$), and $A33^{{\Delta}N{\beta}-cat}$ knock-in mice. Several of these models have provided insights into the mutation spectra induced in vivo by HCAs in target and non-target organs for tumorigenesis, as well as demonstrating enhanced susceptibility to HCA-induced tumors and preneoplastic lesions. This review describes several of the more recent reports in which novel animal models were used to examine HCA-induced mutagenesis and carcinogenesis in vivo, including a number of studies which assessed the inhibitory activities of chemopreventive agents such as 1,2-dithiole-3-thione, conjugated linoleic acids, tea, curcumin, chlorophyllin-chitosan, and sulindac.

• Journal of Animal Reproduction and Biotechnology
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• v.37 no.3
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• pp.176-182
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• 2022

Norflurazon is widely used on agricultural lands and has a high potential to pollute water sources. However, its effects on fish have not been fully elucidated. The purpose of our study was to determine whether norflurazon adversely affects the developmental stage of zebrafish, which are frequently used as a model system to evaluate the environmental impact of pollutants. Norflurazon interfered with the hatching of zebrafish embryos and induced several sublethal deformities including body length reduction, increased yolk sac volume, and enlargement of the pericardial region. We further examined the cardiotoxicity of norflurazon in the flk1:eGFP transgenic zebrafish line. The vascular network, mainly in the brain region, was significantly disrupted in norflurazon-exposed zebrafish. In addition, due to the failure of cardiac looping, norflurazon-exposed zebrafish had an abnormal cardiac structure. These developmental abnormalities were related to the apoptotic process triggered by norflurazon. Overall, the present study demonstrated the non-target toxicity of norflurazon by analyzing the hazardous effects of norflurazon on developing zebrafish.

• Journal of Life Science
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• v.27 no.6
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• pp.617-622
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• 2017

In this study, we sought to develop an effective cloning system by which human cytochrome $b_5$ (cyt $b_5$) is introduced and expressed in zebrafish. First, the 414 bp human cyt $b_5$ gene was amplified from RNA extracts of HeLa cells using RT-PCR, and the amplicon was subsequently sequenced to confirm that it was intact. Next, cyt $b_5$ was cloned into the pEGFP-N3 vector, which also encodes a fluorescent gene. One-cell stage zebrafish embryos were microinjected with the recombinant vector containing the cyt $b_5$ gene. Fluorescence microscopy confirmed high expression of the fluorescent gene in the injected fry compared to the non-fluorescent control fry. Finally, we extracted RNA from the injected fry and performed RT-PCR to determine whether the human cyt $b_5$ gene is expressed in the transgenic zebrafish. Sequencing analysis further confirmed that the cloned human cyt $b_5$ gene was intact. The transgenic zebrafish model produced in this study will be a useful tool to study therapeutic approaches to cure various diseases related to the deficiency of functional human cyt $b_5$ as well as tools for cloning useful genes in fish.

• Korean Journal of Animal Reproduction
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• v.26 no.4
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• pp.385-394
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• 2002

This study was constructed the correlations of the embryonic developmental rates and the frequency of chromosome aberration using ear-skin-fibroblast cell in nuclear transfer (NT) derived embryos. Karyoplast-oocyte complexes were fused and activated simultaneously, then cultured for seven days to assess development. The developmental rates of NT and in vitro fertilization (IVF) embryos were 55.4% vs 63.5%, 31.7% vs 33% and 13.4% vs 16.8% in 2 cell, 8 cell and blastocyst, respectively. Firstly, the frequency of chromosome aberrations were evaluated using fluorescent in situ hybridization (FISH) technique with porcine chromosome 1 submetacentric specific probe. Chromosome aberration was detected at day 3 on the embryo culture, the percentages of chromosomal aneuploidy in NT and IVF embryos at 4-cell stage were 40%, 31.3%, respectively. Secondly, embryonic fragmentation was evaluated at 4-cell stage embryo. Frequency of embryonic fragmentations was in 51.3% of NT, 61.3% of IVF, 28.9% of parthenogenetic activation at 4-cell stage. The proportion of fragmentation in NT embryos was higher than activation embryos. This result indicates that chromosomal abnormalities and embryonic fragments are associated with low developmental rate in porcine NT embryo. It is also suggest that abnormal porcine embryos produced by NT related with lower implantation rate, increased abortion rate and production of abnormal fetuses.

• Korean Journal of Animal Reproduction
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• v.25 no.2
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• pp.171-180
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• 2001

Compared to other gene transfer system, the advantages of retrovirus-mediated gene transfer are technical ease, efficient expression and genetic stability. Despite the high potency of the retrovirus vector system in gene transfer, one of the drawbacks is a difficulty in concentration of virus stock. To overcome this problem, we tested a new retrovirus vector system producing the progeny retrovirus particles encapsidated with VSV-G (vesicular stomatitis virus G glycoprotein). The infectivity of this virus was not sacrificed by ultracentrifugal concentration and the host cell range extended from all mammalian to fish embryos. Virus titer after 1,000 x concentration was more than 10$^{8}$ CFU/ $m\ell$ on most of the target cell lines. We applied this pantropic viruses in transgenic chicken production by injecting the concentrated (100$\times$) stock into subgerminal cavity of stage X chicken embryos. The survival rate of chicken embryos after injection was about 20% and gene integration rate in surviving embryos was scored almost 100%. Analyses of RT-PCR and fluorescence microscopy, however, showed no evidence of the transgene expression.

• Journal of Aquaculture
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• v.8 no.3
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• pp.209-220
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• 1995

The nuclear matrix was isolated from Misgumus mizolepis liver nuclei by low salt extraction and restriction enzyme treatment. The structure was digested with proteinase K. After centrifugation, matrix attachment regions (MARs) were obtained by RNase treatment and phenol-chloroform extraction. The result leads to the appearance of smeared bands in the range of about 0.3-15 kb. pURY19 vector was constructed by inserting 2.13 kb Eco47 III fragment of the yeast uracil 3 gene into the unique Ssp I site of pUC19 plasmid vector as a selection marker. This vector is unable to be maintained in Sacrharomyces cerevisiae by itself since it cannot replicate as an extrachromosomal element. Using this system, we attempted cloning the ARS (autonomously replicating sequence) from M. mizelepis to develop an efficient expression vector for the transgenic fish. pURY19N_{l-62}$ were constructed by inserting MARs in pURY19 plasmid vector and transformation of E. coli $DH5\alpha$. Replication origins (ARS) of M. mizolepis were isolated, which enabled the vector to replicate autonomously in S. cerevisiae. The cloned DNA fragments were sequenced by Sanger's dideoxy-chain termination method. All clones were AT-rich. $pURY19N_6$, one of the clones, expecially contained ARS consensus sequence, Topoisomerase II consensus, near A-box and T-box.