• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.82-82
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• 2003

Offspring have been produced from somatic cells in a number of species. This biotechnology introduced a new phenomenon in reprogramming and differentiation of somatic cell, namely totipotency. However, birth of oversized calves and perinatal abnormalities such as increased gestation length, lack of spontaneous parturition, higher incidence of dystocia, and reduced perinatal viability of offspring are frequently observed in pregnancies of cloned bovine fetuses. Disturbance of feto-placenta has been proposed as likely causes for abnomal growth. However. Little is known the mechanism responsible for the perinatal problems. Therefore, we focused on gestation length in somatic cell nuclear recipient cows. To solve this issues, placental tissues of control and cloned bovine were obtained by a cesarean section (C-section) before 5 days of paturition. Total RNA from control and cloned bovine placenta was extractd by TRIzol reagent. GeneFishing DEG kits (Seegene) were used to identify differentially expression genes. Total RNA (3 ug) were synthesized by M-MLV reverse transcriptase (200 u/ul) with 10 uM dT-annealing control primer (ACP1) at 42C for 90 min. Then, first-strand cDNA (50 ng) was amplified using the 5 uM arbitary ACP (1-20) and 10 uM dT-ACP2 primers. Some specific expression genes were amplified, Now, we are cloning and sequencing. These finding strongly can be support to solve the problems for parturition delay in nuclear transfer cows, suggest that placenta specific proteins are key indicators for the aberration of gestation and placental function in cows.

• Journal of Microbiology and Biotechnology
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• v.33 no.4
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• pp.500-510
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• 2023

In this study, lactic acid bacteria were isolated from 21 top-selling probiotic products on Korean market and their antimicrobial resistance were analyzed. A total 152 strains were claimed to be contained in these products and 70 isolates belonging to three genera (Bifidobacterium, Lactobacillus, and Lactococcus) were obtained from these products. RAPD-PCR showed diversity among isolates of the same species except for two isolates of Lacticaibacillus rhamnosus from two different products. The agar dilution method and the broth dilution method produced different MICs for several antimicrobials. With the agar dilution method, five isolates (three isolates of Bifidobacterium animalis subsp. lactis, one isolate of B. breve, one isolate of B. longum) were susceptible to all nine antimicrobials and 15 isolates were multi-drug resistant. With the broth microdilution method, only two isolates (one isolate of B. breve and one isolate of B. longum) were susceptible while 16 isolates were multi-drug resistant. In this study, only two AMR genes were detected: 1) lnu(A) in one isolate of clindamycin-susceptible and lincomycin-resistant Limosilactobacillus reuteri; and 2) tet(W) in one tetracycline-susceptible isolate of B. longum B1-1 and two tetracycline-susceptible isolates and three tetracycline resistant isolates of B. animalis subsp. lactis. Transfer of these two genes via conjugation with a filter mating technique was not observed. These results suggest a need to monitor antimicrobial resistance in newly registered probiotics as well as probiotics with a long history of use.

• Tuberculosis and Respiratory Diseases
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• v.51 no.2
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• pp.122-134
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• 2001

Background : Some chemotherapeutic drugs induce NF-${\kappa}B$ activation by degrading the $I{\kappa}B{\alpha}$ protein in cancer cells which contributes to anticancer drug resistance. We hypothesized that inhibiting $I{\kappa}B{\alpha}$ degradation would block NF-${\kappa}B$ activation and result in increased tumor cell mortality in response to chemotherapy. Methods : The "superrepressor" form of the NF-${\kappa}B$ inhibitor was transferred by an adenoviral vector (Ad-$I{\kappa}B{\alpha}$-SR) to the human lung cancer cell lines (NCI H157 and NCI H460). With a MIT assay, the level of sensitization to cisplatin and paclitaxel were measured. To confirm the mechanism, an EMSA and Annexin V assay were performed. Results : EMSA showed that $I{\kappa}B{\alpha}$-SR effectively blocked the NF-${\kappa}B$ activation induced by cisplatin. Transduction with Ad-$I{\kappa}B{\alpha}$-SR resulted in an increased sensitivity of the lung cancer cell lines to cisplatin and paclitaxel by a factor of 2~3 in terms of $IC_{50}$. Annexin-V analysis suggests that this increment in chemosensitivity to cisplatin probably occurs through the induction of apoptosis. Conclusion : The blockade of chemotherapeutics induced NF-${\kappa}B$ activation by inducing Ad-$I{\kappa}B{\alpha}$-SR, increased apoptosis and increasing the chemosensitivity of the lung cancer cell lines tested, subsequently. Gene transfer of $I{\kappa}B{\alpha}$-SR appears to be a new therapeutic strategy of chemosensitization in lung cancer.

• Journal of Embryo Transfer
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• v.30 no.3
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• pp.183-188
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• 2015

This study analyzed the coat color-related genes of MC1R, ASIP, ECA3-inversion, and STX17 of 1,462 Jeju horses administered by the Jeju Special Self-Governing Province. This was done to investigate the distributional characteristics of coat color-related genes in the Jeju horse group and the changes of its coat color-related genes by generation. The genotype frequency of the MC1R gene of $E^+/E^+$ and $E^+/E^e$ related to black coat color was 0.122 and 0.447, respectively, while $E^e/E^e$ of the chestnut genotype was 0.429. The genotype frequency of the ASIP gene of $A^A/A^A$, $A^A/A^a$, and $A^a/A^a$ was 0.46, 0.448, and 0.091, respectively, where the genotype frequency of $A^a/A^a$ turned out to be relatively low. The To/To and +/To genotype that manifests the Tobiano shape was 0.001 and 0.119, respectively, with the share of Tobiano shape around 12%. The genotype frequency of G/G and G/g of STX17 related to grey coat color was 0.002 and 0.680, respectively, with the share of grey horses among the Jeju horse group at 68.2%. As for the change of coat color genes by generation, no large changes were observed in the MC1R and ASIP genes. In ECA3-inversion, the To allele that manifests Tobiano significantly decreased following the generational change (p<0.05), while the STX17 G allele related to grey coat color significantly increased following the generational change (p<0.05). It will be necessary to examine the coat color genes when selecting breeding horses so that the diversity of coat colors among the Jeju horse group can be maintained.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.227-233
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• 2017

Pig has been known to be one of the most feasible animals as a bioreactor to produce pharmaceuticals in milk and as a mediator in xenotransplantation research. Previously, we generated transgenic pigs for both purposes, which were expressing Factor 8, vWF, hTPA, and hEPO in milk, along with expression of MCP at GalT gene locus ($GalT^{-MCP/-MCP}$) as well as expressing MCP at GalT gene loci with CD73 expression ($GalT^{-MCP/+}/CD73$). In this study, we performed comparative analyses of sperm parameters between wild type male (WT) pig and those transgenic males to examine the effects of transgenes integrated into the pigs on motility, morphology, viability, and acrosome integrity of the spermatozoa. Our results showed that the rates of actively motile spermatozoa of WT, Factor 8, vWF, hTPA, hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs were 85.0%, 83.3%, 82.5%, 83.3%, 82.5%, 77.5%, and 78.7%, respectively. Whereas, the rates of morphologically normal spermatozoa of WT, Factor 8, vWF, hTPA, hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs were 90.0%, 80.0%, 80.0%, 83.3%, 85.0%, 91.8%, and 80.8%, respectively. In addition, the viability in spermatozoa of WT, Factor 8, vWF, hTPA, hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs were 93.9%, 82.4%, 89.9%, 83.9%, 87.4%, 92.8%, and 83.6%, respectively. The rates of spermatozoa with normal acrosome integrity in WT, Factor 8, vWF, hTPA, hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs were 98.1%, 98.6%, 98.6%, 98.7%, 98.1%, 99.5%, and 95.1%, respectively. There were no significant differences in motility, morphology, viability, and acrosome integrity of the spermatozoa among WT, Factor 8, vWF, hTPA, and hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs. These mean that neither random integration nor targeted integration of the transgene into chromosome of pig effect on characteristics of spermatozoa. Ultimately, the transgenic male pigs subjected in this study could apply to propagate their progenies for production of human therapeutic proteins and advancing the xenotransplantation research.

• Biomolecules & Therapeutics
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• v.30 no.2
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• pp.203-211
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• 2022

Melanogenesis is the production of melanin from tyrosine by a series of enzyme-catalyzed reactions, in which tyrosinase and DOPA oxidase play key roles. The melanin content in the skin determines skin pigmentation. Abnormalities in skin pigmentation lead to various skin pigmentation disorders. Recent research has shown that the expression of EMP2 is much lower in melanoma than in normal melanocytes, but its role in melanogenesis has not yet been elucidated. Therefore, we investigated the role of EMP2 in the melanogenesis of MNT1 human melanoma cells. We examined TRP-1, TRP-2, and TYR expression levels during melanogenesis in MNT1 melanoma cells by gene silencing of EMP2. Western blot and RT-PCR results confirmed that the expression levels of TYR and TRP-2 were decreased when EMP2 expression was knocked down by EMP2 siRNA in MNT1 cells, and these changes were reversed when EMP2 was overexpressed. We verified the EMP2 gene was knocked out of the cell line (EMP2 CRISPR/Cas9) by using a CRISPR/Cas9 system and found that the expression levels of TRP-2 and TYR were significantly lower in the EMP2 CRISPR/Cas9 cell lines. Loss of EMP2 also reduced migration and invasion of MNT1 melanoma cells. In addition, the melanosome transfer from the melanocytes to keratinocytes in the EMP2 KO cells cocultured with keratinocytes was reduced compared to the cells in the control coculture group. In conclusion, these results suggest that EMP2 is involved in melanogenesis via the regulation of TRP-2 expression.

• Korean Journal of Microbiology
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• v.43 no.3
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• pp.151-158
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• 2007

The possibility of inadvertent introduction of therapeutic gene expressing viral vectors has raised safety concerns about germ-line infection. Particularly, for indications such as prostate cancer and ovarian cancer, the proximity of the point of viral administration to organs of the reproductive system raises concerns regarding inadvertent germ-line transmission of genes carried by the virus vector. To evaluate the safety of in vivo adenovirus mediated gene transfer, we explored the biodistribution, persistance and potential germ-line transmission of p53-expressing adenovirus (Ad-CMV-p53). Both male and female Balb/c mice were injected with $1{\times}10^9$ PFU of Ad-CMV-p53. The PCR analysis showed that there were detectable vector sequences in liver, kidney, spleen, seminal vesicle, epididymis, prostate, ovary, and uterus. The RT-PCR analysis for detecting inserted gene, p53 showed that Ad-CMV-p53 viral RNA were present in spleen, prostate and ovary. Direct injected male and female mice of adenovirus vector into testis and ovary were mated and their of offspring were evaluated for germ-line transmission of the adenoviral vector. The PCR and RT-PCR analysis showed no evidence of germline transmission, although vector sequences were detected in DNA extracted from gonadal tissues. Real-time PCR result confirmed a significant decrease of adenovirus in gonad tissues 1 week after injection. We have also analysed the cell specific localization of viral DNA in gonad tissues by using in-situ PCR. Positive signals were detected in interstitial tissue but not in seminiferous tubule in sperm. In the case of ovary, adenovirus signal were localized to the stromal tissue, but no follicular signals were observed. Together, these data provide strong evidence that the risk of the Inadvertent germ-line transmission of vector sequences following intraperitoneal or direct injection into genito-urinary system of adenovirus is extremely low.

• Journal of Animal Science and Technology
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• v.50 no.5
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• pp.641-648
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• 2008

There has been great success for making transgenic animals using somatic cell nuclear transfer(SCNT) up to this time. However, the success rates of the production of live transgenic animals are still very low. The current research has been carried out for delineation of differentially expressed genes between SCNT and normal placenta in cattle. In the present observations, high expression has been observed for CTSZ, LOC509426 and ELF1 genes in normal placenta. On the other hand, TIMP2, PAG1B, PAG-21, LOC782894, SERPINB6 and mKIAA2025 protein were highly expressed in SCNT placenta. Five genes, which were highly expressed in SCNT placenta, have been further investigated using semi-quantitative real-time PCR. The results were similar to that we observed using ACP. In the future, all genes affecting the SCNT and normal placenta have to be discovered and their networks will be fully investigated. The genes were identified in this study would be great help for identifying differential gene expressions in SCNT placenta.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.35 no.2
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• pp.115-121
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• 2002

To develop a new method of conjugation and to determine the distribution of R plasimds, we isolated multi-drug resistant strains from fish pathogenic bacteria in the farms of south and east seacoasts of Korea. Out of the 134 isolates examined, 10 showed resistance to chloramphenicol, tetracycline, streptomycin, ampicillin, colistin, nalidixic acid, oxolinic acid and kanamycin. One out of 10 multi-drug resistance bacteria, Vibfio damsela JE1 (V. damsela JE1), contained transferable R plasmid of chlorarnphenicol- tetracycline resistance genes and other nucleic acids encoding ampicillin and kanamycin resistance. The presence of the R plasmid was confirmed by conjugation using the chromocult medium (CC) as a selective and differential medium for transconiugants with identification based on the growth or colors of the colonies. The frequency of R plasmid transfer with filter mating method was come out much higher than that of broth mating method and appeared to be dependent upon the mating time and temperature. The optimum conditions for filter mating method were found to be 30$^{\circ}C$ and 24hrs as mating temperature and period, respectively, Moreover, donor cells with R plasmid, both isolate and standard bacteria, were shown to have an ability to transfer the plasmid against Escherichia coli K-12 HB101 (E. coli HB101) and Edwardsiella tarda (E. tarda) RE14 at fairly high frequencies, finally, we isolated 3 isolates of Sphingomonas sp., carrying R plasmid from 12 multi-drug resistant bacteria in normal microflora of the flounder (Paralichthys olivaceus) group used for the isolation of V emsela JE1 four months before. The same size and gene transfer chayateristics of R plasimds with those of V damsela JE1 confirmed that normal microflora have the reservoir activity for R plasmid in natural aquatic environment.

• Horticultural Science & Technology
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• v.33 no.6
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• pp.947-954
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• 2015

To select suitable spray chrysanthemum cultivars for Agrobacterium-mediated transformation, thirty-nine (39) spray cultivars bred in the National Institutes of Korea and a standard cultivar Jinba from Japan were collected and tested for regeneration rate and Agrobacterium infection assays. MS medium with $0.5mg{\cdot}L^{-1}$ IAA and $1.0mg{\cdot}L^{-1}$ BAP was used for shoot regeneration from leaf disks and internodes. The shoot regeneration rate in leaf disks was the highest in cultivar BRM, followed by cultivars VS, WW and YTM. The cultivar JB (Jinba) used as a transformation material in previous reports ranked similarly to cultivars PK and SPP. In shoot regeneration from internodes, the shoot regeneration rate was the highest for cultivar PA, followed by cultivar WW. The infection rate of leaves and internodes of 40 chrysanthemum cultivars with agrobacterium was investigated. Cultivars WPP, YNW, VS, PP, WW, FA, PA and YMN showed the highest infection levels in leaves, whereas cultivars WPP, PA, PK and YNW had the highest infection levels in internodes. Considering all of these results, cultivars VS and WW were the most appropriate for gene transformation of chrysanthemum using leaves, while cultivar PA was for internodes.