• Korean Journal of Animal Reproduction
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• v.27 no.2
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• pp.179-185
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• 2003

To determine whether the diphtheria toxin-A (DT) gene disrupts development of thymocytes in transgenic animal, the DT-A gene was used for the production of transgenic mice directed by proximal Ick promoter sequences. Two transgenic founder mice that contained several copies of transgene were produced by DNA microinjection and integration of transgene in transgenic mice was confirmed by PCR and Southern blotting analysis. Transgenic $F_1$ and $F_2$ mice were produced by outbreeding of founder and $F_1$ mice to investigate expression of transgene and phenotypes in transgneic mice. Expression of the diphtheria toxin gene was confirmed in thymus, spleen and liver of transgenic mice by RT-PCR. In circulating blood of transgenic mice, lower number of circulating white blood cells and platelets were observed compared with that of normal mice. In addition, transgneic mice had reduced number of circulating peripheral T-cells analyzed by FACS with anti-CD3 antibody. The data in these transgenic mice indicate that DT gene can play a disruptive role in developing thymocytes of transgenic mice resulted in lower number of T-cells that can be applicable to a wide range of tissues in other animals.

• Journal of Microbiology and Biotechnology
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• v.12 no.4
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• pp.622-627
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• 2002

Diphtheria toxin repressor (DtxR) binds to approximately 30 to 35-bp regions containing an interrupted 9-bp inverted repeat within a 19-bp core sequence. The core sequence is fairly conserved and critical for DtxR binding. The flanking regions that are consisted of 5 to 8 more of nucleotides from the core are also required for DtxR binding. The nucleotides in both flanking regions are A-T rich. To examine whether the A-T nucleotides in both flanking regions from the core have significant roles for DtxR binding, a DNA fragment was constructed based on the diphtheria tox promoter/operator, and DNA fragments with substitution of A and T nucleotides In the flanking regions to G and C were also constructed. To assess the effect of these substitutions on binding of DtxR and repressibility by DtxR, $\beta$-galactosidase activity from lacZ fused to the region was assessed. Gel mobility shift of the region by purified DtxR was also examined. The DNA fragments containing the mutations in the flanking regions still exhibited repression and mobility shift with DtxR. The core segment with the mutation is still, therefore, recognized by DtxR. Nonetheless, the results from the assays indicated that the substitution significantly decreased repression of the operator by DtxR in vivo under high-iron condition and decreased binding of DtxR to the operator. These results suggest that A and T nucleotides fur both flanking regions are preferred for the binding of DtxR.

• Clinical and Experimental Pediatrics
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• v.50 no.4
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• pp.355-362
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• 2007

Purpose : We conducted the study to evaluate the immunogenicity and safety of three component DTaP vaccine ($Infanrix^{(R)}$) in a group of Korean healthy infants on a three-dose primary vaccination. And we compared the immunogenicity of this DTaP vaccine with two component DTaP vaccine which has been widely used in Korea. Methods : We enrolled one hundred fifty one healthy infants aged 8-9 weeks. These infants were vaccinated at age 2, 4 and 6 months of age with three component DTaP vaccine. Solicited adverse events were actively monitored for 72 hours following each vaccination, and all adverse events after each vaccination were observed for three weeks. Anti-diphtheria toxoid Ab., anti-tetanus toxoid Ab., anti-pertussis toxin Ab., anti-filamentous hemagglutinin Ab., and anti-pertactin Ab. were measured using ELISA for assessing immunogenicity of study vaccine in 60 infants. Immunogenicity analysis of two component DTaP vaccine was performed with same methods in 14 infants as control. Results : The seroconversion rates of anti-diphtheria toxoid Ab, anti-tetanus toxoid Ab. anti-filamentous hemagglutinin Ab. were 100% in both group. Seroconversion rate of anti-pertactin Ab in study group was 100%, but the rate in control group was 50%. However, geometric mean concentration of anti-pertussis toxin Ab. was higher in control group. Mild local and systemic reactions were observed within three days after vaccination, and no serious adverse events related study vaccine were happened during study period. Conclusion : Our study results suggest that three component DTaP vaccine ($Infanrix^{(R)}$) is a well-tolerable and high immunogenic vaccine, especially anti-Pertactin Ab. of the study vaccine is very immunogenic. It can be available as routine DTaP vaccination in our infants.

• Horticultural Science & Technology
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• v.18 no.3
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• pp.342-347
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• 2000

The objectives of this study were to investigate the genetic and phenotypic features of male sterile transformants by pollen-specific expression of diphtheria toxin gene and to find out inheritance patterns of transgene to the next generation. When backcrossed (BC) progenies were tested for expression of kanamycin resistance ($Km^R$), 9 lines out of 13 lines, except 4 lines ($BC_{1}5-13,\;BC_{1}5-23,\;BC_{1}5-28,\;BC_{1}5-32$), showed the ratio of $Km^R$ to kanamycin sensitive ($Km^S$), from 1:30 to all $Km^S$. As a result, they were much lower than Mendelian segregation of a dominant gene. To determine whether male sterility is a heritable and stable trait, 5 male sterile plants ($BC_{1}5-13,\;BC_{1}5-14,\;BC_{1}5-23,\;BC_{1}5-32,\;BC_{1}5-33$ lines) which had different transgene copy numbers were backcrossed as female parents with pollens from wild type. To confirm the existence of the DTx-A gene in the genome of the progenies, PCR was conducted using specific primers of the DTx-A coding region. A PCR band of 428 bp was obtained from each generation, which is the predicted size of the DTx-A gene fragment. Trangenes were inherited to the next $BC_4T_0$ progenies and showed male sterility, however, based on the copy numbers of DTx-A gene male sterile plants did not show predicted ratio. When male sterile plants were backcrossed with fertile plants, fruit capsule sizes and seed settings were relatively reduced from those of selfing wild type plants. The fruit sizes and seed settings were reduced in proportion to the increase in the copy number of DTx-A gene.

• Journal of Microbiology and Biotechnology
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• v.28 no.12
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• pp.2113-2120
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• 2018

Cross-reactive material 197 ($CRM_{197}$) is a non-toxic mutant of diphtheria toxin containing a single amino acid substitution of glycine 52 with glutamic acid. $CRM_{197}$ has been used as a carrier protein for poorly immunogenic polysaccharide antigens to improve immune responses. In this study, to develop a sandwich ELISA that can detect $CRM_{197}$ and $CRM_{197}$ conjugate vaccines, we generated a human anti-$CRM_{197}$ monoclonal antibody (mAb) 3F9 using a phage-displayed human synthetic Fab library and produced mouse anti-$CRM_{197}$ polyclonal antibody. The affinity ($K_D$) of 3F9 for $CRM_{197}$ was 3.55 nM, based on Bio-Layer interferometry, and it bound specifically to the B fragment of $CRM_{197}$. The sandwich ELISA was carried out using 3F9 as a capture antibody and the mouse polyclonal antibody as a detection antibody. The detection limit of the sandwich ELISA was <1 ng/ml $CRM_{197}$. In addition, the 3F9 antibody bound to the $CRM_{197}$-polysaccharide conjugates tested in a dose-dependent manner. This ELISA system will be useful for the quantification and characterization of $CRM_{197}$ and $CRM_{197}$ conjugate vaccines. To our knowledge, this study is the first to generate a human monoclonal antibody against $CRM_{197}$ and to develop a sandwich ELISA for $CRM_{197}$ conjugate vaccines.

• Reproductive and Developmental Biology
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• v.38 no.2
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• pp.71-77
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• 2014

The specific genetic modification in porcine somatic cells by gene targeting has been very difficult because of low efficiency of homologous recombination. To improve gene targeting, we designed three kinds of knock-out vectors with ${\alpha}1,3$-galactosyltransferase gene (${\alpha}1,3$-GT gene), DT-A/pGT5'/neo/pGT3', DT-A/NLS/pGT5'/neo/pGT3' and pGT5'/neo/ pGT3'/NLS. The knock-out vectors consisted of a 4.8-kb fragment as the 5' recombination arm (pGT5') and a 1.9-kb fragment as the 3' recombination arm (pGT3'). We used the neomycin resistance gene (neo) as a positive selectable marker and the diphtheria toxin A (DT-A) gene as a negative selectable marker. These vectors have a neo gene insertion in exon 9 for inactivation of ${\alpha}1,3$-GT locus. DT-A/pGT5'/neo/pGT3' vector contain only positive-negative selection marker with conventional targeting vector. DT-A/NLS/pGT5'/neo/pGT3' vector contain positive-negative selection marker and NLS sequences in upstream of 5' recombination arm which enhances nuclear transport of foreign DNA into bovine somatic cells. pGT5'/neo/pGT3'/NLS vector contain only positive selection marker and NLS sequence in downstream of 3' recombination arm, not contain negative selectable marker. For transfection, linearzed vectors were introduced into porcine ear fibroblasts by electroporation. After 48 hours, the transfected cells were selected with $300{\mu}g/ml$ G418 during 12 day. The G418-resistant colonies were picked, of which 5 colonies were positive for ${\alpha}1,3$-GT gene disruption in 3' PCR and southern blot screening. Three knock-out somatic cells were obtained from DT-A/NLS/ pGT5'/neo/pGT3' knock-out vector. Thus, these data indicate that gene targeting vector using nuclear localization signal and negative selection marker improve targeting efficiency in porcine somatic cells.

• Reproductive and Developmental Biology
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• v.32 no.3
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• pp.205-209
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• 2008

This study was carried out to develop knock-in vector for EGFP (enhanced green fluorescent protein) expression in porcine $\beta$-casein locus. For construction of knock-in vector using porcine $\beta$-casein gene, we cloned the $\beta$-casein genome DNA from porcine fetal fibroblast cells, EGFP and SV40 polyA signal using PCR. The knock-in vectors consisted of a 5-kb fragment as the 5' recombination arm and a 2.7-kb fragment as the 3' recombination arm. We used the neomycin resistance gene ($neo^{r}$) as a positive selectable marker and the diphtheria toxin A (DT-A) gene as a negative selectable marker. To demonstrate EGFP expression from knock-in vector, we are transfected knock-in vector that has EGFP gene in murine mammary epithelial cell line HC11 cells with pSV2 neo plasmid. The EGFP expression was detected in HC11 cells transfected knock-in vector. This result demonstrates that this knock-in vector may be used for the development of knock-in transgenic pig.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.11
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• pp.1644-1651
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• 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.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.86-86
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• 2003

Research has been in progress for more than a decade to production of useful proteins by genetic modification in cattle. However, the levels of protein production in transgenic cattle have been reported very low. To enhance protein production in transgenic animal, we tried homologous recombination to donor cells for production of transgenic clone cattle through nuclear transfer procedure. Thus, we constructed the two targeting vectors of human thrombopoietin (TPO) at bovine $\beta$-casein locus using homologous recombination with 13.6 kb and 9.6 kb homology. In two targeting vectors, positive selection was through the neomycin resistance gene and negative selection was by the diphtheria toxin (DT). Gene targeting was attempted in bovine embryonic fibroblasts (bEF) and bovine ear skin fibroblasts (bESF). To determine the most appropriate concentration of neomycin for bEF and bESF, G4l8 resistance was confirmed by culturing the cells in various concentrations of the drug and both of the cells were optimally selected at $900 \mu g/ml$ of neomycin. The transfected bEF and bESF by the targeting vectors were colonized efficiently at the ratio of DNA to transfection reagent such as $4 \mu g$:2 ${mu}ell$ and $1 \mu g$:$2 \mu l$. Comparing number of healthy clones from passage 4 to passage 8, bESF (17%) persist in culture for much longer than bEF (6%). The two gene-targeted bESF clones of 30 random-integrated clones with 9.6 kb homology length were confirmed, however, nothing was out of 72 random integration clones with 13.6 kb homology length, The DT also worked more efficiently in clones transfected with the vector of 9.6 kb homology length. Our data suggests that the choice of donor cell for long culture period should be considered to obtain targeted cell clone, and the gene-targeting frequency and the DT working efficiency are dependent on the length of target homology.