• Molecules and Cells
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• v.40 no.9
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• pp.655-666
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• 2017

We constructed a large $na{\ddot{i}}ve$ human Fab library ($3{\times}10^{10}$ colonies) from the lymphocytes of 809 human donors, assessed available diversities of the heavy-chain variable (VH) and ${\kappa}$ light-chain variable (VK) domain repertoires, and validated the library by selecting Fabs against 10 therapeutically relevant antigens by phage display. We obtained a database of unique 7,373 VH and 41,804 VK sequences by 454 pyrosequencing, and analyzed the repertoires. The distribution of VH and VK subfamilies and germline genes in our antibody repertoires slightly differed from those in earlier published natural antibody libraries. The frequency of somatic hypermutations (SHMs) in heavy-chain complementarity determining region (HCDR)1 and HCDR2 are higher compared with the natural IgM repertoire. Analysis of position-specific SHMs in CDRs indicates that asparagine, threonine, arginine, aspartate and phenylalanine are the most frequent non-germline residues on the antibody-antigen interface and are converted mostly from the germline residues, which are highly represented in germline SHM hotspots. The amino acid composition and length-dependent changes in amino acid frequencies of HCDR3 are similar to those in previous reports, except that frequencies of aspartate and phenylalanine are a little higher in our repertoire. Taken together, the results show that this antibody library shares common features of natural antibody repertoires and also has unique features. The antibody library will be useful in the generation of human antibodies against diverse antigens, and the information about the diversity of natural antibody repertoires will be valuable in the future design of synthetic human antibody libraries with high functional diversity.

• Reproductive and Developmental Biology
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• v.41 no.1
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• pp.7-16
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• 2017

The knock-in efficiency in the fibroblast is very important to produce transgenic domestic animal using nuclear transfer. In this research, we constructed three kinds of different knock-in vectors to study the efficiency of knock-in depending on structure of knock-in vector with different size of homologous arm on the ${\beta}-casein$ gene locus in the somatic cells; DT-A_cEndo Knock-in vector, DT-A_tEndo Knock-in vector I, and DT-A_tEndo Knock-in vector II. The knock-in vector consists of 4.8 kb or 1.06 kb of 5' arm region and 1.8 kb or 0.64 kb of 3' arm region, and neomycin resistance gene(neor) as a positive selection marker gene. The cEndo Knock-in vector had 4.8 kb and 1.8 kb homologous arm. The tEndo Knock-in vector I had 1.06 kb and 0.64 kb homologous arm and tEndo Knock-in vector II had 1.06 kb and 1.8 kb homologous arm. To express endostatin gene as transgene, the F2A sequence was fused to the 5' terminal of endostatin gene and inserted into exon 7 of the ${\beta}-casein$ gene. The knock-in vector and TALEN were introduced into the bovine fibroblast by electroporation. The knock-in efficiencies of cEndo, tEndo I, and tEndo II vector were 4.6%, 2.2% and 4.8%, respectively. These results indicated that size of 3' arm in the knock-in vector is important for TALEN-mediated homologous recombination in the fibroblast. In conclusion, our knock-in system may help to create transgenic dairy cattle expressing human endostatin protein via the endogenous expression system of the bovine ${\beta}-casein$ gene in the mammary gland.

• Reproductive and Developmental Biology
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• v.30 no.3
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• pp.175-180
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• 2006

This study was conducted to examine the effect of pre activation treatment and activation time of recipient cytoplasm on the development of bovine somatic cell nuclear transfer(NT) embryos. Donor cells were transferred and electrofused to enucleated oocytes before(pre-AC) or after activation(post-AC). Activation was induced with a combination of $Ca^{2+}$-ionophore(A23187) and DMAP. NT embryos were cultured in CR1aa containing 3 mg/ml BSA for 9 days. Some NT embryos were fixed at 0.5 to 2.5 hr after fusion(for post-AC) or activation(for pre-AC) for confocal microscopy. Developmental rate to the blastocyst stage was slightly high in the post-AC group(20.6%) compared to that of pre-AC group(15.3%). However, developmental speed of embryos in the pre-AC group was faster than that of embryos in the post-AC group. Development rates to the blastocyst stage were similar among different activation time before fusion(0.5,2 and 4 hr). The result of the present study suggests that development and nuclear morphology are affected f the activation status of the recipient cytoplasm before fusion.

• Journal of Animal Science and Technology
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• v.47 no.1
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• pp.11-18
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• 2005

The objective of this study was to estimate the effects of daily milk yield, somatic cell count(SCC), days in milk(DIM), and parity on the compositions of milk and blood in high or low producing dairy cows. To divide the high or low producing group, there were some restrictions in this study. 235 Holstein dairy cows had a average daily milk yield of 23.2 $\pm$ 6.8 kg were grouped into two classes with low producing(average daily milk 17kg) or high producing(average daily milk 29 kg). The other restrictions were two parities(first and second parity), two SCC groups(under $l{\times}10^5$cells/ml, and $l{\times}10^5$ to $7{\times}10^5$ celis/ml), and three DIM groups(under 80, 81 to 180, and 181 to 305DIM). The blood urea nitrogen(BUN), milk urea nitrogen(MUN) and glucose between two group with high and low somatic cell count were not affected by parity, DIM and SCC. But there were significantly different on BUN and glucose between high and low milk producing(p< 0.01), also was different on glucose between parities(p < 0.05). White blood cell(WBC) and lymphocyte were affected(p< 0.05) by SCC level, protein percent was also affected by DIM(p< 0.01). The least square means of protein in second parity was a 1.3 times higher than that in first parity(p < 0.05), and it showed a higher level in the low producing group than the high producing group(p < 0.0l). WBC and lymphocyte were lower in the $1{\sim}7{\times}10^5$ celis/ml than those under $1{\times}10^5$ celis/ml(p< 0.05). Neutrophil was a higher level in first parity than that in second parity(p < 0.05). Only protein and total solid were affected by parity, the other compositions were not affected by parity, DIM, SCC and milk yields. The results suggested that significant differences were in the blood components such as glucose, WBC, lymphocyte and neutrophil between high and low producing cows. The results also show that more studies are required to clarify the factors and markers related to milk yield, quality and mastitis.

• Korean Journal of Plant Tissue Culture
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• v.24 no.3
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• pp.129-135
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• 1997

Morphogenetic responses were investigated by culturing embryo and leaf explants of Korean wild type tea plant, Camellia sinensis (L.) O. Kuntze. Induction of direct somatic embryogenesis as well as adventitious and/or axillary shoots was obtained from mature zygotic embryo cultures on Murashige and Skoog (MS) basal medium having 5 to $20\mu\textrm{M}$cytokinin a lone. Morphogenetic response was decreased dramatically by the addition of auxins tested. One hundred percent of induced and isolated shoots formed roots after four weeks of culture on half-strength MS or quarter-strength Schenk and Hildebrandt (SH) media supplemented with $10\mu\textrm{M}$indole-3-butyric acid (IBA). Immature zygotic embryos were shown to be a suitable explant for embryogenic callus formation in the presence of 2, 4-dichlorophenoxyacetic acid(2, 4-D) in basal medium. Mature zygotic embryo originated leaves were used to test their ability for mophogenesis by incorporating plant growth regulators such as IBA, naphthyl-1-acetic acid (NAA), and 6-benzylaminopurine (BAP). Apparently, the morphogenetic responses of the cultured explant sources on the types and/or levels of plant growth regulators tested were observed visually.

• Korean Journal of Veterinary Service
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• v.36 no.4
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• pp.263-272
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• 2013

Consistent information on the chemical composition and its seasonal variation of goat udder half milk is limited in Korea. The objective of this study was to analyze the seasonal variation of the chemical composition of goat milk to take establish various parameters into consideration on the pricing of the goat milk. Variations in chemical composition, somatic cell count (SCC) and bacterial count of 1,038 udder half milk samples from 650 heads raised in 7 farms of Jeonnam province were determined by season. Fat, protein, lactose, non-fat solids, milk urea nitrogen (MUN), pH, SCC and bacterial counts were also analyzed. The average composition of the milk was: fat $3.80{\pm}1.36%$, protein $3.23{\pm}0.80%$, lactose $4.39{\pm}0.54%$, total solids $12.18{\pm}1.80%$, non-fat solids $8.38{\pm}0.80%$, and milk urea nitrogen $28.44{\pm}5.00mg/dL$. The average pH was $6.81{\pm}0.24$. The average of SCC and bacterial counts were $2.54{\pm}4.60{\times}10^6cells/mL$ and $1.25{\pm}3.76{\times}10^5CFU/mL$, respectively. Chemical composition, pH, SCC and bacterial counts of dairy goat milk varied widely during the lactation period and by season. The fat concentration was the lowest in spring ($3.39{\pm}1.53%$) and the highest in autumn and winter ($3.98{\pm}1.30%$ and $3.98{\pm}1.48%$). Protein concentration was the lowest during summer ($2.92{\pm}0.48%$) and the highest in winter ($2.92{\pm}0.48%$). Lactose concentration was the lowest in autumn ($4.24{\pm}0.41%$) and the highest in spring ($4.58{\pm}0.35%$). The lowest total solid value was obtained in the spring season ($11.75{\pm}1.80%$) which was then increased in winter ($12.85{\pm}1.96%$). Non-fat solid concentration was the lowest in summer ($8.07{\pm}0.64%$) and the highest in autumn ($8.94{\pm}0.82%$). MUN concentration was the highest in summer ($8.07{\pm}0.64%$), and the pH concentration was the highest in spring at $6.93{\pm}0.27%$. Seasonal variation of SCC and bacterial count were the lowest in spring ($0.94{\pm}1.54{\times}10^6cells/mL$ and $0.22{\pm}0.61{\times}10^5CFU/mL$, respectively) and was the highest in winter ($3.95{\pm}7.14{\times}10^6cells/mL$ and $2.23{\pm}5.54{\times}10^4CFU/mL$, respectively).

• Korean Journal of Veterinary Service
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• v.35 no.4
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• pp.295-305
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• 2012

The aim of this study was to isolate microorganisms from half milk samples of dairy goats by California mastitis test (CMT) during the lactation period and to further investigate the susceptibility of isolated organisms to antimicrobial drugs. From a total of 235 half milk samples with CMT scores of 2 or above from 366 dairy goats distributed throughout Jeonnam province, microorganisms were isolated from 198 (83.5%) samples either singly (99.0%) or in combination (1.0%). The most prevalent microorganism was the coagulase-negative Staphylococcus spp., (44.4%, n=88) followed by Staphylococcus aureus (24.2%, n=48), Escherichia coli (11.1%, n=22) and Streptococcus spp. (7.6%, n=15). Isolated bacteria also included Bacillus spp. (2.5%, n=5), Pseudomonas spp. (2.5%, n=5), Micrococcus spp. (1.5%, n=3), Corynebacterium spp. (1.5%, n=3), Enterococcus facium (1.0%, n=2), Morganella morganii (0.5%, n=1) and Streptococcus agalactiae (0.5%, n=1). During the summer season, a high prevalence of all microorganisms were observed in which Staphylococcus spp. (30.8%), Escherichia coli (8.6%), and Streptococcus spp. (5.6%) were among the most prevalent bacteria isolated. Staphylococcus spp. was also shown to be high in the winter (21.7%). In most samples, the presence of bacterial pathogens in goat milk led to the increase in the total somatic cell count (SCC). Most of the half milk samples of dairy goats with bacterial contamination showed SCC of ${\geq}1{\times}10^6cells/ml$ (90.4%). Minor pathogens (11.4%) were more detected from milk samples with SCC of < $1{\times}10^6cells/ml$ than major pathogens (4.1%), while the major pathogens tended to be higher from samples with SCC of ${\geq}3{\times}10^6cells/ml$. Susceptibility of these bacteria to 12 antimicrobial agents was tested by the Kirby-Bauer disc diffusion method. Results indicated that more than 90% of bacteria isolated from CMT 2+ dairy goat half milk samples were susceptible to trimethoprim/sulfamethoxazole, amoxicillin/clavulanic, enrofloxacin and cephalothin while they were resistant to tetracycline (44.7%).

• 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.

• Korean Journal of Environmental Biology
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• v.29 no.4
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• pp.373-378
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• 2011

Our aim was to investigate the genotoxicity of ambient air in the Krak$\acute{o}$w area after Fukushima Nuclear Power Plant (NPP) accident and compare with results from Chernobyl fallout. For the detection of ambient air genotoxicity the technique for screening gene mutation frequency in somatic cells of the $Tradescantia$ stamen hairs ($Trad$-SH assay) was used. Since 11th of March 2011 (Fukushima NPP accident), several pots containing at least 15 shoots of bioindicating plants were exposed to ambient air at 2 sites in the Krak$\acute{o}$w surrounding area, one in the city center, and about 100 pots in a control site (in the glasshouse of the Institute of Nuclear Physics) Continuous screening of mutations was performed. Progenies of 371,090 cells exposed were analyzed. Mutation frequency obtained in the first 10 days has shown a mean control level (GMF*100=$0.06{\pm}0.01$). At scoring period related to influence of a potential Fukushima fallout, a significant increase of gene mutation frequencies above the control level was observed at each site in the range, 0.10~0.33 depending on the location, (mean value for all sites GMF*100=$0.19{\pm}0.05$) that was associated with a strong expression of toxic effects. In the reported studies following the Chernobyl NPP accident monitoring $in$ $situ$ of the ambient air genotoxicity was performed in the period since April $29^{th}$ till June $3^{rd}$ 1986 also with Trad-SH bioindicator. In general, mutation frequency increases due to Chernobyl fallout(GMF*100=$0.43{\pm}0.02$) were corresponding to fluctuation of radioactivity in the air reported from physical measures, and to published reports about increase in chromosome aberration levels. Although, recent data obtained from monitoring of the ambient air quality in the Krak$\acute{o}$w and surroundings are lower when compared to results reported after Chernobyl NPP accident, though results express a significant increase above the control level and also are corresponding with increased air radioactivity reported from physical measurements. Statistically significant in comparison to control increase in gene mutation rates and more prolonged than that after Chernobyl fallout increase of GMF was observed during the period following the Fukushima NPP failure.

• Development and Reproduction
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• v.4 no.2
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• pp.195-201
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• 2000

Sex differentiation process of the spotted sea bass, Lateolabrax maculatus, was investigated by histological method. The fish samples were collected from just after hatching to 365 days later. The primordial germ cells and genital ridge were appeared separately hanging under air bladder in 30-day larva (total length: 11.7~13.2 mm), and were unified into the undifferentiated gonads in 40-day larva (12.5~14.0 mm). The ovarian differentiation was started in 60-day juvenile (23.6~27.0 mm). The somatic tissues were elongated in tip of both ends of undifferentiated gonad and were fused each other. The complete ovarian cavity was appeared in 80-days juvenile(33.1~42.5 mm). The testicular differentiation was initiated in 70-day juvenile (24.8~31.6 mm). The rudiment of sperm duct was appeared in the center of the undifferentiated gonad. The meiosis of germ cells in the ovary was started in 168-day juvenile (88.0~115.4 mm). In 287-day juvenile (175.1~233.6 mm), the ovary was filled with both of chromatin stage and perinucleolus stage oocytes. The meiosis of male germ cells was started in 245-day juvenile (124.4~168.3 mm). However, the seminiferous tubules of testis were filled with numerous sperm in 365-day juvenile (162.5~253.8 mm). The sex ratio of male and female was 1:1.38. Considering these results, the spotted sea bass was showed differentiated type in sex differentiation and gonochorism in sexuality.