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

• The Korean Journal of Zoology
• /
• v.28 no.3
• /
• pp.166-178
• /
• 1985

In order to express hepatitis B surface antigen $(HB_sAg)$ containing pre-surface antigen region in mammalian calls, 2.7 kb DNA fragment containing pre-surface region-$HB_sAg$ gene poly(A) addition site of HBV genome was cloned into simian virus 40(SV 40) based chimeric vector pSVOB. 2.7 kb DNA fragment was derived from pHBVD 107 containing tandem copies of the HBV genome in a head-to-tail arrangement by Bgl II digestion. Construction of the vector pSVOE involved the incorporation of SV40 sequences spanning the viral origin of replication and 72 bp repeats (enhancer) into a pBR 322 derivative lacking sequences which inhibit replication in mammalian cells. Bam HI linker was inserted at the Pvu II site in the proximity of SV40 late promoter of pSVOE and named as pSVOB. To construct the recombinant plasmid pSVBS, pHBVD 107 was digested with Bgl II to isolate 2.7kb DNA fragment and the fragment was ligated into the Bam HI site of pSVOB by ligation. Preliminary result showed that the recombinant plasmid pSVBS produced $HB_sAg$ in the monkey cell producing large T antigen (COS cell).

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

• The Korean Journal of Food And Nutrition
• /
• v.15 no.2
• /
• pp.112-118
• /
• 2002

Sporulation in the fission yeast Schizosaccharomyces pombe has been regarded as an important model of cellular development and differentiation. S. pombe cells proliferate by mitosis and binary fission on growth medium. Deprivation of nutrients especially nitrogen sources, causes the cessation of mitosis and initiates sexual reproduction by matting between two sexually compatible cell types. Meiosis is then followed in a diploid cell in the absence of nitrogen source. DNA fragment complemented with the mutations of sporulation gene was isolated from the S. pombe gene library constructed in the vector, pDB 248' and designated as pDB(spo5)1. We futher analyzed six recombinant plasmids, pDB(spo5)2, pDB(spo5)3, pDB(spo5)4, pDB(spo5)5, pDB (spo5)6, pDB(spo5)7 and found each of these plasmids is able to rescue the spo5-2, spo5-3, spo5-4, spo5-5, spo5-6, spo5-7 mutations, respectively. Mapping of the integrated plasmid into the homologous site of the S. pombe chromosomes demonstrated that pDB(spo5)1, and pDB(spu5)Rl contained the spo5 gene. Transcripts of spo5 gene were analyzed by Northern hybridization. Two transcripts of 3.2 kb and 2.5kb were detected with 5kb Hind Ⅲ fragment containing a part of the spo5 gene as a probe. The small mRNA(2.5kb) appeared only when a wild-type strain was cultured in the absence of nitrogen source in which condition the large mRNA (3.2kb) was produced constitutively. Appearance of a 2.5kb spo5-mRNA depends upon the function of the meil, mei2 and mei3 genes.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
• /
• v.26 no.1
• /
• pp.53-58
• /
• 2000

Treatment of oral cancers with chemotherapeutic agents are evaluated as an effective method for remission to reduce cancer proliferation nowadays. But, minimization of side-effects such as bone marrow suppression, gastrointestinal toxicity and renal damage is another problem to be solved. Thus, a possible approach to develop a clinically applicable chemotherapeutic agents is to screen anticancer activity among traditional medicinal plants which have been used for thousands of years with very low side-effects in orient. In this study we focused on screening anti-oral cancer activities among 14 traditional medicinal plant extracts that revealed anticancer activities on other solid tumors. The results were as follow : 1. Methanol extract of Lepidium apetalum showed the highest anti-oral cancer activity against A253 cells. At concentration of $4{\mu}g/ml$, the cell viability was 48% under our experimental condition. $IC_{50}$ value obtained was $4{\mu}g/ml$. 2. Methanol extract of Coptis japonica and Solanum nigrum were effective on KB cells. Cell viability observed were 62% and 67% at concentration of $4{\mu}g/ml$, and $IC_{50}$ values were $12{\mu}g/ml$ and $10{\mu}g/ml$ respectively. 3. When the methanol extract of Lonicera caerule was combined with $2{\mu}g/ml$ of cisplatin, the anticancer activity was synergistically increased. One hundred ${\mu}g/ml$ of Lonicera caerule showed 92%(alone) or 59%(combined with cisplatin) cell viabilities. $IC_{50}$ value of Lonicera caerule extract against KB cells was reduced from $301{\mu}g/ml$ to $126{\mu}g/ml$ when combined with $2{\mu}g/ml$ of cisplatin. 4. Medicinal plant extracts effective on both A253 and KB cells were Coptis japonica, Lepidium apetalum, Solanum nigrum, Caesalpiniae Lignum, Curcuma aromatica.

• The Journal of Korea Institute of Information, Electronics, and Communication Technology
• /
• v.10 no.2
• /
• pp.176-183
• /
• 2017

There is a problem of long erase and program times in testing large-density memories. Also, there is a need of testing the VT voltages of EEPROM cells at each step during the reliability test. In this paper, a cell verification module is designed for a 512kb EEPROM and a CG (control gate) driver is proposed for measuring the VT voltages of a split gate EEPROM having negative erase VT voltages. In the proposed cell verification module, asymmetric isolated HV (high-voltage) NMOS devices are used to apply negative voltages of -3V to 0V in measuring erase VT voltages. Since erasing and programming can be done in units of even pages, odd pages, or a chip in the test time reduction mode, test time can be reduced to 2ms in testing the chip from 4ms in testing the even and the odd pages.

• Microbiology and Biotechnology Letters
• /
• v.36 no.4
• /
• pp.307-313
• /
• 2008

The yeast surface expression system, pCTXYN (6.8 kb), of Bacillus endoxylanase gene (xynB, 642 bp) was constructed and introduced into Saccharomyces cerevisiae EBY100 cell. The transformed yeast cell showing the highest endoxylanase activity was selected through the active staining of colonies grown on YPDG medium containing xylan. With the yeast transformant, EBY100/pCTXYN, grown on galactose containing medium, it was found that the endoxylanase was successfully displayed on the yeast cell surface and the xylooligosaccharides were efficiently produced from xylan. The most of endoxylanase activity was detected in the cell fraction and reached about 1.9 unit/mL after 48 h cultivation. The optimized conditions for xylooligosaccharides production from xylan were determined as follows: substrate and its concentration, oat spelt xylan 6%; concentration of yeast whole-cell, 5 unit/mL; temperature, $50^{\circ}C$, and reaction time $2{\sim}4\;h$. When the oat spelts xylan and corncob xylan were hydrolyzed by treatment with cell surface-displayed endoxylanase, xylotriose was formed as a main product.

• Korean Journal of Microbiology
• /
• v.52 no.3
• /
• pp.249-253
• /
• 2016

The inky cap, Coprinellus congregatus, produces several laccase isozymes during its life cycle: both hyphal tip laccase and sclerotial laccase are involved in the fungal development. When this fungus was transferred to an acid liquid medium (pH 4.0-4.5), a new laccase was synthesized and secreted into the culture supernatant. In order to examine its regulation by external pH, green fluorescent protein gene was ligated at the downstream of the promoters having different lengths. These expression vectors having different promoter lengths were inserted into the fungal transformation vector, pBARGEM7-1. These expression vectors were introduced to the mating type a1 and a2 monokaryons, and the transformants were selected by the phosphinothricin resistance. Transformant a1 (a1TF) and transformant a2 (a2TF) were mated with each other to generate homozygotic dikaryon transformants. All these transformants were grown in neutral liquid medium for 5 days, and then the whole cell homogenates were transferred to the acidic liquid medium (pH 4.1). After 36 h incubation at $25^{\circ}C$, cells were harvested for the analysis of GFP expression. GFP expression was detected in the transformant having full-length promoter (2.0 kb), but other transformants having shorter length promoter (shorter than 1.29 kb) failed to show the fluorescence. Therefore, the acid-responsive element in the laccase promoter should be localized between -2.0 kb ~ -1.29 kb region.

• Journal of Physiology & Pathology in Korean Medicine
• /
• v.16 no.6
• /
• pp.1270-1276
• /
• 2002

Compounds 1 - 12 were tested for their growth inhibitory effects against tumor cell lines using two different 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and sulforhodamine B protein (SRB) assays and antimicrobial activity. The cytotoxic activity of methyl-4-[{(2E)-3,7-dimethyl-2,6-octadienyl}oxy]-3-methoxy benzoate (1) exhibit more active than that of 5-fluorouracil (11) on human oral epithelioid carcinoma (KB, ATCC No. OCL 17) cell lines. But this compound (1) on human skin melanoma (SK-MEL-3, HBT 69) cell lines shows less active than that of adriamycin (12). However, compound 9 showed the antimicrobial activity against S. epidermidis (MIC, 15.625 ㎍/㎖), S. aureus, C. albicans (MIC, 31.25 ㎍/㎖), S. mutans, S. typhimurium, P. putida (MIC. 125 ㎍/㎖) and P. aeruginosa (MIC, 500 ㎍/㎖).

• Food Science and Biotechnology
• /
• v.16 no.4
• /
• pp.537-542
• /
• 2007

Sophora flavescens AITON (Leguminosae) is a typical traditional Korean medical herb considered to exhibit antibacterial, anti-inflammatory, and antipyretic effects, and is also used for the treatment of skin and mucosal ulcers, sores, diarrhea, gastrointestinal hemorrhage, arrhythmia, and eczema. In this study, the compound sophoraflavanone G was isolated from the dried roots of S. flavescens by bioassay-guided fractionation. We then investigated the effects of various concentrations of sophoraflavanone G on cell viability and the induction of apoptosis in KB cells after an incubation of 24 hr. The results were determined by the following methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-terazolium bromide (MTT) assay, Hoechst-33258 dye staining, flow cytometry (cell cycle), and Western blotting for caspase-3 and poly (ADP-ribose) polymerase (PARP). We found sophoraflavanone G induced the apoptosis of KB cells in a dose-dependent manner that was verified by DNA fragmentation, apoptotic bodies, the sub-G1 ratio, caspase-3 activity, and cleavage of PARP. These results suggest that sophoraflavanone G has potent anti-proliferative effects on human oral epidermoid carcinoma cells, with the induction of apoptosis.