• Preventive Nutrition and Food Science
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• 제5권3호
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• pp.153-159
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• 2000

A 4.4 kb DNA fragment encompassing lacA (galactoside acetyltransferase) and lacZ($\beta$-galactosidase) genes from Lactococus lactis ssp. lactis ATCC 7962 (L. lactis 7962) was introduced ito a Lac strain, Lactococcus lactis ssp. lactis MG1363 (L. lactis MG1363) by using a lactococcal expression vector, pMG36e and expression level of lacZ was examined. Growth rates and $\beta$-galactosidase ($\beta$-gal) activities of MG1363 cells carrying recombinant plasmid, pMLZ3, on M17 broth containing different carbon sources (1%, w/v) were examined. Contrary to the expectations, MG1363 [pMLZ3] grown on lactose showed the lowest enzyme activity (17 units) and cells grown on galactose had the highest $\beta$-gal activity (41 units). Cells grown on glucose had intermediate activity (33 units). These activities are about one tenth of the values observed in L. lactis 7962 where lacZ is present as a single-copy gene in the chromosome. When the cellular concentrations of lacZ transcript were examined using slot blot hybridization, it was found that MG1363[pMLZ3] produced sufficient amounts of transcript. These results indicate that either proteolytic degradation of $\beta$-gal or other regulatory mechanism prevent the translation or accumulation of $\beta$-gal in L. lactis MG1363 cells. In regard to regulation, the presence of the ccpA gene in L. lactis MG1363 was confirmed by Southern blot.

• Biotechnology and Bioprocess Engineering:BBE
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• 제11권4호
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• pp.319-324
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• 2006

Although the sera used in animal cell culture media provide the macromolecules, nutrients, hormones, and growth factors necessary to support cell growth, it could also be an obstacle to the production of recombinant proteins in animal cell culture systems used in many sectors of the biotechnology industry. For this reason, many research groups, including our laboratory, have been trying to develop serum-free media (SFM) or serum-supplemented media (SSM) for special or multi-purpose cell lines. The Chinese hamster ovary (CHO) cell, for example, is frequently used to produce proteins and is especially valuable in the large-scale production of pharmaceutically important proteins, yet information about its genome is lacking. Also, SFMs have only been evaluated by comparing growth patterns for cells grown in SFMs with those grown in SSM or by measuring the titer of the target protein obtained from cells grown in each type of medium. These are not reliable methods of obtaining the type of information needed to determine whether an SFM should be replaced with an SSM. We carried out a cDNA microarray analysis to evaluate MED-3, an SFM developed in our laboratory, as a CHO culture medium When CHO cells were cultured in MED-3 instead of an SSM, several genes associated with cell growth were down-regulated, although this change diminished over time. We found that the insulin-like growth factor (IGF) gene was representative of the proteins that were down-regulated in cells cultured in MED-3. When several key supplements - including insulin, transferrin, ethanolamine, and selenium - were removed from MED-3, the IGF expression was consistently down- regulated and cell growth decreased proportionately. Based on these results, we concluded that when an SFM is used as a culture medium, it is important to supplement it with substances that can help the cells maintain a high level of IGF expression. The data presented in this study, therefore, might provide useful information for the design and development of SFM or SSM, as well as for the design of genome-based studies of CHO cells to determine how they can be used optimally for protein production in pharmaceutical and biomedical research.

• Biotechnology and Bioprocess Engineering:BBE
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• 제9권4호
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• pp.292-296
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• 2004

The recombinant soluble human tumor necrosis factor-alpha (hTNF-$\alpha$) was expressed in a yeast Saccharomyces cerevisiae and its cytotoxicity was evaluated. A cDNA encoding hTNF-$\alpha$ was placed under the control of two different promoters: a glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter and a yeast hybrid ADH2-GPD promoter, consisting of alcohol dehydrogenase II (ADH2) and the GPD promoter. A Northern blot analysis revealed that, although variation in the expression level of hTNF-$\alpha$ existed among transformants, the higher expression was obtained with the GPD promoter. Expressed hTNF-$\alpha$ protein (rhTNF-$\alpha$) was successfully secreted into the culture medium, producing 2.5 mg per liter of culture filtrate, with no changes in cell growth. The bioassay for observing the cytotoxicity to the murine L929 fibroblast cell line, with serial dilution of rhTNF-$\alpha$, indicated that the secreted rhTNF-$\alpha$ was bioactive and its dose-response was improved eight to ten times over that of the E. coli-derived rhTNF-$\alpha$.

• BMB Reports
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• 제43권8호
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• pp.573-578
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• 2010

Several organophosphorus (OP) insecticides can selectively kill the silkworm maggot, Exorista sorbillans (Es) (Diptera: Tachinidae), while not obviously affecting the host (Bombyx mori) larvae, but the mechanism is not yet clear. In this study, the cDNA encoding an acetylcholinesterase (AChE) from the field Es was isolated. One point mutation (Gly353Ala) was identified. The Es-353G AChE and Es-353A AChE were expressed in baculovirus-insect cell system, respectively. The inhibition results showed that for eserine and Chlorpyrifos, Es-353A AChE was significantly less sensitive than Es-353G AChE. Meanwhile, comparison of the I50 values of eserine, dichlorvos, Chlorpyrifos and omethoate of recombinant Es AChEs with its host (Bombyx mori) AChEs indicated that, both Es AChEs are more sensitive than B. mori AChEs. The results give an insight of the mechanism that some OP insecticides can selectively kills Es while without distinct effect on its host, B. mori.

• Molecules and Cells
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• 제23권1호
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• pp.17-22
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• 2007

We have expressed human erythropoietin (EPO) in transgenic mice using a recombinant EPO cDNA combined with a partial TPO construct. The gene was microinjected using standard techniques and five mice were detected as transgenic by PCR and further used as founders. The life span of the transgenic founders was much shorter than that of their normal littermates. Most of the tissues of the transgenic founders contained human EPO transcripts as judged by RT-PCR. Especially high expression levels were seen in the liver and lung. EPO protein levels in serum were examined by ELISA and ranged from 266-414 mIU/ml. The number of red blood cell, white blood cell and hemoglobin in the hEPO transgenic mice was higher than in normal mice. These results indicate that overexpression of hEPO is deleterious and can provoke lung failure and erythrocytosis.

• 한국수정란이식학회지
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• 제30권4호
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• pp.299-303
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• 2015

KO mice provide an excellent tool to determine roles of specific genes in biomedical filed. Traditionally, knockout mice were generated by homologous recombination in embryonic stem cells. Recently, engineered nucleases, such as zinc finger nuclease, transcription activator-like effector nuclease and clustered regularly interspaced short palindromic repeats (CRISPR), were used to produce knockout mice. This new technology is useful because of high efficiency and ability to generate biallelic mutation in founder mice. Until now, most of knockout mice produced using engineered nucleases were C57BL/6 strain. In the present study we used CRISPR-Cas9 system to generate knockout mice in FVB strain. We designed and synthesized single guide RNA (sgRNA) of CRISPR system for targeting gene, Abtb2. Mouse zygote were obtained from superovulated FVB female mice at 8-10 weeks of age. The sgRNA was injected into pronuclear of the mouse zygote with recombinant Cas9 protein. The microinjected zygotes were cultured for an additional day and only cleaved embryos were selected. The selected embryos were surgically transferred to oviduct of surrogate mother and offsprings were obtained. Genomic DNA were isolated from the offsprings and the target sequence was amplified using PCR. In T7E1 assay, 46.7% among the offsprings were founded as mutants. The PCR products were purified and sequences were analyzed. Most of the mutations were founded as deletion of few sequences at the target site, however, not identical among the each offspring. In conclusion, we found that CRISPR system is very efficient to generate knockout mice in FVB strain.

• Journal of Plant Biotechnology
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• 제2권2호
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• pp.79-82
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• 2000

We have isolated a cDNA encoding a calcium-dependent protein kinase (CDPK) in Nicotiana tabacum, which was designated NtCDPK1. Accumulation of the NtCDPK1 mRNA was stimulated by various stimuli, including phytohormones, CaCl$_2$ wounding, fungal elicitors, chitin and methyl jasmonate. The NtCDPK1 gene encodes a functional Ser/Thr protein kinase of which phosphorylation activity is strongly induced by calcium. By analyzing expression of the NtCDPK1-GFP fusion protein and by immunoblotting with antibody which reacts with NtCDPK1, we found that NtCDPK1 is localized in membrane and nucleus in plant cells. Silencing expression of the NtCDPK1 transgene resulted in marked decrease of lateral root development in the transgenic tobacco plants. Yeast two hybrid screening using NtCDPK1 as a bait identified a tobacco homologue of proteasome regulatory subunit 21D7, designated Nt21D7. The 21D7 mRNA has been shown to be predominantly expressed in proliferating tissues in the cell cycledependent manner in carrot. The recombinant NtCDPK1 protein associated with Nt21D7 in vitro, and could phosphorylate the Nt21D7 protein in vitro in the presence of calcium, suggesting that Nt21D7 protein is a natural substrate of NtCDPK1 in tobacco. These results suggest that NtCDPK1 may regulate tell proliferation processes, such as lateral root formation, by regulating specificity and/or activity of proteasome-mediated protein degradation pathway.

• Journal of Microbiology
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• 제37권3호
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• pp.168-174
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• 1999

Escherichia coli O157 is an important pathogenic organism which causes diarrhea, haemorrhagic colitis, and haemolytic ureamic syndrome (HUS) in human. E. coli O157 KNIH317 was isolated form patients suffering with HUS in Korea. We designed a primer set for cloning shiga-like toxin (slt) gene. The amplified PCR product was used to Southern and colony hybridization as a probe. As a result, we cloned 4.5-kb KpnI fragment containing the slt gene encoding shiga-like toxin from chromosomal DNA of E. coli O157 KNIH317. This recombinant plasmid was named pOVT45. E. coli XL1-Blue harboring pOVT45 showed cytotoxicity in Vero cells. We sequenced the slt gene of this strain. The A-subunit gene of the slt was composed of 960 base pairs with ATG initiation codon and TAA terminationcodon. The B-subunit was composed of 270 base paris with ATG initiation codon and TGA termination codon. Nucleotide sequence comparison of the slt gene exhibited 100%, 98.4%, 93.7%, and 93.7% identity with that of shiga-like toxin type II (sltII) of E. coli bacteriophage 933W, variant slt of E. coli, slt of E. coli, and variant sltII of E. coli, respectively. From these results, it was concluded that the cloned slt gene belongs to SltII family and that the strain used in this study may be a lysogeny of E. coli bcteriphage 933W.

• Journal of Microbiology and Biotechnology
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• 제20권4호
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• pp.767-774
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• 2010

In this study, the problems of high caloric content, increased maturation time, and off-flavors in commercial beer manufacture arising from residual sugar, diacetyl, and acetaldehyde levels were addressed. A recombinant industrial brewing yeast strain (TQ1) was generated from T1 [Lipomyces starkeyi dextranase gene (LSD1) introduced, ${\alpha}$-acetohydroxyacid synthase gene (ILV2) disrupted] by introducing Saccharomyces cerevisiae glucoamylase (SGA1) and a strong promoter (PGK1), while disrupting the gene coding alcohol dehydrogenase (ADH2). The highest glucoamylase activity for TQ1 was 93.26 U/ml compared with host strain T1 (12.36 U/ml) and wild-type industrial yeast strain YSF5 (10.39 U/ml), respectively. European Brewery Convention (EBC) tube fermentation tests comparing the fermentation broths of TQ1 with T1 and YSF5 showed that the real extracts were reduced by 15.79% and 22.47%; the main residual maltotriose concentrations were reduced by 13.75% and 18.82%; the caloric contents were reduced by 27.18 and 35.39 calories per 12 oz. Owing to the disruption of the ADH2 gene in TQ1, the off-flavor acetaldehyde concentrations in the fermentation broth were 9.43% and 13.28%, respectively, lower than that of T1 and YSF5. No heterologous DNA sequences or drug resistance genes were introduced into TQ1. Hence, the gene manipulations in this work properly solved the addressed problems in commercial beer manufacture.

• 한국식품위생안전성학회지
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• 제18권1호
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• pp.25-29
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• 2003

리스테리아의 p60단백질은 Listeria속 고유의 단백질로써, 주요 세포외 단백질이기에, 식품에서 이 세균의 존재를 밝혀주는 중요한 지시단백질로 사용된다. 본 연구는 재조합 DNA 방법으로 재조합-p60 단백질을 대장균에서 생산하였으며, 아밀로오스 컬럼 크로마토그라피의 방법으로 정제하여, Listeria spp.의 p60에 특이적 항체를 생산하는 단일클론을 선별하였다. 생산된 항p60 항체 1H4는 병원성 Listeria 균주인 L. monocytogenes, L. ivanovii, L. weishi meri II 특이적으로 강한 항체반응을 보였으며, Listeria속의 비병원성균인 L. innocua등과는 상대적으로 매우 약한 항체반응을 보였으며, 타 세균의 단백질과는 거의 반응하지 않았다. 1H4항체는 복수생산법에 의해 대량생산되었으며, 이 항체의 특이성은 면역학적 방법에 의한 리스테리아 검출키트의 개발에 유용하게 사용될 수 있을 것이다.