• Korean Journal of Animal Reproduction
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• v.19 no.4
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• pp.283-291
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• 1996

Transgenic animal을 응용할 수 있는 분야에서는 이식유전자의 기능을 정확하게 규명하고 이를 바탕으로 실질적인 유전적인 개량을 이루기 위해서 이식유전자의 발현을 조절할 수 있는 정교한 system이 필요하다. 유전자의 미세주입법에 의해 transgenic animal을 생산할 수 있는데 이용되고 있는 tissue-specific promoter에 의한 이식유전자의 발현조절은 필요로 하는 시기나 양 등을 인위적으로 조절하고자 하는데 한계점을 갖고 있다. 이러한 이식유전자 발현의 문제점을 극복하기 위해 효모의 recombinase나 미생물의 repressor 단백질과 이들의 binding site인 operator sequence를 이용하여 인위적으로 이식유전자의 발현을 조절할 수 있는 system이 개발되고 있다. Cre/loxP system은 site-specific recombination에 의해 DNA sequence를 제거함으로서 이식유전자의 발현을 조절할 수 있다. 이식유전자 발현의 장소와 양을 조절하기 위해서는 미생물이 이용하고 있는 repressor와 이들의 operator sequence를 적용하여 ligand binary system이 개발되었다. Lac repressor system에서는 isopropyl-$\beta$-D-thiogalactoside (IPTG)가 이식유전자 발현을 조절할 수 있는 positive regulator로서 작용하고, tetracycline-VP16 system에서는 tetracycline이나 유사물질들이 negative regulator로서 이용할 수 있다. 이러한 binary system은 transgenic animal에서 이식유전자 발현의 장소와 시기 또한 양을 효과적으로 조절하는데 적용할 수 있는 것으로 나타났다. 따라서 기존의 binary system과 함께 새로운 regulatory system의 장점을 이용하여 보다 완벽한 이식유전자의 인위적인 조절 system을 이룩함으로서 transgenic animal technology의 실용화를 앞당길 것으로 기대된다.

• Korean Journal of Environmental Agriculture
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• v.30 no.1
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• pp.82-88
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• 2011

BACKGROUND: Tetracyclines (TCs) are mainly regulated as parent compounds by bioactivity-based screening methods in food. Especially with respect to antimicrobial residues, their metabolites/epimers are also highly concerning chemicals and traditionally applied microbial detection methods are needed to improve with validation for regulatory control. METHODS AND RESULTS: Detection capability and biological activity of tetracycline (TC), chlortetracycline (CTC), oxytetracycline (OTC) and their epimers; anhydrotetracycline (ATC), epianhydrotetracycline (EATC), epitetracycline (ETC), 4-epi-chlortetracycline (ECTC), 4-epianydrochlotetra-cycline (EACTC), 4-epioxychlortetracycline (EOTC), were measured by microbial growth inhibition screening method of Korea Food Code. CONCLUSION(S): Limited detection capabilities were found, B. megarerium and B. subtilis showed for TC and CTC, and B. subtilis for OTC. Biological potency of each epimer was also presented against various microorganisms, at the level from 50% to 96%, comparing with parent TCs. It is recommended that more advanced microbial screening methods with validation are needed, and biologically active epimers are to be considered as marker residues for MRL setting of regulatory control purpose.

• Korean Journal of Microbiology
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• v.51 no.1
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• pp.39-47
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• 2015

Secondary metabolism in actinomycetes has been known to be controlled by a small molecule, ${\gamma}$-butyrolactone autoregulator, the binding of which to each corresponding receptor leads to the regulation of the transcriptional expression of the secondary metabolites. We expected that expression of an autoregulator receptor or a pleiotropic regulator in a non-host was to be gained insight of effective production of new metabolic materials. In order to study the function of the receptor protein (seaR), which is isolated from Saccharopolyspora erythraea, we introduced the seaR gene to Streptomyces coelicolor A3(2) as host strains. An effective transformation procedure for S. coelicolor A3(2) was established based on transconjugation by Escherichia coli ET12567/pUZ8002 with a ${\varphi}C31$-derived integration vector, pSET152, which contained int, oriT, attP and $ermEp^*$ (erythromycin promotor). Therefore, the pEV615 was introduced into S. coelicolor A3(2) by conjugation and integrated at the attB locus in the chromosome of the recipients by the ${\varphi}C31$ integrase (int) function. Exconjugant of S. coelicolor A3(2) containing the seaR gene was confirmed by PCR and transcriptional expression of the seaR gene in the transformant was analyzed by RT-PCR. In case of S. coelicolor A3(2), a phenotype microarray was used to analyze the phenotype of transformant compared with wild type by seaR expression. After that, in order to confirm the accuracy of the results obtained from the phenotype microarray, an antimicrobial susceptibility test was carried out. This test indicated that sensitivity of the transformant was higher than wild type in tetracycline case. These results indicated that some biosynthesis genes or resistance genes for tetracycline biosynthesis in transformant might be repressed by seaR expression. Therefore, subsequent experiments, analysis of transcriptional pattern of genes for tetracycline production or resistance, are needed to confirm whether biosynthesis genes or resistance genes for tetracycline are repressed or not.

• Reproductive and Developmental Biology
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• v.31 no.3
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• pp.161-167
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• 2007

In this study, we constructed and tested retrovirus vectors designed to express the human thrombopoietin gene under the control of the tetracycline-inducible promoters. To increase the hTPO gene expression at him-on state, WPRE sequence was also introduced into retrovirus vector at downstream region of either the hTPO gene or the sequence encoding reverse tetracycline-controlled transactivator (rtTA). Primary culture cells (PFF, porcine fetal fibroblast; CEF, chicken embryonic fibroblast) infected with the recombinant retrovirus were cultured in the medium supplemented with or without doxycycline for 48hr, and induction efficiency was measured by comparing the hTPO gene expression level using RT-PCR, western blot and ELISA. Higher hPTO expression and tighter expression control were observed from the vector in which the WPRE sequence was placed at downstream of the hTPO (in CEF) or rtTA(in PFF) gene. This resulting tetracycline inducible vector system may be helpful in solving serious physiological disturbance problems which have been a major obstacle in successful production of transgenic animals.

• Journal of Microbiology and Biotechnology
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• v.15 no.4
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• pp.880-886
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• 2005

Using carotenoid genes of Erwinia herbicola, metabolic engineering was carried out for lycopene production with the pAC-LYCO4 plasmid, which was composed of a chromosomal DNA fragment of E. herbicola containing the crtE, crtB, and crtI genes under the control of the tetracycline promoter and the ipi gene of Haematococcus pluvialis with the trc promoter. Plasmid pAC-LYCm4 was constructed for efficient expression of the four exogenous genes using a strong RBS sequence and the same tetracycline promoter. The optimized expression construct of pAC-LYCm4 increased Iycopene production three times as compared with pAC-LYCO4. pAC-LYCm5 containing ispA behind the four exogenous genes was constructed. There was no significant difference in Iycopene production and cell growth between pAC-LYCm4 and pAC-LYCm5. FPP synthase encoded by ispA was not rate-limiting for Iycopene production. Each gene of crtE, crtB, crtI, and ipi was overexpressed, using pBAD-crtE, pBAD-crtIB, and pBAD-ipiHPI, in addition to their expression from pAC-LYCm4. However, there was no increase oflycopene production with the additional overexpression of each exogenous gene. The four exogenous genes appeared to be not rate-limiting in cells harboring pAC-LYCm4. When pDdxs, pBAD24 containing dxs, was introduced into cells harboring lycopene synthetic plasmids, lycopene production of pAC-LYCO4, pAC-LYCm4, and pAC-LYCm5 was increased by 4.7-, 2.2-, and 2.2-fold, respectively. Lycopene production of pBAD-DXm4 containing crtE, crtB, crtI, ipi, and dxs was 5.2 mg/g dry cell weight with $0.2\%$ arabinose, which was 8.7-fold higher than that of the initial strain with pAC-LYC04. Therefore, the present study showed that proper regulation of a metabolically engineered pathway is important for Iycopene production.