• Journal of Microbiology and Biotechnology
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• v.17 no.1
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• pp.74-80
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• 2007

An enantioselective lipase gene (esf) for the kinetic resolution of optically active (S)-flurbiprofen was cloned from the new strain Serratia marcescens ES-2. The esf gene was composed of a 1,845-bp open reading frame encoding 614 amino acid residues with a calculated molecular mass of 64,978 Da. The lipase expressed in E. coli was purified by a three-step procedure, and it showed preferential substrate specificity toward the medium-chain-length fatty acids. The esf gene encoding the enantioselective lipase was reintroduced into the parent strain S. marcescens ES-2 for secretory overexpression. The transformant S. marcescens BESF secreted up to 217kU/ml of the enantioselective lipase, about 54-fold more than the parent strain, after supplementing 3.0% Triton X-207. The kinetic resolution of (S)-flurbiprofen was carried out even at an extremely high (R,S)-flurbiprofen ethyl ester [(R,S)-FEE] concentration of 500 mM, 130 kU of the S. marcescens ES-2 lipase per mmol of (R,S)-FEE, and 1,000 mM of succinyl ${\beta}-cyclodextrin$ as the dispenser at $37^{\circ}C$ for 12h, achieving the high enantiomeric excess and conversion yield of 98% and 48%, respectively.

• 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.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.55-55
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• 2003

The random insertion of useful gene in genome has been a common method to produce transgenic animals. This method is inefficient for induction of high levels gene expression in transgenic animals. To improve this limit, we tried to develop the system which target the gene at the specific genomic region. Thus, in our experiment, the vector system to target the human thrombopoietin (TPO) gene was developed. Targeting vector including TPO, neo and DT genes was transfrcted into bovine embryonic fibroblasts (bEF) or bovine ear skin fibroblasts (bESF). First of all, we determined concentration of the geneticin (G418) for selection of transfected cell lines. Our results showed that 1200 and 900 $\mu\textrm{g}$/ml of G418 were the most proper for selection of transfscted bEF and bESF cells. In this study, lipofectamine was used as a transfection reagent. Thus, the proper ratio of DNA:lipofectamine for transfection was also required to elevate targeting efficiency in primary mammalian cells. Our result indicates that the most proper ratios of DNA:lipofectamine were 4:2 and 1:2 in bEF and bESF cells. According to the optimized these conditions, single colonies were picked following transfection and were analyzed by PCR. More than 90% of the single colonies have TPO gene. However, there were no colonies with targeted TPO at the specific genomic region. Therefore, further experiments to select the specifically targeted colonies and to find more efficient methods such as reducing selection time and shortening a size of TPO gene are required.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.33-45
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• 2011

Heat shock protein 90 (Hsp90) is ATPase-directed molecular chaperon and affects survival of cancer cell. Inhibitory effect of Hsp90 by inducing cell cycle arrest and apoptosis in the cancer cell was reported. However, its role during oocyte maturation and early embryo development is very insufficient. In this study, we traced the effects of Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), on meiotic maturation and early embryonic development in pigs. We also investigated several indicators of developmental potential, including structural integrity, gene expression (Hsp90-, cell cycle-, and apoptosis-related genes), and apoptosis, which are affected by 17-AAG. Then, we examined the roles of Hsp90 inhibitor on viability of primary cells in pigs. Porcine oocytes were cultured in the NCSU-23 medium with or without 17-AAG for 44 h. The proportion of GV arrested oocytes was significantly different between the 17-AAG treated and untreated group (78.2 vs 34.8%, p<0.05). After completion of meiotic maturation, the proportion of MII oocytes was lower in the 17-AAG treated group than in the control group (27.9 vs 71.0%, p<0.05). After IVF, the percentage of penetrated oocytes was significantly lower in the 17-AAG treated group (25.2%), resulting in lower normal pronucleus formation (2PN of 14.6%). Therefore, the inhibition of meiotic progression by Hsp90 inhibitor played a critical role in fertilization status. Porcine embryo were cultured in the PZM-3 medium with or without 17-AAG for 6 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without 17-AAG. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 17-AAG treated group compared with control (7.5 vs 4.4, respectively). Blastocysts that developed in the 17-AAG treated group had low structural integrity and high apoptotic nuclei than those of the untreated control, resulting in decrease the embryonic qualities of preimplantation porcine blastocysts. The mRNA expressions of cell cycle-related genes were down-regulated in the 17-AAG treated group compared with control. Also, the expression of the pro-apoptotic gene Bax increased in 17-AAG treated group, whereas expression of the anti-apoptotic gene Bel-XL decreased. However, the expression of ER stress-related genes did not changed by 17-AAG. Cultured pESF cells were treated with or without 17-AAG and used for MIT assay. The results showed that viability of pESF cells were decreased by treatment of 17-AAG ($2{\mu}M$) for 24 hr. These results indicated that 17-AAG decreased cell proliferation and increased cell death. Expression patterns Hsp90 complex genes (Hsp70 and p23), cell cycle-related genes (cdc2 and cdc25c) and apoptosis-related genes (Bax and Bcl-XL) were significantly changed by using RT-PCR analysis. The spliced form of pXbp-1 product (pXbp-1s) was detected in the tunicamycin (TM) treated cells, but it is not detected in 17-AAG treated cells. In conclusion, Hsp90 appears to play a direct role in porcine early embryo developmental competence including structural integrity of blastocysts. Also, these results indicate that Hsp90 is closely associated with cell cycle- and apoptosis-related genes expression in developing porcine embryos.