• Mycobiology
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• 제44권2호
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• pp.105-111
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• 2016

Paclitaxel (taxol) has long been used as a potent anticancer agent for the treatment of many cancers. Ever since the fungal species Taxomyces andreanae was first shown to produce taxol in 1993, many endophytic fungal species have been recognized as taxol accumulators. In this study, we analyzed the taxol-producing capacity of different Colletotrichum spp. to determine the distribution of a taxol biosynthetic gene within this genus. Distribution of the taxadiene synthase (TS) gene, which cyclizes geranylgeranyl diphosphate to produce taxadiene, was analyzed in 12 Colletotrichum spp., of which 8 were found to contain the unique skeletal core structure of paclitaxel. However, distribution of the gene was not limited to closely related species. The production of taxol by Colletotrichum dematium, which causes pepper anthracnose, depended on the method in which the fungus was stored, with the highest production being in samples stored under mineral oil. Based on its distribution among Colletotrichum spp., the TS gene was either integrated into or deleted from the bacterial genome in a species-specific manner. In addition to their taxol-producing capacity, the simple genome structure and easy gene manipulation of these endophytic fungal species make them valuable resources for identifying genes in the taxol biosynthetic pathway.

• Journal of Microbiology and Biotechnology
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• 제22권9호
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• pp.1279-1287
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• 2012

We established an efficient transformation method for thermophile Geobacillus kaustophilus HTA426 using conjugative transfer from Escherichia coli of host-mimicking plasmids that imitate DNA methylation of strain HTA426 to circumvent its DNA restriction barriers. Two conjugative plasmids, pSTE33T and pUCG18T, capable of shuttling between E. coli and Geobacillus spp., were constructed. The plasmids were first introduced into E. coli BR408, which expressed one inherent DNA methylase gene (dam) and two heterologous methylase genes from strain HTA426 (GK1380-GK1381 and GK0343-GK0344). The plasmids were then directly transferred from E. coli cells to strain HTA426 by conjugative transfer using pUB307 or pRK2013 as a helper plasmid. pUCG18T was introduced very efficiently (transfer efficiency, $10^{-5}-10^{-3}\;recipient^{-1}$). pSTE33T showed lower efficiency ($10^{-7}-10^{-6}\;recipient^{-1}$) but had a high copy number and high segregational stability. Methylase genes in the donor substantially affected the transfer efficiency, demonstrating that the host-mimicking strategy contributes to efficient transformation. The transformation method, along with the two distinguishing plasmids, increases the potential of G. kaustophilus HTA426 as a thermophilic host to be used in various applications and as a model for biological studies of this genus. Our results also demonstrate that conjugative transfer is a promising approach for introducing exogenous DNA into thermophiles.

• 한국수정란이식학회지
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• 제12권1호
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• pp.91-102
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• 1997

This study was carried out to enhance the efficiency of Korean Native cattle embryos and establish the techniques for producing the twin calves. Bisected embryos without zona pellucida which were divided by simple method not using holding pipette or whole two embryos were transferred to recipients.The pedigrees of monozygotic twin calves produced by transfer of bisected pair embryos were identified. The results obtained were as follows ; The average successful bisection rate was 89.16%. The embryos of blastocyst stage (91.66%) were bisected successfully at significantly (P<0.05) higher rate, compared with the morula stage embryos (86.66%). The average survival rate of bisected embryos following 24 hours culture was 59.02%. The survival rate of morula stage embryos (62.50%) was significantly (P<0.05) higher than that of blastocyst stage embryos (55.5%). For the production of monozygotic twin calves, ten pairs of flesh or frozen demi-em- lymphocytes antigen, the twin calves produced by transfer of bisected pair embryos of Korean Native cattle were identified in pedigrees and confirmed as monozygotes.

• 한국수정란이식학회지
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• 제30권4호
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• pp.305-313
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• 2015

Xenotransplantation of pig islet regarded as a good alternative to allotransplantation. However, cellular death mediated by hypoxia-reoxygenation injury after transplantation disturb success of this technique. In the present study, we produce transgenic pig expressing human heme oxygenase 1 (HO1) genes to overcome cellular death for improving efficiency of islet xenotransplantation. Particularly, Korean miniature pig breed, Micro-Pig, was used in the present study. Somatic cell nuclear transfer (SCNT) technique was used to produce the HO1 transgenic pig. Six alive transgenic piglets were produced and all the transgenic pigs were founded to have transgene in their genomic DNA and the gene was expressed in all tested organs. Also, in vitro cultured fibroblasts derived from the HO1 transgenic pig showed low reactive oxygen species level, improved cell viability and reduced apoptosis level.

• Journal of Computing Science and Engineering
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• 제5권3호
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• pp.257-268
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• 2011

Machine learning and data mining have found many applications in biological domains, where we look to build predictive models based on labeled training data. However, in practice, high quality labeled data is scarce, and to label new data incurs high costs. Transfer and multitask learning offer an attractive alternative, by allowing useful knowledge to be extracted and transferred from data in auxiliary domains helps counter the lack of data problem in the target domain. In this article, we survey recent advances in transfer and multitask learning for bioinformatics applications. In particular, we survey several key bioinformatics application areas, including sequence classification, gene expression data analysis, biological network reconstruction and biomedical applications.

• IMMUNE NETWORK
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• 제6권4호
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• pp.192-198
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• 2006

Background: Investigating strategy to enhance efficiency of gene transfer via adenovirus is critical to sustain gene expression in targeted cells or tissues to regulate immune responses. However, the use of adenovirus as a gene delivery method has been limited by the native tropism of the virus. In this study, the critical parameter is to improve the efficient binding of viral particles to the plasma membrane prior to cellular uptake. Methods: Human immunodeficiency virus (HIV-1) trans-acting activator of transcription (TAT), a protein transduction domain, was fused to the ectodomain of the coxsackie-adenovirus receptor (CAR). The CAR-TAT protein was produced from a Drosophila Schneider 2 cells (S2) transfected with CAR-TAT genes. The function of CARTAT was analyzed the efficiency of adenoviral gene transfer by flow cytometry, and then immunizing AdVGFP with CAR-TAT was transduced on dendritic cells (DCs). Results: S2 transfectants secreting CAR-TAT fusion protein has been stable over a period of 6 months and its expression was verified by western blot. Addition of CAR-TAT induced higher transduction efficiency for AdVGFP at every MOI tested. When mice were vaccinated with DC of which adenoviral transduction was mediated by CAR-TAT, the number of IFN-${\gamma}$ secreting T-cells was increased as compared with those DCs transduced without CAR-TAT. Conclusion: Our data provide evidence that CAR-TAT fusion protein enhances adenoviral transduction and immunogenecity of transgenes on DCs and may influence on the development of adenoviral-mediated anti-tumor immunotherapy.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.56-56
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• 2001

The exogenous gene transfer by intracytoplasmic sperm injection (ICSI) procedure has been recently used to produce transgenic mice and pigs. Sperm-mediated DNA transfer has the potential to markedly simplify the generation of transgenic animals. This method may serve as an alternative to the pronucleus injection of DNA for the production of transgenic pigs. Therefore, in this study, we investigated the expression of transgene after co-injection of spermatozoon or sperm head with green fluorescent protein (GFP) gene into in vitro matured porcine oocytes. Spermatozoon and sperm head, that was obtained by sonication, were treated with 0.03% Triton X-100 to remove the membrane. They were preincubated with linearized pEGFP-N1 for 1 min, and then embryos cultured NCSU23 medium for 2.5 days after co-injected of sperm and DNA. We monitored expression of GFP in embryos under epifluorescent microscope. The remove of sperm membrane did not alter the developmental competence of embryos after ICSI. At 7 days following injection, the rates of blastocysts following injection of intact sperm (15.0%), and of sperm with disrupted membrane (14.2%) were higher than that following IVF (10.0%). Porcine oocytes injected with sperm which co-cultured with DNA concentration of 1, 0.1, and 0.01 ng were 60, 65.7 and 75% and 18.5, 37.4 and 22.2% for rates of cleavage and GFP expression, respectively. In vitro matured porcine oocytes injected with sperm and isolated sperm head resulted in 69 and 59.7% of cleavage rates, respectively The rates of embryo GFP expressed did not significantly different between sperm (20.4%) and sperm head (20.0%) injection. The transgenic embryos with the clusters of positive blastomeres were observed under fluorescent microscope. Most of embryos expressed GFP gene showed mosaicism. They showed GFP expression at 1/4, 2/4 and 3/4 of blastomeres at the 4-cell stage. Among these 4-cell embryos, the expression rate of 1/4 blastomere group (54.6%) was higher than the other groups (15.3-30.7%). These results indicate that membrane disrupted sperm could attach with exogenous DNA, and that this procedure may be useful to introduce foreign gene into porcine oocytes. Therefore, our data suggest that the ICSI car be a useful tool to efficiently produce transgenic pig as well as other mammals.

• 한국미생물·생명공학회지
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• 제32권3호
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• pp.199-205
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• 2004

Bifidobacterium longum SJ32 균주로부터 cloning한 sucrose phosphorylase 유전자를 포함하는 8.6 kb의 EclRI 단편의 염기서열을 결정하였다. 5개의 open reading frame이 존재하였고, 상동성 검색의 결과, sucrose대사에 관여하는 sucrose phosphorylase (ScrP), sucrose transporter (ScrT), GalR-LacI-type transcriptional regulator (ScrR) 유전자의 존재를 확인하였다. SJ32 균주의 scrPTR 유전자군은 다른 B. longum균주의 scrPTR과 배열이 동일하고, 각 유전자 산물이 아미노산 수준에서 94%이상의 상동성을 가지고 있지만, 주변 유전자는 다르게 나타나 B. longum균주 간의 scrPTR 유전자군의 horizontal transfer를 추정하게 한다. 대장균에서의 scrP와 scrT의 동시 발현은 세포 내로의 sucrose 유입을 증가시켜 sucrose phosphorylase의 활성 증가에 영향을 주는 것으로 나타나, scrT가 sucrose transporter유전자임을 뒷받침한다. 기존에 보고된 B. longum NCC2705균주의 유전체로부터 scrPTR외에도 sucrose대사에 관여하는 다양한 sucrose multiple transport system에 관여하는 유전자의 존재가 확인되어, B. longum이 다양한 sucrose유입체계를 보유하고 있음이 추정된다.

• 한국수정란이식학회지
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• 제30권2호
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• pp.99-107
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• 2015

The aim of the present study was to identify coat color associated genes that are differentially expressed in mature Korean brindle cattle (KBC) with different coat colors and in Hanwoo cows. KBC calves, before and after coat color appearance, were included. Total cellular RNA was isolated from the tail hair cells and used for microarray. The number of expressed coat color associated genes/probes was 5813 in mature KBC and Hanwoo cows. Among the expressed coat color associated genes/probes, 167 genes were the coat color associated genes listed in the Gene card database and 125 genes were the pigment and melanocyte genes listed in the Gene ontology_bovine database. There were 23 genes/probes commonly listed in both databases and their expressions were further studied. Out of the 23 genes/probes, MLPH, PMEL, TYR and TYRP1 genes were expressed at least two fold higher (p<0.01) levels in KBC with brindle color than either Hanwoo or KBC with brown color. TYRP1 expression was 22.96 or 19.89 fold higher (p<0.01) in KBC with brindle color than either Hanwoo or KBC with brown color, respectively, which was the biggest fold difference. The hierarchical clustering analysis indicated that MLPH, PMEL, TYR and TYRP1 were the highly expressed genes in mature cattle. There were only a few genes differentially expressed after coat color appearance in KBC calves. Studies on the regulation and mechanism of gene expression of highly expressed genes would be next steps to better understand coat color determination and to improve brindle coat color appearance in KBC.

• Reproductive and Developmental Biology
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• 제35권1호
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• pp.67-75
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• 2011

The faulty regulation of imprinting gene lead to the abnormal development of reconstructed embryo after nuclear transfer. However, the correlation between the imprinting status of donor cell and preimplantation stage of embryo development is not yet clear. In this study, to determine this correlation, we used the porcine spermatogonial stem cell (pSSC) and fetal fibroblast (pFF) as donor cells. As the results, the isolated cells with laminin matrix selection strongly expressed the GFR ${\alpha}$-1 and PLZF genes of SSCs specific markers. The pSSCs were maintained to 12 passages and positive for the pluripotent marker including OCT4, SSEA1 and NANOG. The methylation analysis of H19 DMR of pSSCs revealed that the zinc finger protein binding sites CTCF3 of H19 DMRs displayed an androgenic imprinting pattern (92.7%). Also, to investigate the reprogramming potential of pSSCs as donor cell, we compared the development rate and methylation status of H19 gene between the reconstructed embryos from pFF and pSSC. This result showed no significant differences of the development rate between the pFFs ($11.2{\pm}0.8%$) and SSCs ($13.3{\pm}1.1%$). However, interestingly, while the CTCF3 methylation status of pFF-NT blastocyst was decreased (36.3%), and the CTCF3 methylation status of pSSC-NT blastocyst was maintained. Therefore, this result suggested that the genomic imprinting status of pSSCs is more effective than that of normal somatic cells for the normal development because the maintenance of imprinting pattern is very important in early embryo stage.