• Development and Reproduction
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• v.7 no.2
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• pp.61-68
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• 2003

Zygote genome should entail a complex process of epigenetic reprogramming including a global DNA demethylation to reach a totipotency or pluripotency during early mammalian development. In this study, we have analyzed methylation patterns in cloned bovine embryos to monitor the epigenetic reprogramming process of donor genomic DNA. Aberrant DNA methylation patterns were observed in various genomic regions of cloned embryos except single-copy gene sequences. The overall genomic methylation status of cloned embryos was quite different from that of normal embryos produced in viかo or in vivo. Abnormal methylation profiles were also specifically represented in trophectoderm cells of cloned embryos, which probably result in widespread gene dysregulation in extraembryonic region or placental dysfunction familiar to cloned animals. Our findings suggest that developmental failures of cloned embryos are due to incomplete epigenetic reprogramming of donor genomic DNA. Understanding the epigenetic reprogramming processes of donor genome will clearly define the faulty development of cloned embryos.

• KSBB Journal
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• v.19 no.5
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• pp.341-347
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• 2004

Several gene delivery therapies are being developed for treatment of serum protein deficiency. EPO is one of the most promising therapeutic agent for this treatment which is currently being investigated in depth. This study has the ultimate purpose of improving the gene delivery system for an increase of red blood cell production. A plasmid DNA was constructed smaller than other plasmids for an increase in penetration into animal cells, and two genes were cloned into each vector as a co-delivery system to express erythropoietin, and interluekin-3 or thrombopoietin, which can act on erythroid cell, thus activating hematopoiesis synergically. This co-delivery system has an advantage of decreasing the labour required for industrial production of DNA vaccine. A new plasmid vector, pVAC, in size 2.9 kb, was constructed with the essential parts from PUC 19 and pSectagB, which is much smaller than other plasmid vector and is the size of 2.9 kb. Co-delivery system was constituted by cloning human erythropoietin with each of human interluekin-3 gene or human thrombopoietin gene into both pVAC and pSectagB. As a result, the transfection efficiency of pVAC was higer than that of pSectagB in vitro, and hematocrit level of the mice injected with pVAC is higher than that of other mice. And co-delivery system, made of several plasmid DNAs, was expressed in vitro.

• Journal of Plant Biotechnology
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• v.30 no.4
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• pp.307-313
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• 2003

Fluorescent differential display (FDD) is a method for identifying differentially expressed genes in eukaryotic cells. The mRNA FDD technology works by systematic amplification of the 3' terminal regions of mRNAs. This method involve the reverse transcription using anchored primers designed to bind 5'boundary of the poly A tails, followed by polymerase chain reaction (PCR) amplification with additional upstream primers of arbitrary sequences. The amplified cDNA subpopulations are separated by denaturing polyacrylamide electrophoresis. To identify the genes involved in the development of first trap leaf, we applied a FDD method using mRNAs from leaf base, first trap leaf and flower tissue, respectively. We screened several genes that expressed specifically in first trap leaf. Nucleotide sequence analysis of these genes revealed that these were protease inhibitor (PI), myo-inositol-1-phosphate synthase and lipocalin-type prostaglandin D synthase. Northern blot analysis showed that these genes were expressed specifically in first trap leaf (in vivo and in vitro). FDD could prove to be useful for simultaneous scanning of transcripts from multiple cDNA samples and faster selection of differentially expressed transcripts of interest.

• Journal of Microbiology and Biotechnology
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• v.20 no.12
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• pp.1689-1695
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• 2010

Pantothenate kinase (PanK) catalyzes the first step in the biosynthesis of the essential and ubiquitous cofactor coenzyme A (CoA) in all organisms. Here, we report the identification, cloning, and characterization of panK-sp from Streptomyces peucetius ATCC 27952. The gene encoded a protein of 332 amino acids with a calculated molecular mass of 36.8 kDa and high homology with PanK from S. avermitilis and S. coelicolor A3(2). To elucidate the putative function of PanK-sp, it was cloned into pET32a(+) to construct pPKSP32, and the PanK-sp was then expressed in E. coli BL21(DE3) as a His-tag fusion protein and purified by immobilized metal affinity chromatography. The enzyme assay of PanK-sp was carried out as a coupling assay. The gradual decrease in NADH concentration with time clearly indicated the phosphorylating activity of PanK-sp. Furthermore, the ca. 1.4-fold increase of DXR and the ca. 1.5-fold increase of actinorhodin by in vivo overexpression of panK-sp, constructed in pIBR25 under the control of a strong $ermE^*$ promoter, established its positive role in secondary metabolite production from S. peucetius and S. coelicolor, respectively.

• Journal of Life Science
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• v.19 no.5
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• pp.566-572
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• 2009

A recombinant plasmid, pDH3, was obtained from the genomic library of Serattia marcescens KCTC 2172, and several recombinant subclones constructed from pDH3. The nucleotide sequence of a 5,137 bp segment, pPH4, was determined and three open reading frames were detected. The three ORFs encoded the phosphate specific transport (pst) operon, which was pstC, pstA, and pstB, with the same direction of transcription. Comparison of the pst operon of S. marcescens with that of other organisms revealed that the genes for pstS and phoU were missing. A potential CRP bonding site and pho box sequence was found in the upstream of the putative promoter at the regulatory region. Analysis of the nucleotide sequence showed that homology in amino acid sequences between the PstC protein and Yersinia sp., Vibrio sp., and Pseudomonas sp. were 49, 37 and 33%, respectively. The PstA protein and Yersinia sp., Vibrio sp., and Pseudomonas sp. showed homologies of 64, 51, and 47%, respectively. PstB protein and Methanocaldococcus sp., E. coli, and Mycoplasma sp. showed homologies of 60, 50, and 48%, respectively. The pst genes could be expressed in vivo and positively regulated by cAMP-CRP. The E. coli strain harboring plasmid pPH7, with pst genes, increased with the transport of phosphate.

• Proceedings of the Ginseng society Conference
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• 1978.09a
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• pp.43-49
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• 1978

This study was devised to observe the cytotoxic activity of extracts of Panax ginseng root against some cancer cells and to purify the crude extract. Three kinds of cancer cells(leukemic cells L5178Y, HeLa cells and Sarcoma 180 cells) and mouse embryo cells (as normal cells) were used for this study. The ginseng roots were extracted with petroleum ether in soxhlet apparatus, and the crude extracts were purified by the silicic acid column chromatography and thin-layer chromatography methods. The results obtained are summarized as follows; 1. Eight to ten mg of the petroleum ether extract (crude extract) were obtained from 1 g of Panax ginseng root, and its activities per mg were about 1,000 units. 2. Doubling time of the L5178Y cells was increased to two fold by 24 hours incubation in culture medium containing about one ${\mu}g$ of extract per ml, and eight and ten folds higher concentration of ginseng extract were required for the Sarcoma 180 cells and HeLa cells, respectively, than for the leukemic cells(L5178Y) to inhibit the cellular growth to the same degree. 3. When the L5178Y cells were exposed to medium containing various concentration of the extract for 24 hours before initiation of the soft agar cloning procedure, about $99\%$ of the L5178Y cells were killed at concentration of 8 units per ml. 4. The growth rate of mouse embryo cell (as normal cell) was not affected by the culture with media containing various amounts (1.45 to 30.0 ${\mu}g/ml$) of the extract. 5. The crude extract could be purified about four times by silicic acid column chromatography using several solvent systems, and one spot of active compound could be obtained on the thin-layer chromatogram. 6. In the Swiss mice inoculated with Sarcoma 180 cells, a survival time of the experimental group (injection group of active compound) was extended more. 1.5 to 2.0 times than the control group's(no injection group).

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.49-49
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• 2003

본 연구에서는 vesicular stomatitis virus G glycoprotein (VSV-G)를 envelope로 가지는 pantropic retrovirus vector system을 이용하여 재조합 human FSH 유전자가 전이된 형질전환 닭을 생산하고자 하였다. Human FSH $\alpha$ 및 $\beta$ 유전자와 CTP linker는 human pituitary gland cDNA library에서 RT-PCR 방법을 이용하여 cloning하였으며, 각각의 fragment는 FSH$\beta$-CTP-FSH$\alpha$ 순서의 단일사슬로 연결하였다. 연결된 FSH$\beta$-CTP-FSH$\alpha$는 retroviral vector 내의 $\beta$-actin promoter의 조절 하에 도입한 후, PT67 packaging cell line에 transfection하여 virus를 생산하였으며 생산된 virus는 pantropic한 virus producing cell인 GP293에 infection하여 FSH 유전자가 도입된 virus를 생산하였다. FSH 유전자의 발현을 in vitro에서 확인하기 위하여 CHO (chinese hamster ovary) 세포에 virus를 감염시킨 후, 세포의 배양액을 취하여 electrochemilumine-scence immunoassay 방법으로 정량하였다. In vitro에서 전이 후 발현이 확인된 FSH 외래유전자의 retroviral vector virus를 초원심분리로 고농축하여 stageX의 계란의 배반엽 층에 주입하였으며, 그 결과 18%의 부화율과 91%의 부화한 닭의 유전자 전이율을 확인할 수 있었다. 전이된 유전자의 확인은 FSH$\beta$와 Neo 유전자에 대한 primer를 이용한 RT-PCR의 방법을 이용하였다. In vitro에서와는 달리 in vivo에서는 FSH 유전자의 전이는 확인되었으나 발현을 확인하지는 못하였는데, 이는 적은 수의 실험군이 형질전환율에 비해 상대적이지 못하였거나, 외래 유전자인 FSH의 발현에 의한 생리적인 부작용이 유발되어 해당개체가 부화되지 못한 것으로 추정된다. 본 문제점을 해결하기 위하여 실험군의 수를 늘리고 외래 유전자에 대한 controllable expression system이 보완될 필요성이 요구되며, 이러한 점이 해결된다면 높은 유전자 전이율에 기인하여 retrovirus를 이용한 형질전환 방법은 형질전환 가금의 생산에 있어서 매우 효율적이고 주목할 만한 방법으로 사료된다.

• Animal Bioscience
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• v.35 no.9
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• pp.1360-1366
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• 2022

Objective: The present study analyzed the influence of co-transferring embryos with high and low cloning efficiencies produced via somatic cell nuclear transfer (SCNT) on pregnancy outcomes in dogs. Methods: Cloned dogs were produced by SCNT using donor cells derived from a Tibetan Mastiff (TM) and Toy Poodle (TP). The in vivo developmental capacity of cloned embryos was evaluated. The pregnancy and parturition rates were determined following single transfer of 284 fused oocytes into 21 surrogates and co-transfer of 47 fused oocytes into four surrogates. Results: When cloned embryos produced using a single type of donor cell were transferred into surrogates, the pregnancy and live birth rates were significantly higher following transfer of embryos produced using TP donor cells than following transfer of embryos produced using TM donor cells. Next, pregnancy and live birth rates were compared following single and co-transfer of these cloned embryos. The pregnancy and live birth rates were similar upon co-transfer of embryos and single transfer of embryos produced using TP donor cells but were significantly lower upon single transfer of embryos produced using TM donor cells. Furthermore, the parturition rate for TM dogs and the percentage of these dogs that remained alive until weaning was significantly higher upon co-transfer than upon single transfer of embryos. However, there was no difference between the two embryo transfer methods for TP dogs. The mean birth weight of cloned TM dogs was significantly higher upon single transfer than upon co-transfer of embryos. However, the body weight of TM dogs did not significantly differ between the two embryo transfer methods after day 5. Conclusion: For cloned embryos with a lower developmental competence, the parturition rate and percentage of dogs that remain alive until weaning are increased when they are co-transferred with cloned embryos with a greater developmental competence.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.30 no.6
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• pp.984-991
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• 1997

New tumor suppressor gene, snm23, homologous to human nm23/NDP kinase (human nucleoside diphosphate kinase) gene whose product has a tumor metastasis inhibitory activity, was first cloned from Korean tiger shark (Scyliorhinus forazame) skin cDNA library constructed by using a $\lambda$ ZAP-II cDNA synthesis kit. About $1\times10^5$ plaques were screened and several positive plaques were isolated and confirmed by second screening. The phagemid containing a positive clone from the Uni-Zap XR vector was excised in vivo and the gene containing the tumor metastasis suppressor protein was named as snm23. Cloned gene, snm23, was sequenced with ABI-PRISM 310 Genetic Analyzer. The nucleotide and deduced amino acid sequences of snm23 have shown an open reading frame consisting of 450 base pairs that correspond to a protein of 150 amino acid residues, with a calculated molecular mass of 16.8 kDa. Sequence comparison of snm23 with human nm23/NDP kinase was performed by using Blast protein data base of National Center for Biotechnology Information. In order to determine tissue specificity, reverse transcription-polymerase chain reaction (RT-PCR) was used. Good expression level of snm23/NDP kinase was detected at the tissues from skin, cartilage, and liver of Korean tiger shark.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.117-117
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• 2002

The oocytes from most of animal species accumulate genetic information and other necessary materials during oogenesis for the later use in the early development. Over the years oocyte maturation has been studied extensively both in vitro and in vivo. Particularly, maturation of follicular oocyte in vitro becomes one of the important tools for the studies of basic cell biology, the in vitro technology of animal production, and in particular, the somatic cell cloning by nuclear transfer. We examined meiotic maturation and cumulus expansion in the presence of translation or transcription inhibitors for varying periods of in viかo maturation (IVM) of pig oocyte. In Experiment 1, the results revealed that translation and transcription inhibitors inhibited cumulus expansion and meiotic maturation during 35h of IVM. However, 50 to 60% of the oocytes underwent nuclear maturation without cumulus expansion during 75h of IVM. The rest of the oocytes were arrested at metaphase I (40-50%) in the presence of the inhibitors. In Experiment II, the OCCs were exposed to the drugs only for 15h to examine translation and transcription inhibitors on cumulus expansion and meiotic maturation. Transcription inhibitors for 15h did not arrest meiotic maturation when the oocytes were cultured for subsequent, necessary period of IVM, whereas cumulus expansion was completely inhibited, suggesting that initial 15h is critical transcription activity far cumulus expansion. Translation inhibitors for 15h exposure did not alter cumulus expansion and meiotic maturation during subsequent culture in the absence of the drugs. In Experiment III, the OCCs were exposed to the drugs only for later 30h to examine the influence of transcription and translation inhibitors on oocyte maturation. Interestingly, all meiotic maturation underwent normally with full expansion of cumulus. Similar results were obtained from Experiment IV where 5h of exposure from 15 to 20h of IVM culture to the drugs was performed and subsequently cultured for same period in fresh medium. Taken there results together, both transcription and translation are necessary for nuclear maturation and cumulus expansion, and first 15h IVM for cumulus expansion is critical. The arrested oocytes by the drugs were still capable of undergoing nuclear maturation, although cumulus expansion was affected.