• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.61-61
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• 2002

비 반추동물 특히 돼지의 경우 섬유소 소화율이 극히 낮은 것으로 알려져 있다. 따라서 본 연구는 섬유소분해효소유전자 (CelD) 를 췌장특이 프로모터인 rat Elastase Ⅰ 하류에 연결하여 구축된 벡터를 수정란내 미세주입한 후 생산된 형질전환 돼지를 이용하여 섬유소 소화능력을 측정하기 위하여 실시하였다. 먼저 35두의 수란돈으로 부터 65 두의 산자를 생산하였으며, PCR 분석 결과 4두가 형질전환돼지로 판명되었으며 이중 3두가 죽고, 생존한 l두를 다시 southern blot 분석결과 형질 전환된 것으로 판단하여 같은 복의 6두와 함께 섬유소 소화율을 측정하기 위하여 분리사육을 실시하였다. (중략)

• Journal of Plant Biotechnology
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• v.42 no.3
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• pp.180-185
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• 2015

Leaf discs from 'Yeobong' and 'Maehyang' strawberry plants were used as explants for transformation. The Agrobacterium tumefaciens strain EHA105 harboring the monellin gene under the control of the CaMV 35S promoter was used in co-cultivation experiments. The frequencies of callus formation and plant regeneration from leaf explants after co-cultivation in 'Yeobong' were higher than those of 'Maehyang'. These transgenic plants showed normal growth patterns and flowering. PCR and Southern hybridization confirmed that 1 to 2 copies of the monellin gene were integrated into genome of the transgenic strawberry plants. Northern blot analysis confirm that the transcripts were expressed in transgenic strawberry plants. Although long-term subcultured transgenic strawberry plants showed a phenomenon to escape the transgene, the transformation system established in this study provides new opportunities for genetic improvement of strawberry plants.

• Journal of Plant Biotechnology
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• v.35 no.1
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• pp.87-94
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• 2008

Porcine epidemic diarrhea virus (PEDV) is an infectious and highly contagious virus of swine. In order to develop the transgenic tobacco culture cells producing PEDV antigen protein, four vectors expressing PEDV spike protein (SP) gene under the control of a CaMV 35S promoter were constructed. Four fragments of the SP region of PEDV, SP1 (444 bp, 1487-1930 bp), SP2 (1.7 kb, 2300-3987 bp), SP3 (1.4 kb, 1559-2950 bp), and SP4 (2.6 kb, 9-2643 bp) were amplified by PCR and then C-MYC tag was fused to the end of each SP gene, respectively. These cassettes are inserted into the pCAMBIA2300 (named as 35S::SP1-M, 35S::SP2-M 35S::SP3-M, and 35S::SP4-M, respectively). Tobacco (cv. BY-2) cultured cells were transformed by co-cultivation with Agrobacterium tumefaciens harboring expression vector. We selected kanamycin-resistant calli and checked for the presence of the introduced SP gene using PCR, resulting 70% of them showed the foreign gene. We selected the lines with high-level expression of PEDV antigen protein based on dot blot analysis. Southern blot analysis confirmed that the PEDV SP gene was integrated into the genome of the tobacco cultured cells. Northern blot analysis showed that the introduced gene was highly expressed in transgenic cultured cells. Transgenic tobacco cultured cells-derived antigen induced immunogenicity in mice as determined by a plaque reduction neutralization assay. These results suggest that the vectors expressing PEDV spike protein gene in this study will be useful for the development of transgenic plants and cultured cells producing PEDV antigene protein.

• Korean Journal of Microbiology
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• v.32 no.1
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• pp.34-39
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• 1994

The gene encoding ${\alpha}$-amylase of Schwanniomyces castellii was cloned in Saccharomyces cerevisiae. The 5.0-kilobase insert was shown to direct the synthesis of ${\alpha}$-amylase. Southern blot analysis confirmed that this ${\alpha}$-amylase gene was derived from the genomic DNA of Sch. castellii. Immunoblot analysis showed that ${\alpha}$-amylase production from S. cerevisiae transformant was less than that of donor strain. The ${\alpha}$-amylase secreted from S. cerevisiae transformant was shown to be indistinguishable from that of Sch. castellii on the basis of molecular weight and enzyme properties.

• Journal of Animal Science and Technology
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• v.45 no.2
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• pp.169-182
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• 2003

To understand the structure and regulation of bovine ADRP (Adipocyte Differentiation Related Protein) gene, we have isolated the genomic clone of bovine ADRP and determined its sequence. A genomic Southern blot analysis confirmed that ADRP gene is present as a single copy in bovine genome and the ADRP gene spans 12 kb. Bovine ADRP genomic clone, HwADRPg-1, had 8 exons and 7 introns, and all splicing sites conformed to the GT/AG rule with the exon-intron boundaries located exactly. Analysis of the upstream 649 bp of the sequence of HwADRPg-1 showed that it does not contain any canonical TATAA boxes; however Sp1 binding sites and CAAT boxes are found. The promoter contained potential binding sites for AP-1, AP-2 and several putative transcription factor binding sites. The 5'-flanking region of HwADRPg-1 contained muscle specific transcription activator Myo G and C/EBP (CCAAT/ enhancer binding protein) recognizing site. These results suppose that the Myo G transcription activator regulate the transcription of bovine ADRP gene in muscular tissue and its transcriptional activity was triggered by degree of muscular development. Our results provide the necessary analysis for other flanking sequences are needed in addition to the proximal cis elements of this promoter to confer adipocyte differentiation-dependent or growth-dependent transcriptional control.

• Korean Journal of Plant Resources
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• v.20 no.5
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• pp.389-397
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• 2007

Using the active-increment of Tos17 copies in the genome of Oryza sativa L., there were many studies about induction and selection of new mutants. This study mainly focuses on the induction for retrotransposon(Tos17) activity in the callus of Ilpumbyeo(Oryza sativa L.) according to varied culture period and condition. The objectives of this study are obtaining various mutants($M_1$) through plant regeneration, identification of the mutation relation with Tos17, and subsequent phenotyping of the mutants($M_2,\;M_3$). A total of 371 $M_1$ mutants was obtained. The degree of Tos17 activity obtained regeneration plants with each different culture period was evaluated by Southern blot analysis. The result showed that control Ilpumbyeo rice has 5 numbers of copies and the band numbers obtained 7, 8, 9.5, 12, 6, 13.5, 17.5 from culture period of 1, 2, 3, 5, 6, 7, 8 month, respectively. In this study, the result showed that most effectual culture period for activity of Tos17 in Ilpumbyeo rice is 5 month. Hereafter, collections and analysis of various recombination plants will act on an important factor in multiplication and preservation of $M_2$ and $M_3$ generation. And an urgent and important subject is a development of screening method for selection of diverse mutants.

• Food Science of Animal Resources
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• v.25 no.2
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• pp.218-225
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• 2005

This study was performed to determine sequence variation and RFLP of the mt DNA D-loop region using Southern blot hybridization analysis and to develop mt DNA marker affecting milk production traits in Hanwoo cows. The PCR was used to amplify an 1142 bp fragment within the D-loop region of mt DNA using specific primers. Mt DNA were digested with seven restriction enzymes and hybridized using DIG-labeled D-loop probe. The mt DNA RFLP polymorphisms were observed in the four enzymes, BamHI, RsaI, XbaI and HpaII. Nucleotide substitutions were detected at positions 441 (G/C), 469 (T/C), 503 (C/T), 569 (G/A), 614 (C/A) and 644 (C/T) of the mt DNA D-loop region between two selected lines. Significant relationship between the XbaI RFLP type and breeding value was found(p<0.05). Cows with A type had higher estimated breeding values than those with B type (P<0.05) between high and low milk production lines. Therefore, the RFLP marker of mt DNA could be used as a selection assisted tool for individuals with high milk producing ability in Hanwoo.

• Journal of Plant Biotechnology
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• v.32 no.4
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• pp.243-250
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• 2005

The coat protein gene (AF296280) of the Korean isolate Potato leafroll virus (PLRV) was cloned and the open reading frame (627 bp) was transformed into potato (Solanum tuberosum cv. Superior). Out of seventeen individual transgenic lines, five lines were identified to confer resistance to PLRV through the five generation's selection program in the greenhouse as well as isolated trial field. Successful introduction and genetic stability of coat protein gene in the genome of potato were confirmed by polymerase chain reaction (PCR), Southern blot hybridization and northern blot hybridization. Some of the transgenic lines were highly resistant to PLRV but did not show any resistance to less homologous Potato virus Y (PVY). Our results suggest that the resistance to PLRV is due to homology dependent gene silencing by sense strand coat protein gene. In addition, the results of field test through five generations showed that there were no significant differences comparing to nontransgenic potatoes in the morphological aspect of shoot as well as tuber, Ho remarkable differences were also observed in the major agronomic characters and yields except for the resistance to PLRV.

• Journal of Plant Biotechnology
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• v.32 no.3
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• pp.209-215
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• 2005

Lactoferrin is an iron-binding glycoprotein with many biological roles, including the protection against microbial and virus infection, stimulation of the immune system. We developed the transgenic Siberian ginseng (Acanthopanax senticosus) cell cultures producing the human lactoferrin (hLf) protein following Agrobacterium tumefaciens-mediated transformation. A construct containing a targeting signal peptide from tobacco endoplasmic reticulum fused to hLf cDNA under the control of an oxidative stress-inducible SWPA2 promoter was engineered. Transgenic Siberian ginseng cultured cells to produce a recombinant hLf protein were successfully generated and confirmed by PCR and Southern blot analysis. ELISA and western blot analysis showed that full length-hLf protein was synthesized in the transgenic cells. The production of hLf increased proportionally to cell growth and reached a maximal (up to 3% of total soluble proteins) at the stationary phase. These results suggest that the transgenic Siberian ginseng cultured cells in this study will be biotechnologically useful for the commercial production of medicinal plant cell cultures to produce hLf protein.

• Korean Journal of Veterinary Research
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• v.35 no.3
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• pp.515-530
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• 1995

Coronaviridae에 속하는 transmissible gastroenteritis virus(TGEV)와 porcine epidemic diarrhea virus(PEDV)를 specific하게 detection할 수 있는 방법을 개발하고자 본 연구를 수행하였다. 두 바이러스 모두 RNA 바이러스이기 때문에 reverse transcription-polymerase chain reaction(RT-PCR)으로 nucleocapsid(N) protein gene의 cDNA를 증폭시켰다. SmaI으로 처리한 pTZ19R에 ligation시킨 후 염기서열을 밝히고자 sequencing하였다. 각각의 prototype virus와 비교하여 상동성을 밝혔다. 두 바이러스에 대한 cDNA probe를 제작하여 Southern blot hybridization을 실시하였다. TGEV의 경우 백신주인 P45와 병독주인 Miller strain을 사용하였다. cDNA를 증폭시키기 위해 N1/N1R과 N2/N2R 두 가지 primer를 이용한 결과, N1/N1R primer의 경우 586bp 크기의 PCR product를 얻을 수 있었고, N2/N2R primers로 582bp의 cDNA를 증폭시킬 수 있었다. PEDV 실험을 위하여 PED 임상 증상을 나타내는 분변을 이용하여 RT-PCR을 실시하였다. P2/P2R primer로 753bp의 PCR product를 얻을 수 있었다. TGEV의 두 가지 strain의 N protein gene을 sequencing하여 prototype인 Purdue strain과 염기서열 상동성을 조사한 결과, 97%이상의 높은 homology를 나타내었다. PED-V 역시 N protein gene을 sequencing하여 CV777과 염기서열 상동성을 조사한 결과 97%이상의 homology로 PEDV임을 알 수 있었다. TGEV와 PEDV의 염기서열을 비교한 결과 29%의 낮은 homology를 관찰할 수 있었다. 두 가지 바이러스의 N protein gene에 대한 cDNA probe를 제작하여 Southern blot hybridization을 한 결과, 각 바이러스에 매우 특이적 반응을 나타내었다.