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

• The Microorganisms and Industry
• /
• v.16 no.3
• /
• pp.44-47
• /
• 1990

Avery, MacLeod, McCarty가 1944년에 세균에서 유전적 재조합이 이루어진다는 사실을 발견한 후 세균은 진화 학자들과 미생물 학자들로 부터 주목을 받기 시작했다. 왜냐하면 세균은 자연생태계에서 또다른 형태의 유전적 적응성을 지니고 그러한 돌영변이 중 일부는 모세포(parent cell)보다도 주변환경에 더 잘 적응되었기 때문이다. 이제까지 GEM들이 생태계에서 유전자들 전이시키는 빈도가 대부분 낮았고, 토양이나 다른 자연생태계애서 유전자의 전이가 지속적으로 일어난다는 실험적 증거는 없었지만 이들을 생태계에 방출한 결과 유전자 전이가 몇몇 실험에서 확인된 적이 있어 토양에서의 유전적 전이의 가능성을 강하게 암시하고 있다. 하지만 어떻게 전이되고 토양의 물리, 화학및 전이에 필요한 최소한의 donor와 recipient의 수와 정확한 감지 방법등이 아직까지 밝혀져 있지 않은 상태이다. 더우기 토양환경에서 미생물의 활성과 생태, 군집 동태에 영향을 줄 수 있는 기능을 나타내려면 얼마만큼의 재조합 세균이 단위면적당 필요한지도 밝혀지지 않은 상태이다. 따라서 이러한 여러가지 문제점을 밝히는 것은 학문적인면 뿐만 아니라 진화론적인 이론에도 도움이 되며 GEM들이 토양이나 다른 생태계에 유출되었을 때의 환경영향평가나 규제를 하는데에도 도움이 될 수 있다고 본다.

• Korean Journal of Poultry Science
• /
• v.39 no.3
• /
• pp.241-244
• /
• 2012

This study was performed to analyze the generational transmission and the expression of the foreign gene in the GFP transgenic chickens. The transmission rate and the expression of the GFP gene was investigated from the GFP transgenic rooster (G2) as the first founder to the ninth (G8). Analysis of GFP expression in hatched chickens was used the UV lamp. When GFP was expressed in the wings, bill and legs of a chick, the bird only was selected as a transgenic chick. The average transmission rate of the overall transgenic was 38~58%. These results showed that the transmission of the GFP gene in the transgenic chickens in accordance with the laws of Mendel's continues to the next generation without gene silencing.

• Reproductive and Developmental Biology
• /
• v.28 no.3
• /
• pp.203-207
• /
• 2004

The transgenic mice carrying human Interleukin-10 (hIL-10) gene in conjunction with bovine (3 -casein promoter express hIL-10 in milk during lactation. In this study, stability of germ line transmission and expression of hIL-10 transgene integrated into host chromosome were monitored up to generation F8 of transgenic mice. When male mouse of generation F8 was crossbred with normal females, approximately half of offspring (50.9±5.8%) were identified as transgenic mice. Generation F9 to F15 mice also showed similar transmission rates (66.0±20.1%, 61.5±16.7%, 41.1±8.4%, 40.7±20.3%, 61.3±10.8%, 49.2±18.8% and 43.8±25.9%, respectively), implying that hIL-10 transgene can be transmitted stably up to long term generation in the transgenic mice. Expression levels of human IL-10 from milk of generation F9 to F14 mice were 3.6± 1.2 mg/ml, 4.2±0.9 mg/ml, 5.7±1.5 mg/ml, 6.3±3.5 mg/ml, 6.8±4.5 mg/ml and 6.8±3.1 mg/ml, respectively, which was showed high-level expression compared with that of generation F1 (1.6 mg/ml) mice. In conclusion, our results suggest that transgenic mice can be continuously passed their transgenes to the progeny through the breeding program with the same productivity of human IL-10 protein in their milk.

• Journal of Life Science
• /
• v.14 no.6 s.67
• /
• pp.1009-1017
• /
• 2004

Sialyltransferases cloned so far show the remarkable tissue-specific expression, which is correlated with the existence of cell type-specific sialylated sugar structure in glycoconjugates. In the previous studies, we found various mRNA isoforms of human sialyltransferases generated by alternative splicing and alternative promoter utilization. To understand the regulatory mechanisms for specific expression of human sialyltransferase genes and for production of their mRNA isoforms, in this study, we have isolated and characterized five kinds of human sialyltransferase genes: hST3Gal II, hST8Sia II, hST8Sia III, hST8Sia IV, and hST8Sia V. The hST3Gal II gene is composed of six exons, which span over 17kb, with exons ranging in size from 46 to over 1017 bp. The hST8Sia III gene comprises over 10 kb, and consists of only four exons, which is much smaller and simpler than other human sialyltransferase genes. In contrast, three genes (hST8Sia II, hST8Sia IV and hST8Sia V) span more than 70 kb, and comprise five or more exons. All exon-intron boundaries follow the GT-AG rule. In particular, the sialylmotif L, which is a highly conserved region in all cloned sialyltransferases, was found in one exon of hST8Sia III, whereas this motif is encoded by discrete exons in the other human sialyltransferases. Exon structures of these sialyltransferase genes show the structural diversity, as found in other human sialyltransferase genes reported so far. We determined the transcription start site of hST3Gal II gene by the 5'-RACE and cap site hunting experiments.

• Microbiology and Biotechnology Letters
• /
• v.20 no.4
• /
• pp.483-490
• /
• 1992

In order to understand the transfer behavior of a particular gene in water environments, kanamycin resistance ($Km^r$) gene was tracked by Southern hybridization with DNA probe in its conjugal transfer. A $Km^r$ natural bacterial isolate and genetically modified microorganisms (GMMs) constructed from the isolate were used as donor for conjugal transfer of the $Km^r$ gene. The transfer frequencies of the $Km^r$ gene from GMM strains were generally 10 to 100 times higher than those from the natural isolate. The conjugants obtained from GMM strains in LB broth had more plasmids newly appeared, and particularly the conjugants in A Wand FW waters revealed more rearrangement in their plasmids as a function of conjugation time. When plasmids of the conjugants obtained in LB broth were Southern hybridized with DNA probe of the $Km^r$ gene, the $Km^r$ plasmids in the conjugants were detected at the same position of the plasmids in donor cells, in spite of the fact that the plasmids were highly rearranged in conjugant cells. But the $Km^r$ plasmids in the donor of DKI and DKC601, and DKC600 were not identified in the conjugants obtained after 50 h conjugation in AW and after 30 h in AW, respectively. The size of the $Km^r$ plasmids showing hybridization signal were a little changed in the conjugants obtained in A Wand FW waters. Therefore, the method of Southern hybridization with DNA probe was proved to be very specific and useful for tracking of particular genes in water environments.

• Korean Journal of Breeding Science
• /
• v.42 no.5
• /
• pp.540-546
• /
• 2010

This study was conducted to understand the role of soybean pathogenesis-related 10 (GmPR10) gene in salt tolerance and to develop salt-tolerant rice using GmPR10 cDNA. GmPR10 transgene was expressed constitutively in the shoot and root of the $T_1$ transgenic rice plants. Interestingly, however, the levels of the transgene expression were increased temporally up to over four- to five-fold in the shoot and root by 125 mM NaCl treatment, peaking at six hours after the treatment and decreasing thereafter. Electrolyte leakage of leaf cells under 125 mM NaCl treatment was lower in all the transgenic lines than in the control variety, Dongjin-byeo. Ability of seedlings to recover from 125 mM NaCl treatment for two weeks was higher in the transgenic plants than in the control plants. These results demonstrated that GmPR10 had function to increase cell integrity and promote growth under the saline stress imposed by NaCl. The transgenic line GmPR10-3 which showed highest ability to recover from the saline stress could be used as a potential source for salt tolerance in rice breeding programs.

• Journal of Sericultural and Entomological Science
• /
• v.37 no.1
• /
• pp.46-51
• /
• 1995

In order to develope baculovirus expression vector system, we constructed new transfer vector of nuclear polyhedrosis virus of the silkworm, Bombyx mori. The promoter region containing only adenine of translation start codon of polyhedrin gene was cloned by polymerase chain reaction technique. And the 5' and 3' leader regions of polyhedrin gene was sequentially cloned. The polyhedrin coding gene was deleted from the ＋2 to the ＋597 position. As the result, we constructed new transfer vector which has EcoRI, SacI and KpnI sites for the cloning sites of foreign gene. New transfer vector was named as pBmKSKl. Escherichia coli $\beta$-galactosidase gene as foreign gene was inserted into pBmKSKl, under the control of the polyhedrin promoter and expressed in B. mori cells. The result showed that the new transfer vector pBmKSK1 is functional.

• Research in Plant Disease
• /
• v.15 no.3
• /
• pp.202-208
• /
• 2009

The genetically modified leaffolder-resistant (Cry1Ac1) rice plant was evaluated for the changes of resistance by comparing the occurrence of major diseases with a japonica type Korean rice variety, Nakdong which was the mother plant of the transgenic rice event, in greenhouse and field conditions. There was no difference in the occurrence of sheath blight and Helminthosporium blight between the two varieties in the fields. We couldn't find any difference of resistance for fungal blast and bacterial leaf blight by artificial inoculation in greenhouse. There was also no difference in the susceptibility to sheath blight in artificial inoculation tests confirming the results in the fields. The possibility of gene transfer of Bar and Cry1Ac1 from the genetically modified rice plant to naturally infected pathogens such as Fusarium moniliforme and Pyricularia oryzae in the field conditions was tested by PCR. And the possible transfer of those genes by continuous inoculation of Xanthomonas oryzae pv. oryzae and Rhizoctonia solani was also tested. However, we couldn't find any possibility of transfer of the genes in natural and artificial conditions.

• Korean Journal of Plant Resources
• /
• v.19 no.5
• /
• pp.612-616
• /
• 2006

In recent years, there has been increasing concerns in gene flow from GM crops to wild or weedy relatives as a potential risk in the commercialization of GM crops. To access the possibility of the environmental impacts by GM rice, small-scale experiments of gene transfer were carried out. Herbicide and drought stress resistant GM rice and non-GM rice Nakdongbyeo, wild rice Oryza nivara, and weedy rice Sharebyeo were used for artificial pollination experiments and bar gene was used as a tractable marker after pollination. The harvested putative hybrid seeds after artificial pollination were germinated and true hybrid plants were selected by basta treatment. The hybrid plants were verified again by PCR amplification of bar and trehalose-6-phosphate phosphatase (TPP) genes and RAPD PCR analysis.