• 한국수정란이식학회지
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• 제14권1호
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• pp.1-8
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• 1999

The efficiency of transgenic livestock animal production may be improved by early selection of transgenci preimplantation embryos. To examine the possibility of GFP gene as a non-invasive marker for the early screening of transgenic embryo, the GFP gene was microinjected into rabbit zygotes and the later stages of preimplantation embryos were examined for the expression of GFP. The presence of injected DNA was detected by PCR analysis and the expression of GFP was detected by observing green fluorescence in embryos under a fluorescent microscope. Out of 108 GFP gene-injected rabbit zygotes, seventy three(67.6%) were fluorescence-positive. When 11 fluroresecence-positive blastocysts were analyzed for the presence of GFP gene by PCR, 6(54.5%) were positive, and all of the 8 flrouescence-negative blastocysts were also negative by PCR. The results indicate that the screening of transgene in rabbit embryos by PCR analysis and GFP detection could be a promising method for the preselection of transgenic embryos.

• BMB Reports
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• 제39권1호
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• pp.61-67
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• 2006

Molecular co-suppression phenomena are important to consider in transgene experiments. Embryogenic cells were obtained from immature cotyledons and engineered with two different gene constructs (pHV and pHVS) through particle bombardment. Both constructs contain a gene conferring resistance to hygromycin (hpt) as a selective marker and a modified glycinin (11S globulin) gene (V3-1) as a target. sGFP(S65T) as a reporter gene was, however, inserted into the flanking region of the V3-1 gene (pHVS). Fluorescence microscopic screening after the selection of hygromycin, identified clearly the expression of sGFP(S65T) in the transformed soybean embryos bombarded with the pHVS construct. Stable integration of the transgenes was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. Seeds of transgenic plants obtained from the pHV construct frequently lacked an accumulation of endogenous glycinin, which is encoded by homologous genes to the target gene V3-1. Most of the transgenic plants expressing sGFP(S65T) showed highly accumulation of glycinin. The expression of sGFP(S65T) and V3-1 inherits into the next generations. sGFP(S65T) as a reporter gene may be useful to increase the transformation efficiency of transgenic soybean with avoiding gene co-suppression.

• Journal of Microbiology
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• 제42권4호
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• pp.340-345
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• 2004

To investigate the co-expression and crystallization of a fusion gene between the Bacillus thuringiensis crystal protein and a foreign protein in B. thuringiensis, the expression of the Cry1Ac fused with green fluorescent protein (GFP) genes in a B. thuringiensis $Cry^-B$ strain was examined. The cry1Ac gene was cloned in the B. thuringiensis-E. coli shuttle vector, pHT3101, under the control of the native cry1Ac gene promoter, while the GFP gene was inserted into the XhoI site upstream of the proteolytic cleavage site, in the middle region of the crylAc gene (pProAc-GFP). The B. thuringiensis $Cry^-B$ strain carrying pProAc-GFP (ProAc-GFP/CB) did not produce any inclusion bodies. However, the transformed strain expressed fusion protein forms although the expression level was relatively low. Furthermore, an immu­noblot analysis using GFP and Cry1Ac antibodies showed that the fusion protein was not a single spe­cies, but rather multiple forms. In addition, the N-terminal fragment of Cry1Ac and a non-fused GFP were also found in the B. thuringiensis $Cry^-B$ strain after autolysis. The sporulated cells before autolysis and the spore-crystal mixture after autolysis of ProAc-GFP/CB exhibited insecticidal activities against Plutella xylostella larvae. Accordingly, the current results suggest that a fusion crystal protein produced by the transfomant, ProAc-GFP/CB, can be functionally expressed but easily degraded in B. thuring­iensis.

• The Plant Pathology Journal
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• 제24권3호
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• pp.296-304
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• 2008

The transient and rapid expression system of a foreign protein in planta is a very useful technique in biotechnology application. We have investigated optimum condition of Agrobacterium-infiltration technique in which expression level of foreign proteins were maximized without detrimental effects on plants using GFP and Cucumber mosaic virus 2b protein, which is known as an enhancer of gene expression and a suppressor of post-transcriptional gene silencing(PTGS). The optimum expression level of both RNA and protein of GFP with minimum leaf impairment was obtained at $OD_{600}$=0.2 of Agrobactrium inocula. The steady-state levels of GFP RNA and protein generally peaked at 3 and 7 days post-infiltration(dpi), respectively. In the presence of 2b, both the magnitude and duration of GFP expression was highly increased and we could detect GFP level until 17 dpi. On the other hands, the 2b-mediated higher accumulation of foreign proteins resulted in the repression of normal leaf growth, possibly due to the limitation of supply of energy or materials required for growth maintenance. Using this Agrobacterium-infiltration system with 2b and GFP, we tested a hypothesis for the threshold model of PTGS initiation. Four GFP transgenic lines of N. benthamiana, which shows different expression level of GFP were tested to determine the threshold level for PTGS initiation. Agrobacterium-infiltration of GFP into those GFP-transgenic plants resulted in the co-silencing of the transgenic GFP. It was found that very low concentration of Agrobacterium with GFP and GFP+2b($OD_{600}$=0.002-0.02) which could not phenotypically induce an additive GFP expression, was enough to trigger PTGS pathway in all GFP transgenic plants. This strongly indicates that each GFP-transgenic plant should be expressing the transgenic GFP at its own pre-determined level and there was no buffer zone of additive GFP-expression to the threshold. In other words, the PTGS seems to be immediately activated as a self-defensive mechanism if an internal balance of gene expression is broken.

• 한국가금학회지
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• 제39권3호
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• pp.241-244
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• 2012

본 연구는 형질전환 닭에서 주입한 외래 유전자의 세대간 전이와 발현 양상을 조사하고자 하였다. 외래 유전자 전이 양상 조사는 제3세대(G2) GFP 형질전환 수탉을 최초 부계로 사용하여 최종적으로 제9세대(G8) 형질전환 닭을 연속적으로 생산하면서 GFP 유전자 전이 양상을 조사하였다. 형질전환 병아리 유전 분석은 자외선 램프 아래에서 부화한 병아리들의 날개, 부리와 다리에서 녹색형광단백질을 발현하는 병아리들만을 형질전환으로 선발하였다. 형질전환 닭에서 외래 유전자 전이율은 대략 38~58%이었다. 이는 유전자 전이가 멘델의 유전 법칙을 따르고 있음을 보여주고 있다. 연구 결과는 GFP 유전자가 유전자 침묵 없이 멘델의 유전 법칙에 따라 다음 세대로 계속 전이와 발현된다는 것을 보여 주고 있다.

• IMMUNE NETWORK
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• 제4권3호
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• pp.190-197
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• 2004

Background: Rheumatoid arthritis (RA) is an autoimmune disorder characterized by chronic synovial inflammation which leads to joint destruction. Gene therapy of RA targets the players of inflammation or articular destruction. However, viral vectors have safety problems and side effects, while non-viral vectors suffer from inefficient gene transfer and fast loss of gene expression. To overcome the limits of non-vial vectors, an EBV-based plasmid which is known to exert prolonged high level gene expression can be used. Methods: pEBVGFP, pEBVIL-10, and pEBVvIL-10 were constructed by cloning GFP, IL-10, and vIL-10 genes into an EBV-based plasmid, respectively. The pGFP was used as a control plasmid. Each constructs were lipofected into HIG-82 rabbit synoviocytes. The expression of GFP was monitored by FACS and confocal microscopy. IL-10 and vIL-10 expressions were measured by ELISA. Results: GFP expression 2 days after transfection was achieved in 33.2% of cells. GFP-expressing cells transfected with pGFP decreased rapidly from 4 days after transfection and disappeared completely by 11 days. Cells transfected with pEBVGFP began to decrease slowly from 4 days. But GFP expression was detected for over 35 days. In addition, HIG-82 cells transfected with pEBVIL-10 ($44.6{\pm}1.5ng/ml$) or pEBVvIL-10 ($51.0{\pm}5.7ng/ml$) secreted these cytokines at high levels. High level cytokine production by hygromycin selection was maintained at least for up to 26 days after transfection. Conclusion: These results suggest that the EBV-based plasmid has a potential to improve non-viral gene transfer system and may be applicable to treat RA without the drawbacks of viral vectors.

• 한국자원식물학회지
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• 제21권4호
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• pp.249-253
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• 2008

식물의 미토콘드리아에서 eukaryotic 유전자 발현하는 양상을 관찰한다는 것은 매우 중요하게 알여져 왔다. 본 실험에서는 식물의 미토콘드리아에서 발현하는 유전자와 GFP가 미토콘드리아에 발현하는지를 구명하였다. 미토콘드리아(mt)에서 발현 하는 AtBI-1 유전자와 GFP 유전자를 35S promoter를 가진 pBin vector에 재조합한 후, 유전자총을 이용한 형질전환법으로 담배의 잎과 cotyledon에 형질전환하여 재분화 된 shoot를 얻었다. mt에서 그 유전자가 발현 되는 것을 현미경하에서 GFP가 발광하는 것으로 확인하였다. 또한 PCR분석과 Southern분석에서도 미토콘드리아에서 AtBI-1 유전자가 발현함을 확인하였다. 따라서 본 연구의 결과로 mt에 관련된 유전자를 식물의 조직에 형질전환 하여 1개 이상의 유전자가 식물의 mt에 삽입되어 그 유전자의 특성이 발현되는데 이용되어 질수 있을것이라 생각된다.

• Asian-Australasian Journal of Animal Sciences
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• 제13권10호
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• pp.1367-1372
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• 2000

The present study was conducted to evaluate the efficiency of transgenic rabbit production by DNA microinjection using EGFP (Enhanced Green Fluorescent Protein) gene. In this experiment EGFP coding sequences fused to CMV promoter were microinjected into rabbit one-cell embryos, and then GFP expression and gene integration were evaluated in preimplantation embryos and fetuses recovered on day 15 of pregnancy to determine efficiency of transgenic rabbit production. Effect of DNA concentration was also tested on development in vitro following microinjection and transgene integration in fetuses. Development of embryos in vitro was decreased by DNA microinjection, but the rates of pregnancy and implantation were not significantly affected by microinjection. As development progressed in vitro percentage of GFP expression in rabbit embryos was decreased, resulting GFP expression detected in 37.5% of blastocysts. The efficiencies for production of transgenic fetuses were 4.0% and 7.6%, respectively, when $10ng/{\mu}l$ and $20ng/{\mu}l$ of DNA concentration were microinjected. Transgenic fetuses were confirmed by GFP expression and PCR analysis of fetus genomic DNA. These results indicated that DNA microinjection itself damaged embryo development and DNA concentration affected the efficiency of transgenic rabbit production.

• 한국어병학회지
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• 제16권2호
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• pp.69-73
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• 2003

In this study, GFP reporter gene was transfected into a fibroblast cell line RTG-2 using a polycationic transfection reagent (Superfect) and showed a successful expression of GFP. The transfection efficiency by Superfect was compared to the commonly used transfection method, i.e. DNA-calcium phosphate coprecipitatlon. Transfection by Superfect was more effective than calcium phosphate coprecipltation method (frequency of cell expressing orr was 11.3% and 3.5%, respectively). The optimal expression of GFP and {\beta}-galactosidase was observed when $5-6\;{\mu}{\ell}$ of Superfect per ${\mu}g$ DNA was used for transfcction, 1:5-6 ratio between DNA(${\mu}g$) and Superfect ($\mu\ell$).

• 한국어병학회지
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• 제33권2호
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• pp.163-169
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• 2020

Snakehead rhabdovirus (SHRV) is different from other fish novirhabdoviruses such as viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), and hirame rhabdovirus (HIRRV) in that it replicates at high temperatures. Therefore, the delivery of foreign proteins to fish living at high water temperature would be possible by using recombinant SHRVs. In the present study, to evaluate the possible use of SHRV as a vehicle for foreign proteins delivery, we generated a recombinant SHRV that contains an enhanced-GFP (eGFP) gene between nucleoprotein (N) and phosphoprotein (P) genes (rSHRV-A-eGFP), and another recombinant SHRV expressing two heterologous genes by inserting an eGFP gene between N and P genes, and mCherry gene between P and M genes (rSHRV-AeGFP-BmCherry). Epithelioma papulosum cyprini (EPC) cells infected with the recombinant SHRVs showed strong fluorescence(s), suggesting the possible availability of recombinant SHRVs for the development of combined vaccines by expressing multiple foreign antigens.