• Reproductive and Developmental Biology
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• 제32권2호
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• pp.135-140
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• 2008

Recent studies on nuclear transfer and induced pluripotent stem cells have demonstrated that differentiated somatic cells can be returned to the undifferentiated state by reversing their developmental process. These epigenetically reprogrammed somatic cells may again be differentiated into various cell types, and used for cell replacement therapies through autologous transplantation to treat many degenerative diseases. To date, however, reprogramming of somatic cells into undifferentiated cells has been extremely inefficient. Hence, reliable markers to identify the event of reprogramming would assist effective selection of reprogrammed cells. In this study, a transgene construct encoding enhanced green fluorescent protein (EGFP) under the regulation of human Oct4 promoter was developed as a reporter for the reprogramming of somatic cells. Microinjection of the transgene construct into pronuclei of fertilized mouse eggs resulted in the emission of green fluorescence, suggesting that the undifferentiated cytoplasmic environment provided by fertilized eggs induces the expression of EGFP. Next, the transgene construct was introduced into human embryonic fibroblasts, and the nuclei from these cells were transferred into enucleated porcine oocytes. Along with their in vitro development, nuclear transfer embryos emitted green fluorescence, suggesting the reprogramming of donor nuclei in nuclear transfer embryos. The results of the present study demonstrate that expression of the transgene under the regulation of human Oct4 promoter coincides with epigenetic reprogramming, and may be used as a convenient marker that non-invasively reflects reprogramming of somatic cells.

• Fisheries and Aquatic Sciences
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• 제4권1호
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• pp.39-46
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• 2001

To examine the effect of copy number-dependent transgenic genotype on the expression of foreign gene, stable hemizygous and homozygous transgenic breeding line was established using artificial parthenogenesis. For this purpose, induced diploid gynogenetic transgenesis was optimized in mud loach (Misgurnus mizolepis) using UV-irradiated cyprinid loach (M. anguillicaudatus) sperm and thermal shocks. Optimum UV range for inactivation of cyprinid loach sperm was between 3,150 to $4,050\;ergs/mm^2$ The UV-irradiated sperm were inseminated into eggs from recessive color strain (yellow) or heterozygous transgenic mud loach containing CAT gene. Cold shock at $2^{\circ}C$ for 60 min, 5 min post fertilization successfully restored the diploidy of eggs inseminated with UV-irradiated sperm. Restoration to diploidy was confirmed by flow cytometry and gynogenetic status was verified by examining maternal exclusive inheritance of multi-locus DNA fingerprints, body color and transgenic marker. Putative isogenic transgenic fish clearly showed homozygous status at trans gene locus based on Southern blot hybridization and progeny testing. Further, such homozygous gynogenetic diploids revealed the increased levels of transgene expression, when compared to those of heterozygous (hemizygous) transgenic fish.

• 한국가축번식학회지
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• 제20권1호
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• pp.19-26
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• 1996

본 연구는 체외에서 생산된 돼지수정란에 외래유전자를 미세주입후 체외 배 발달과 유전자 발현을 조사하기 위하여 실시하였다. 체외수정후 18∼20 시간 사이에 LacZ 유전자와 산양 성장호르몬 유전자를 미세주입하였으며, 체외 배 발달율과 유전자 발현은 미세주입후 9일간 체외배양을 실시한 다음 조사하였다. 돼지수정란을 원심분리하여 전핵을 관찰한 결과 60.3%의 난자에서 전핵이 가시화되었다. 또한 유전자가 미세주입된 수정란중 상실배와 배반포까지 발달한 비율은 각각 8.6, 9.1%로 대조구의 발달율 19.0, 20.8%보다 유의하게 낮았다. 그러나, NCSU23 배양액에 4일간 배양후 EMEM 배양액으로 교체하여 배양한 결과, 배반포 및 부화배반포까지 높은 발달율(19.4%)을 나타내었다. X-gal 염색의 결과로서, LacZ의 발현을 나타낸 수정란의 비율은 상살배, 배반포 단계에서 40.0, 42.9%로 나타났으나, 이들 형질전환 수정란의 대부분은 mosaic 현상이 관찰되었다. 또한 PCR 부분에서, gGH 유전자가 도입된 수정란의 비율은 상실배, 배반포단계에서 45.0, 44.4%로 X-gal 염색의 결과와 유의한 차이가 없었다. 따라서 본 실험에서 얻어진 결과들은 체외에서 생산된 돼지수정란은 미세주입후에도 배반포 및 부화배반포까지 성공적으로 발달할 수 있다는 것을 입증하였다. 또한 체외성숙, 수정된 돼지수정란을 이용하여 형질전환 돼지 생산의 가능성을 시사하고 있다.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2000년도 추계학술대회 및 정기총회
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• pp.27-33
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• 2000

• 한국산림과학회지
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• 제84권1호
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• pp.77-86
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• 1995

임목을 대상으로 삽입된 외래 유전자의 공간적, 시기별 발현 특성을 이해하기 위한 기초연구로서 온실에서 생육 중인 형질전환된 2년생 잡종 포플러 (Populus alba X P. grandidentata) Hansen 클론을 대상으로 삽입된 외래 유전자의 발현정도를 각 기관별로 조사하였다. Agrobacterium binary vector pRT45, pRT102 및 pRT104에 의해서 형질전환된 3계통의 형질전환체 Tr15, Tr345, Tr665 모두는 선발가능한 표식 유전자로서 nos promoter-NPT II 유전자가 대상 식물체의 genome에 삽입되어 있으며, 그외에, pin2 promoter-CAT 유전자(pRT45), nos promoter-PIN2 유전자(pRT102), Cauliflower Mosaic Virus 35s promoter-PIN2 유전자(pRT104)가 3계통의 형질전환체에 제각각 삽입되어 있는 잡종 포플러이다. 이들 3계통의 형질전환 포플러 식물체의 DNA를 PCR 검정 기법을 이용하여 분석해 본 결과 선발 가능한 표식 유전자인 NPT-II가 삽입되어 있음이 입증되었다 발현 정도를 비교 분석하기 위해서 NPT-ELISA 검정을 실시하였다. 삽입된 NPT II 유전자는 형질전환된 포플러의 잎, 엽병, 형성층 조직, 줄기의 목질부, 뿌리에서 발현되었으며, 발현 정도는 형질전환된 식물체의 계통에 따라서, 그리고 형진전환된 식물체의 부위에 따라서 다양하게 나타났다. pRT45에 의해서 형질전환된 Tr15 형질전환체의 경우, 늙은 잎과 엽병에서 NPT II 유전자가 가장 높은 수준으로 발현되었으며, 어린 잎과 뿌리 조직에서 가장 낮게 발현되었다. 삽입된 외래 유전자가 각 식물체간에, 각 기관에 따라서 각각 상이한 발현 정도를 나타내는 이와 같은 결과는 형질전환된 식물체에 대한 효과적인 선발과정이 요구됨을 의미함은 물론이고, 형질전환 식물체의 발달 과정에 따라서 삽입된 외래 유전자가 공간적, 시기적으로 각각 다르게 발현할 수 있다는 것을 나타낸다.

• Reproductive and Developmental Biology
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• 제33권1호
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• pp.63-69
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• 2009

In this study we tried to construct a more efficient tetracycline-inducible gene expression system by replacing previous key elements with more advance ones. At the beginning, we substituted PGK (phophoglycerate kinase) promoter for CMV (cytomegalovirus) promoter to control "$rtTA2^sM2$" which has been known for high induction efficiency in response to tetracycline. With this modification, expression of the EGFP marker gene under the induction condition was significantly increased. Next, we replaced "TRE" fragment with a modified version named "TRE-tighf" which has been reported to have higher affinity and specificity to the transactivator by minor base change of the "TRE" DNA fragment sequence. Use of "TRE-tighf" instead of "TRE" resulted in more than 10 fold increment in terms of induction efficiency and significant decrement of background expression in non-inducible condition. By combining PGK promoter and "TRE-tight" fragment, we could upgrade previous tetracycline-inducible system to show more stringent turn on/off gene switch ability and stronger expression of the gene of our interest. Use of this newly developed system must be very helpful to the studies of gene expression, especially to the transgenic animal study in which non-controllable constitutive expression of the transgene has been one of the urgent problems to be solved.

• 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.

• 한국가축번식학회지
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• 제18권2호
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• pp.133-139
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• 1994

Two human lactoferrin expression vectors(pCChcLf and pCChcLf-1) were constructed using rat $\beta$-casein gene and human lactoferrin cDNA. The recombinant DNAs containing human lactoferrin cDNA were microinjected into the fertilized eggs of hybrid mice (BDF1 : C57BL$\times$DBA) and the DNA-injected eggs were treansferred into the oviducts of foster mothers. Genomic DNAs were isolated from the tails of mice born from the microinjected eggs and analyzed by Southern blot analysis. As a result, 5 and 9 transgenic mice with CChcLf and CChcLf-1 gene were produced, respectively. To determine tissue-specificity of transgene expression, Northern blot analysis was performed. Female transgenic mice were killed at day 10 of lactation and total RNAs from various tissues were isolated. Based on Northern blot analysis, it was shown that transgene was mainly expressed in the mammary glands of transgenic mice. In addition, the human lactoferrin in milk was detected by enzyme-linked immunosorbent assay. For this study, milk was obtained from the mammary glands of the transgenic mice at day 10 of lactation. In line #2 of CChcLf and line #7 of CChcLf-1 transgenic mice, human lactoferrin was secreted into the milk at concentration levels of 340ng/ml and 60ng/ml, respectively.

• Journal of Plant Biotechnology
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• 제40권1호
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• pp.49-54
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• 2013

C3HC4-type RING zinc finger proteins essential in the regulation of plant processes, including responses to abiotic stresses. We previously isolated and examined the C3HC4-type RING zinc finger protein (BrRZFP1) from Brassica rapa under abiotic stresses. To elucidate the role of the BrRZFP1 transcription factor in gene regulation, we transformed tobacco plants with the BrRZFP1 gene. Plants were regenerated from 82 independently transformed callus lines of tobacco and analysed for transgene expression. Transgene integration and expression was confirmed by Southern and RT-PCR analyses, respectively. T2 plants displayed more tolerance to the bacterial pathogens Pectobacterium carotovorum and Ralstonia solanacearum, and the tolerance levels were correlated with BrRZFP1 expression levels. These results suggest that the transcription factor BrRZFP1 is an important determinant of stress response in plants and its overexpression in plants could increase biotic stress resistance.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.54-54
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• 2003

The activation of protooncogenes or the inactivation of their gene products may be a specific and effective functional study for human neoplasia. To examine this possibility, we have used the tetracycline regulatory system to generate transgenic mice that conditionally express the HccR-2 protooncogene in vivo. The new human cervical cancer protooncogene (HccR-2) was detected from cervical cancer cell line. To elucidate its biological functions, we generated transgenic mice that expressed the HccR-2 gene. The sustained expression of the HccR-2 transgene culminated chronic neutrophilic leukemia (CNL). CNL is a rare chronic myeloproliferative disorder that presents as a sustained, mature neutrophilic leukocytosis with few or no circulating immature granulocytes, the absence of peripheral blood monocytosis, basophilia, or eosinophilia, and infiltration of neutrophils at the liver, spleen and kidney. Mice expressing the HccR-2 and tetracycline-transactivating protein (tTa) transgene were found to have altered myeloid development that was characterized by increased percentages of mature neutrophil and band form neutrophil in the peripheral blood, liver and spleen. Activation of the transgene causes CNL. In our model, expression of HccR-2 transgene mice was similar in many respects to the human CNL. This model will be valuable not only for investigating the biological properties of the HccR-2 and other protooncogenes in vivo but also for analyzing the mechanism involved in the progression of CNL.