• BMB Reports
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• v.31 no.4
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• pp.413-417
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• 1998

Transgenic mice containing a bovine ${\beta}-Casein/Human$ lysozyme fusion gene (pBZ) were generated in order to produce human lysozyme in their milk. The expression vector was a quadripartite fusion consisting of a 2 kb upstream DNA of the bovine ${\beta}-casein$ gene, human lysozyme gene, intron II of the rabbit ${\beta}-globin$ gene, and the polyadenylation/termination signals of SV40 DNA. Fertilized mouse zygotes were microinjected with pBZ, then transferred into the oviduct of foster mothers. Out of 20 mice born, 11 survived until postweaning and three were identified as positivetransgenic by Southern blot analysis (one male and two females). The founder mice were mated to BCFl mice to produce transgenic progeny. It was confirmed by RT-PCR and Northern blot analyses that the transgene was specifically expressed in the mammary gland of the founder mice. Furthermore, the artificial introns within the transgenic RNA was proven to be correctly spliced out as judged by RT-PCR analysis. These results indicated that transgenic mice generated in this study properly expressed the human lysozyme RNA in their mammary gland.

• Plant Biotechnology Reports
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• v.4 no.3
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• pp.185-192
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• 2010

The Arabidopsis ${\omega}$-3 fatty acid desaturase (AtFAD7) catalyzes the synthesis of trienoic fatty acids (TA). A transgenic tobacco line, T15, was produced by a sense AtFAD7 construct and showed a cosuppression-like phenotype, namely extremely low TA levels. The sequence similarity between AtFAD7 and a tobacco ortholog gene, NtFAD7, was moderate (about 69%) in the coding sequences. AtFAD7 siRNAs accumulated at a high level, and both AtFAD7 and NtFAD7 mRNAs are degraded in T15 plants. The low-TA phenotype in T15 was dependent on a tobacco RNA-dependent RNA polymerase6 (NtRDR6). We also produced tobacco RNAi plants targeting AtFAD7 gene sequences. The AtFAD7 siRNA level was trace, which was associated with a slight reduction in leaf TA level. Unexpectedly, this RNAi plant showed an increased NtFAD7 transcript level. To investigate the effect of translational inhibition on stability of the NtFAD7 mRNAs, leaves of the wild-type tobacco plants were treated with a translational inhibitor, cycloheximide. The level of NtFAD7 mRNAs significantly increased after cycloheximde treatment. These results suggest that the translational inhibition by low levels of AtFAD7 siRNAs or by cycloheximide increased stability of NtFAD7 mRNA. The degree of silencing by an RNAi construct targeting the AtFAD7 gene was increased by co-existence of the AtFAD7 transgene, where NtRDR6-dependent amplification of siRNAs occurred. These results indicate that NtRDR6 can emphasize silencing effects in both cosuppression and RNAi.

• Journal of Plant Biotechnology
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• v.37 no.3
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• pp.275-282
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• 2010

Chloroplast transformation in higher plants offers many attractive advantages over nuclear transformation, including a high-level accumulation of foreign proteins, multi-gene expression in single transformation event via transgene stacking in operons and no position effect due to site-specific integration of transgenes by homologous recombination. Most importantly, chloroplast transgenic plants are eco-friendly because their transgenes are maternally inheritance in most crop plants. However, chloroplast transformation system has limited success in crops alike nuclear transformation. In the past two decades, great progress has been made to overcome the limitations of chloroplast transformation, thus expending chloroplast bioreactor to several important crops including soybean, carrot, lettuce, and oilseed. Therefore, it has become possible that chloroplast transformation of crops can be used not only for the improvement of agronomic traits, but also for the production of vaccines and high valuable therapeutic proteins in pharmaceutical industry.

• Journal of Plant Biotechnology
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• v.47 no.2
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• pp.172-178
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• 2020

Fruit-specific promoters play an important role in the improvement of traits, such as fruit quality through genetic engineering. In tomato, the development of fruit-specific promoters was previously reported, but less attention has been paid to the promoters involved in the fruit development stage. In this study, we characterized the gene expression patterns of tomato alcohol dehydrogenase 2 (SlAdh2) in various tissues of wild-type tomato (cv. Ailsa Craig). Our findings revealed that SlAdh2 expression levels were higher in the developing fruit than in the leaves, stems, and flowers. The ProSlAdh2 region, which is expressed at different stages of fruit development, was isolated from tomato genomic DNA. Following this, it was fused with a β-glucuronidase reporter gene (GUS) and introduced into wild-type tomato using Agrobacterium-mediated transformation to evaluate promoter activity in the various tissues of transgenic tomato. The ProSlAdh2:GUS promoter exhibited strong activity in the fruit and weak activity in the stems, but displayed undetectable activity in the leaves and flowers. Interestingly, the promoter was active from the appearance of the green fruit (1 cm in size) to the well-ripened stage in transgenic tomatoes, indicating its suitability for transgene expression during fruit development and ripening. Thus, our findings suggest that ProSlAdh2 may serve as a potential fruit-specific promoter for genetic-based improvement of tomato fruit quality.

• Toxicological Research
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• v.17
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• pp.293-297
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• 2001

The international pharmaceutical and regulatory communities had been recognizing the limited utility of conventional rodent carcinogenicity study particularly on the second species, mouse, after intense investigation of carcinogenicity data base worldwide, and a new scheme for carcinogenicity testing for pharmaceuticals was proposed at the Expert Working Group on Safety in the International Conference on Harmonization (ICH) in 1996. CB6F 1-Tg rasH2 mouse carrying human prototype c-Ha-ras gene with its own promoter/enhancer is one oj the new carcinogenicity assay model for human cancer risk assessment. Studies have been conducted since 1992 to validate the transgenic (Tg) mice for rapid carcinogenicity test-ing, short term (26 weeks) studies with genotoxic (by Salmonella), non-genotoxic carcinogens, genotoxic non-carcinogens, non-genotoxic non-carcinogens revealed relatively high concordance oj the response of the Tg mouse with classical bioassay across classes of carcinogenic agents. Mechanistic basis for carcinogensis in the model are being elucidated in terms of the role of overexpression and/or point mutation of the transgene. This report review the initial studies of validation of the model and preliminary results of on-going ILSI HESI ACT project will be presented.

• Development and Reproduction
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• v.14 no.1
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• pp.13-19
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• 2010

We developed a novel dicistronic system for the expression of target cDNA sequences in the milk of transgenic animals using goat beta-casein/hGH fusion construct, pGbc5.5hGH (Lee, 2006) and internal ribosome entry site (IRES) sequences of encephalomyocarditis virus (EMCV). Granulocyte colony-stimulating factor (hG-CSF) cDNA was linked to 3' untranslated region of hGH gene in the pGbc5.5hGH via EMCV IRES sequences. Transgenic mice were generated by microinjection and transgene expression was examined in the milk and mammary gland of transgenic mice at 10 days of lactation. Northern blot analysis showed that hGH gene and hG-CSF cDNA were transcribed as a single dicistronic mRNA. The hG-CSF and hGH proteins were independently translated from the dicistronic mRNA and secreted into the milk of transgenic mice. The highest concentration of hG-CSF and hGH in the milk of transgenic mice were $237{\mu}g/m{\ell}$ and $8,990{\mu}g/m{\ell}$, respectively. In contrast, another hG-CSF expression cassette, in which hG-CSF genomic sequences were inserted into a commercial milk-specific expression vector (pBC1), generated a lower level ($91{\mu}g/m{\ell}$) of hG-CSF expression in the milk of transgenic mice. These results demonstrated that the novel pGbc5.5hGH-based dicistronic construct could be useful for an efficient cDNA expression in the milk of transgenic animals.

• Reproductive and Developmental Biology
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• v.28 no.3
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• pp.191-196
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• 2004

In this study, to construct more effective retrovirus system, we compared four internal promoters (RSV: Rous sarcoma virus, UbC: Ubiquitin C, β-actin, CMV: human cytomegalovirus) in the retrovirus vector by measuring GFP (green fluorescent protein) expression. The effect of WPRE (woodchuck hepatitis virus posttrans-criptional regulatory element) sequence on transgene expression was also investigated. Experiments were conducted with cells derived from three different species (human, pig and chicken) and evaluated the activity of each promoter and the effect of WPRE sequence by fluorometry and Western blotting. In all cells tested, RSV and CMV promoters were superior to UbC and β-actin promoters, and RSV promoter was the best one in chicken cells. The boosting effect of WPRE sequence on GFP expression was evident in human and porcine cells but not in chicken cells.

• Reproductive and Developmental Biology
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• v.28 no.3
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• pp.197-202
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• 2004

Human PTH (parathyroid hormone) is known to be efficacious for curing osteoporesis. In this study, we attempted to construct genetically modified porcine cell lines that can ultimately be used for donor cells in nuclear transfer-mediated transgenesis. By using retrovirus vectors carrying tetracycline-regulatory expression system and WPRE (woodchuck hepatitis virus posttranscriptional regulatory element) sequence, we could control PTH expression with tetracycline and boost the promoter activity. Considering that low or constitutive expression of the transgene has been one of major problems that needs to be solved in transgenic animal studies, our results could be helpful in successful production of transgenic pigs as bioreactors.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.77-77
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• 2003

Chimeric animals are referred to as an organism composed of tissues derived from more than one species. In order to examine if a pluripotency of embryonic stem cells can cross the limitation of a species, we tried to establish human-mouse chimeric animals. Human embryonic stem cells were genetically modified to express eGFP using eukaryonic expression vector pcDNA 3.1 (In Vitrogene) for an easy identification. After selection with neomycin, approximately 15 cells were implanted into mouse blastocoele cavity. Ten chimeric blastocysts were transferred to one of the uterine horn of 2.5 days pesudopregnent ICR female. Out of 272 blastocysts transferred to pseudopregnant recipients 20 live newborn were obtained after 20 days. When newborn were obtained, pups were quickly removed immersed into 4% PFA. By histological examination using fluorescent microscope, green fluorescence was observed from the liver, heart, and spleen in newborn mice. Three weeks after born, presence of eGFP sequence within mouse genome (tail and kidney) was reconfirmed by PCR. eGFP sequence was amplified from the progenies of the animal suggesting a genetic transmission of the transgene. These chimeric mice having human cells at the beginning of development, are expected to recognize human cells as “self”, therefore, human cells or tissues will be able to escape the immunological surveillance of the host if grafted into the animal. These animals will serve as a good model system for studying the graft rejection in tissue transplantation and the potential of the cells to work well in many human disease.

• Korean Journal of Animal Reproduction
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• v.27 no.4
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• pp.317-324
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• 2003

The present study were performed to analysis the hematocrit and the red blood cells content into the blood plasma of the transgenic pigs harboring recombinent human erythropoietin gene (rhEPO). Mouse whey acidic protein (mWAP) linked to rhEPO gene was microinjected into pronuclei of porcine one-cell zygotes. After delivered of offspring, PCR analyses identified one mWAP-rhEPO transgenic founder offspring(F$_{0}$). The first generation of transgenic pig (F$_{0}$) harboring mWAP-hEPO appeared to be a male, and the second generation (F$_1$) pigs were made by natural mating of F$_{0}$ with domestic swine, and male and female transgenic pigs (F$_1$) were identified by PCR. The blood samples from transgenic and normal pigs were collected for 50 days during lactation and were counted the red blood cell (RBC) numbers and Hematocrit (HCT) content into the blood. The transgenic pigs expressing rhEPO in their blood gave rise to higher RBC numbers and HCT contents than control animals. rhEPO was secreted both in the blood and milk of genetically engineered pigs harboring rhEPO gene. Therefore, this study provides a model regarding the production of transgenic pig carrying hEPO transgene for biomedical research.earch.