• Development and Reproduction
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• v.13 no.3
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• pp.191-198
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• 2009

In an attempt to simultaneously produce two human proteins, hGH and hG-CSF, in the milk of transgenic mice, we constructed goat $\beta$-casein-directed hGH and hG-CSF expression cassettes individually and generated transgenic mice by co-injecting them into mouse zygotes. Out of 33 transgenic mice, 29 were identified as double transgenic harboring both transgenes on their genome. All analyzed double transgenic females secreted both hGH and hG-CSF in their milks. Concentrations ranged from 2.1 to $12.4\;mg/m{\ell}$ for hGH and from 0.04 to $0.13\;mg/m{\ell}$ for hG-CSF. hG-CSF level was much lower than hGH level but very similar to that of single hG-CSF mice, which were introduced with hG-CSF cassette alone. In order to address the causes of concentration difference between hGH and hG-CSF in milk, we examined mRNA level of hGH and hG-CSF in the mammary glands of double transgenic mice and tissue specificity of hG-CSF mRNA expression in both double and single transgenic mice. Likewise protein levels in milk, hGH mRNA level was much higher than hG-CSF mRNA, and hG-CSF mRNA expression was definitely specific to the mammary glands of both double and single transgenic mice. These results demonstrated that two transgenes have distinct transcriptional potentials without interaction each other in double transgenic mice although two transgenes co-integrated into same genomic sites and their expressions were directed by the same goat $\beta$-casein promoter. Therefore goat $\beta$-casein promoter is very useful for the multiple production of human proteins in the milk of transgenic animals.

• Reproductive and Developmental Biology
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• v.32 no.1
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• pp.45-49
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• 2008

We have previously produced transgenic (TG) mice expressing the human lactoferrin (hLF), interleukin-10 (hIL-10), and thrombopoietin (hTPO) proteins in the milk. In this study, we examined whether simple crossbreeding between two kids of a single transgenic mouse can produce double transgenics co-expressing two human proteins.. The hLF male, and the hIL-10 male were crossbred with the hIL-10 and hTPO females, and the hTPO female, respectively. PCR analysis for genotyping showed 32%, 23% and 24% double transgenic rates for hLF/hIL-10, hLF/hTPO, and hIL-10/hTPO transgenes, respectively. We analyzed the expression levels of the human proteins from double transgenic mice and compared those with their single transgenic siblings. All double transgenic co-expressed two human proteins at comparable levels to singles', unless hTPO was not co-expressed: for hLF, 1.1 mg/ml in hLF/hIL-10, whereas 0.5 mg/ml in hLF/hTPO; for hIL-10, 4.1 mg/ml in hIL-10/hLF, whereas 1.4 mg/ml in hIL-10/hTPO. Ihe downregulation of hTPO to half level of singles' was observed in double transgenic mice. The possible reason why hTPO co-expressed might lead to down-regulation of another human protein was discussed. These results suggested that double transgenic generated by crossbreeding between two singles' could be useful system for bioreactor.

• Biomolecules & Therapeutics
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• v.22 no.3
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• pp.232-238
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• 2014

Alzheimer's disease (AD) is the most common neurodegenerative disease without known ways to cure. A key neuropathologic manifestation of the disease is extracellular deposition of beta-amyloid peptide (Ab). Specific mechanisms underlying the development of the disease have not yet been fully understood. In this study, we investigated effects of 4-O-methylhonokiol on memory dysfunction in APP/PS1 double transgenic mice. 4-O-methylhonokiol (1 mg/kg for 3 month) significantly reduced deficit in learning and memory of the transgenic mice, as determined by the Morris water maze test and step-through passive avoidance test. Our biochemical analysis suggested that 4-O-methylhonokiol ameliorated $A{\beta}$ accumulation in the cortex and hippocampus via reduction in beta-site APP-cleaving enzyme 1 expression. In addition, 4-O-methylhonokiol attenuated lipid peroxidation and elevated glutathione peroxidase activity in the double transgenic mice brains. Thus, suppressive effects of 4-O-methylhonokiol on $A{\beta}$ generation and oxidative stress in the brains of transgenic mice may be responsible for the enhancement in cognitive function. These results suggest that the natural compound has potential to intervene memory deficit and progressive neurodegeneration in AD patients.

• Journal of Embryo Transfer
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• v.31 no.1
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• pp.1-7
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• 2016

Xenotransplantation involves multiple steps of immune rejection. The present study was designed to produce nuclear transfer embryos, prior to the production of transgenic pigs, using fibroblasts carrying transgenes human complement regulatory protein hCD59 and interleukin-18 binding protein (hIL-18BP) to reduce hyperacute rejection (HAR) and cellular rejection in pig-to-human xenotransplantation. In addition to the hCD59-mediated reduction of HAR, hIL-18BP may prevent cellular rejection by inhibiting the activation of natural killer cells, activated T-cell proliferation, and induction of $IFN-{\gamma}$. Transgene construct including hCD59 and ILI-18BP was introduced into miniature pig fetal fibroblasts. After antibiotic selection of double transgenic fibroblasts, integration of the transgene was screened by PCR, and the transgene expression was confirmed by RT-PCR. Treatment of human serum did not affect the survival of double-transgenic fibroblasts, whereas the treatment significantly reduced the survival of non-transgenic fibroblasts (p<0.01), suggesting alleviation of HAR. Among 337 reconstituted oocytes produced by nuclear transfer using the double transgenic fibroblasts, 28 (15.3%) developed to the blastocyst stage. Analysis of individual embryos indicated that 53.6% (15/28) of embryos contained the transgene. The result of the present study demonstrates the resistance of hCD59 and IL-18BP double-transgenic fibroblasts against HAR, and the usefulness of the transgenic approach may be predicted by RT-PCR and cytolytic assessment prior to actual production of transgenic pigs. Further study on the transfer of these embryos to surrogates may produce transgenic clone miniature pigs expressing hCD59 and hIL-18BP for xenotransplantation.

• Journal of Environmental Science International
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• v.10 no.1
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• pp.79-84
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• 2001

To assess resistance of transgenic tobacco plants which overexpress superoxide dismutase (SOD) and ascorbate peroxidase (APX) in chloroplasts to water stress, changes in leaf water potential, turgor potential, stomatal conductance and transpiration rate were measured. Leaf water potential in all plants remained high up to day 4 after withholding water but thereafter decreased markedly. In spite of a remarkable decrease in leaf water potential, some of transgenic plants maintained higher turgor potential compared with control plant on day 12. In particular, the transgenic plant expressing MnSOD showed an outstanding maintenance in turgor pressure by osmotic adjustment throughout the experiment, resulting in high stomatal conductance and transpiration rate. However, among transgenic plants, osmotic potential was reduced more effectively in multiple transformants such as the double transformant expressing both MnSOD and APX, and the triple transformant expressing CuznSOD, MnSOD and APX than single transformants. Consequently, further research is needed to get general agreement on the tolerance of transgenic plants to water stress at different growth stages for each transgenic plant.

• Journal of Embryo Transfer
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• v.27 no.1
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• pp.9-14
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• 2012

This study was conducted to analyze the transgenic efficiency and sex ratio in ${\alpha}$-1,3-galactosyltransferase (GalT) knock-out (KO) transgenic pigs according to generation. GalT KO piglets were produced by artificial insemination or natural mating. The transgenic confirmation of GalT KO was evaluated by PCR amplification using specific primers. After electrophoresis, three types of bands were detected such as 2.3 kb single band (Wild), 2.3 and 3.6kb double bands (GalT KO -/+; heterozygote), and 3.6kb single band (GalT KO -/-; homozygote). Transgenic efficiency in F1 generation was 64.5% (23/35) of GalT KO (-/+). In F2 generation, GalT KO transgenic efficiency was 36.4% (21/57, Wild), 47.5% (28/57, GalT KO -/+), and 16.1% (8/57, GalT KO -/-), respectively. Interestingly, no homozygote piglets were born in 6 deliveries among total 11 deliveries, although they were pregnant between male (M) and female (F) $F_1$ heterozygote. In the 5 litters including at least one GalT KO -/- piglet, the transgenic efficiency was 13.3% (2/24, Wild), 51.3% (14/24, GalT KO -/+), and 35.3% (8/24, GalT KO -/-), respectively. The sex ratio of M and F was 40:60 in $F_1$ and 49:51 in $F_2$ generation, respectively. Based on these results, GalT KO transgenic pigs have had a reproductive ability with a normal range of transgenic efficiency and sex ratio.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.79.1-79
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• 2003

Two pathogen-induced hot pepper transcription factors (CaNACl and CapIfl) were introduced into‘MicroTom’tomato by Agrobacterium tumefaciens-mediated transformation. We used to nptII containing kanamycin resistance gene as a selection marker. Both transformed and non-transformed plants were transferred to pot after rooting test in vitro. To approximate the levels of caNACl transcript in leaves of wild-type and transgenic plants, RNA blots were hybridized with double-stranded full-length CaNACl probe at moderate stringency, Although the relative signal strength for hybridization fluctuated among the samples on different blots, transgenic plant lines N-1, N-2 and N-3 consistently displayed increased levels of CaNACl transcript relative to other transgenic lines and wild-type plants. Of all the transgenic lines examined, line N-7 had the least amount of CaNACl transcript. Role of these transcription factors in pathogen defense will be examined by overexpression in tomato.

• Journal of Plant Biotechnology
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• v.36 no.4
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• pp.366-372
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• 2009

This study was conducted to develop new lines expressing the characteristic of early flowering by introducing MdMADS2 gene in chrysanthemum (Dendranthema grandiflorum (Ramat.) Kitamura) ‘Zinba'. Transformation of chrysanthemum was conducted by Agrobacterium tumefaciens LBA4404 harboring the binary vector containing MdMADS2 controlled by double CaMV 35S promoters. Ninety three shoots were regenerated from 1,463 leaf segment explants cultured on the first selection medium (MS basal salts + 1.0 mg/L BA + 0.5 mg/L IAA + 10 mg/L kanamycin + 400 mg/L cefotaxime, pH 5.8) after co-cultivation, and 20 out of the 93 shoots rooted on the second selection medium containing 20 mg/L kanamycin and 400 mg/L cefotaxime. Many escapes (98.6%) were removed on the selection stage for rooting. Nineteen lines were confirmed as transgenic plant with transgene by PCR analysis. Six transgenic plants flowered 2-11 days earlier than non-transgenic plant without big change of phenotype, and especially, 3 (Mo-7, Mo-11, Mo-17) out of 6 transgenic lines showed a significant reduction in days to flowering compared to non-transgenic plant. Introduction and expression of MdMADS2 gene in them were confirmed by Southern and real-time PCR analyses, respectively.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.47
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• pp.175-185
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• 2002

Rapeseed(Brassica napus L.) is an important oil crop as a vegetable oil, concentrated feed and industrial materials. The name "canola" was registered in 1979 by the Western Canadian Oilseed Crushers Association to describe "double-low" varieties. Double low indicates that the processed oil contains less than 2% erucic-acid and the meal less than 3mg/g of glucosinolates. Today annual worldwide production of rapeseed is approximately 35 million tons on 24 million hectares. China accounts for 33% of the world production and the European Economic Community for nearly 32%. Canola ranks 3rd in production among the world's oilseed crops following soybeans, sunflowers, peanuts and cottonseed. The recent advances in genomics and in gene function studies has allowed us to understand the detailed genetic basis of many complex traits, such as flowering time, height, and disease resistance. The manipulation of seed oil content via transgene insertion has been one of the earliest successful applications of modern biotechnology in agriculture. For example, the first transgenic crop with a modified seed composition to be approved for unrestricted commercial cultivation in the US was a lauric oil, rape-seed, grown in 1995. There were also some significant early successes, mostly notably the achievement of 40% to 60% lauric acid content in rapeseed oil, which normally accumulates little or no lauric acid. The name "$\textrm{Laurical}^{TM}$" was registered in 1995 by Calgene Inc. Nevertheless, attempts to achieve high levels of other novel fatty acids in seed oils have met with much less success and there have been several reports that the presence of novel fatty acids in transgenic plants can sometimes lead to the induction of catabolic pathways which break down the novel fatty acid, i.e. the plant recognizes the "strange" fatty acid and, far from tolerating it, may even actively eliminate it from the seed oil. It is likely that, in the future, transgenic oil crops and newly domesticated oil crops will both be developed in order to provide the increased amount and diversity of oils which will be required for both edible and industrial use. It is important that we recognize that both approaches have both positive and negative points. It will be a combination of these two strategies that is most likely to supply the increasing demands for plant oils in the 21st century and beyond.ant oils in the 21st century and beyond.

• Plant Biotechnology Reports
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• v.3 no.2
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• pp.139-145
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• 2009

DNA methylation is known to play an important role in the regulation of gene expression in eukaryotes. In this study, we isolated NtMET1 from Nicotiana tabacum cv. Havana (SR1) and obtain transgenic plants that reduced MET1 expression level with the double-strand RNA (dsRNA) MET1 gene. Transgenic tobacco plants showed dwarf and abnormal flower development when compared with the wild type. Using methylation-sensitive amplified polymorphism (MSAP) analysis, the patterns of cytosine methylation in transformed plants and the wild type were compared. MseI/HpaII selection primers showed an interesting polymorphism, and 153 DNA bands of interest were detected. Among these, 30 selective fragments were sequenced and analyzed with a BLAST search by successful MSAP modifications. The homology search showed that the transposons and tandem repeated sequences were related to the phenotypes. These results suggested that the decreased degree of methylation by dsRNA strategy caused abnormal growth and development in N. tabacum.