• 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.

• 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.

• YAKHAK HOEJI
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• v.49 no.3
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• pp.217-224
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• 2005

Human Immunodeficiency Virus type 1 (HIV-l) based lentivirus vector has demonstrated great potential as gene therapy vectors mediating efficient gene delivery and long-term transgene expression in both dividing and nondividing cells. However, for clinical studies it must be confirmed that vector preparations are safe and not contaminated by replication competent lentivirus (RCL) related to the parental pathogenic virus, HIV-l. In this study, we would like to establish the method for titration and RCL detection of lentivirus vector. The titration was determined by vector expression containing the green fluorescent protein, GFP in transduced cells. The titer was $1{\times}10^7$ Transducing Unit/ml in the GFP expression assay and $8.9{\times}10^7$ molecules/ml in the real-time PCR. Also, for the detection of RCL, we have used a combination method of PCR and p24 antigen detection. First, PBS/psi and VSV-G region in the genomic DNA of transduced cells was detected by PCR assay. Second, transfer and expression of the HIV-1 gag gene was detected by p24 ELISA. In an attempt to amplify any RCL, the transduced cells were cultured for 3 weeks (amplification phase) and the supernatant of amplified transduced cell was used for the second transduction to determine whether a true RCL was present (indicator phase). Analysis of cells and supernatant at day 6 in indicator phase were negative for PBS/psi, VSV-G, and p24 antigen. These results suggest that they are not mobilized and therefore there are no RCL in amplification phase. Thus, real-time PCR is a reliable and sensitive method for titration and RCL detection of lentivirus vector.

• IMMUNE NETWORK
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• v.11 no.1
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• pp.1-10
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• 2011

Interleukin-7 (IL-7) is an essential cytokine for T cells. However, IL-7 is not produced by T cells themselves such that T cells are dependent on extrinsic IL-7. In fact, in the absence of IL-7, T cell development in the thymus as well as survival of naive T cells in the periphery is severely impaired. Furthermore, modulating IL-7 availability in vivo either by genetic means or other experimental approaches determines the size, composition and function of the T cell pool. Consequently, understanding IL-7 expression is critical for understanding T cell immunity. Until most recently, however, the spatiotemporal expression of in vivo IL-7 has remained obscured. Shortage of such information was partly due to scarce expression of IL-7 itself but mainly due to the lack of adequate reagents to monitor IL-7 expression in vivo. This situation dramatically changed with a recent rush of four independent studies that describe the generation and characterization of IL-7 reporter mice, all utilizing bacterial artificial chromosome transgene technology. The emerging consensus of these studies confirmed thymic stromal cells as the major producers of IL-7 but also identified IL-7 reporter activities in various peripheral tissues including skin, intestine and lymph nodes. Strikingly, developmental and environmental cues actively modulated IL-7 reporter activities in vivo suggesting that IL-7 regulation might be a new mechanism of shaping T cell development and homeostasis. Collectively, the availability of these new tools opens up new venues to assess unanswered questions in IL-7 biology in T cells and beyond.

• Korean Journal of Environmental Agriculture
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• v.40 no.3
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• pp.186-193
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• 2021

BACKGROUND: Before generating transgenic plant using the CRISPR-Cas9 system, the efficiency test of sgRNAs is recommended to reduce the time and effort for plant transformation and regeneration process. The efficiency of the sgRNA can be measured through the transient expression of sgRNA and Cas9 gene in tomato cotyledon; however, we found that the calculated efficiency showed a large variation. It is necessary to increase the precision of the experiment to obtain reliable sgRNA efficiency data from transient expression. METHODS AND RESULTS: The cotyledon of 11^th, 15^th, 19^th, and 23^rd-day-old tomato (Solanum lycopersicum cv. Micro-Tom) were used for expressing CRISPR-Cas9 transiently. The agrobacterium harboring sgRNA for targeting ALS2 gene of tomato was injected through the stomata of leaf adaxial side and the genomic DNA was extracted in 5 days after injection. The target gene edition was identified by amplifying DNA fragment of target region and analyzing with Illumina sequencing method. The target gene editing efficiency was calculated by counting base deletion and insertion events from total target sequence read. CONCLUSION: The CRISPR-Cas9 editing efficiency varied with tomato cotyledon age. The highest efficiency was observed at the 19-day-old cotyledons. Both the median and mean were the highest at this stage and the sample variability was also minimized. We found that the transgene of CRISPR-Cas9 system was strongly correlated with plant leaf development and suggested the optimum cotyledon leaf age for Agrobacterium-mediated transfection in tomato.

• The Plant Pathology Journal
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• v.24 no.3
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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.

• Korean Journal of Animal Reproduction
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• v.19 no.1
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• pp.35-41
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• 1995

In this study, we have tested whether the retrovirus vector system is applicable in transgenic cattle production. To overcome low infectivity of currently available retrovirus vector system we have directly microinjected retrovirus-producing cells into the perivitelline space of the day 1.5 embryos. The virus-producing cell line was designed to release replication-defective retrovirus encapsidated with Gibbon ape leukemia virus (GaLV) envelope protein. E. coli LacZ gene was used as a marker gene to facilitate evaluation of the transgene expression and X-gal staining at morula or blastocyst stage resulted in expression of E. coli LacZ gene The results in these experiments were summarized as follows : 1. The lowest concentration of polybrene necessary for efficient virus infection was Sf' g/ml. 2. Development rate from day 1.5 embryos microinjected with virus-producing cells to the morulae /blastocysts was 29%. 3. 21% of the morulae /blastocysts were LacZ+. 4. There was no evidence that the retrovirus-producing cells used in this study produced replication-competent retrovirus.

• Korean Journal of Animal Reproduction
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• v.27 no.1
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• pp.15-23
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• 2003

Lactadherin (formerly known as BA46), a major glycoprotein of the human milk fat globule membrane, is abundant in human breast milk and breast carcinomas and may prevent symptomatic rotavirus infections. In this study, under the control of tissue specific and hormonal inducible mouse whey acidic protein (WAP) promote., the expression pattern of lactadherin (Ltd) in lactogenic hormone-dependent mouse mammary epithelial cell line HC11 were tested. pLNWLtd construct containing 2.4 kilobases of the WAP promoter and 1.5 kilobases of human lactadherin gene was stably transfered into HC11 cells using retroviral vector system. Integration and expression level of the transgene was estimated using PCR and RT-PCR, respectively. Prominent induction of Ltd gene under the WAP promoter was accomplished in the presence of insulin, hydrocortisone and prolactin. Compared to the control (cells cultured with insulin alone), however we observed that the WAP promoter was leaky. These data indicate that luther studies are needed in finding an appropriate promoter other than WAP promoter because of its leakiness.

• 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.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.239-245
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• 2011

The overexpression of Phosphoprotein Enriched in Astrocytes (PEA15) gene is commonly found in human diabetic patients. The overexpression of this gene in skeletal muscle and fat tissues have been reported to cause insulin resistance, thereby impairing insulin stimulated glucose uptake. We introduced a gene of mouse PEA15 (mPEA15) and enhanced green fluorescent protein (EGFP) into fertilized one cell pig zygotes using microinjection, and produced a piglet that showed overexpression of mPEA15 in the muscle tissues and expression of EGFP in the ear tissues and hooves. RT-PCR RFLP, southern blot and FISH analysis showed that the tissues carried the transgene. Real-time RT-PCR and western blots demonstrated that PEA15 gene was overexpressed in the various tissues and muscle tissues, respectively. These fads suggest that expression vector system is normally expressed in the transgenic (TG) pigs. To use as animal diseases model for type 2 diabetes, further study is necessary to confirm whether diabetes occur in these TG pigs, especially insulin resistance.