• Molecules and Cells
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• v.37 no.5
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• pp.426-434
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• 2014

Peptidylarginine deiminase type 2 (PADI2) deiminates (or citrullinates) arginine residues in protein to citrulline residues in a $Ca^{2+}$-dependent manner, and is found in lymphocytes and macrophages. Vimentin is an intermediate filament protein and a well-known substrate of PADI2. Citrullinated vimentin is found in ionomycin-induced macrophage apoptosis. Citrullinated vimentin is the target of anti-Sa antibodies, which are specific to rheumatoid arthritis, and play a critical role in the pathogenesis of the disease. To investigate the role of PADI2 in apoptosis, we generated a Jurkat cell line that overexpressed the PADI2 transgene from a tetracycline-inducible promoter, and used a combination of 12-O-tetradecanoylphorbol-13-acetate and ionomycin to activate Jurkat cells. We found that PADI2 overexpression reduced the cell viability of activated Jurkat cells in1a dose- and time-dependent manner. The PADI2-overexpressed and -activated Jurkat cells presented typical manifestations of apoptosis, and exhibited greater levels of citrullinated proteins, including citrullinated vimentin. Vimentin overexpression rescued a portion of the cells from apoptosis. In conclusion, PADI2 overexpression induces apoptosis in activated Jurkat cells. Vimentin is involved in PADI2-induced apoptosis. Moreover, PADI2-overexpressed Jurkat cells secreted greater levels of vimentin after activation, and expressed more vimentin on their cell surfaces when undergoing apoptosis. Through artificially highlighting PADI2 and vimentin, we demonstrated that PADI2 and vimentin participate in the apoptotic mechanisms of activated T lymphocytes. The secretion and surface expression of vimentin are possible ways of autoantigen presentation to the immune system.

• Journal of Embryo Transfer
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• v.30 no.3
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• pp.207-211
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• 2015

Many pronuclear stage eggs were used to generate transgenic mice (Tg) by microinjection. In this study, we used in vitro fertilized mouse eggs, followed by ultrarapid freezing to establish a simple procedure for production of Tg mice. We produced in vitro fertilized mouse eggs and cryopreserved them by ultrarapid freezing method. A total of 139 cryopreserved-thawed pronuclear eggs, of which 101 (72.6%) were survived following microinjection of chicken ${\beta}-actin$ promoter-driven firefly improved luciferase cDNA (${\beta}-act/luc^+$) and were transferred into 5 recipients. All recipients became pregnant and gave birth to a total of 15 (14.8%) pups. As a control, same DNA construction (${\beta}-act/luc^+$) was also injected into 450 in vitro fertilized eggs, of which 338 (75.1%) were survived and then were transferred into 14 recipients. Eleven (78%) mice became pregnant and littered a total of 54 (19.1%) pups. Southern blotting analysis of Tg mice indicated that one (1/15, 6.6%) and three (3/54, 5.5%) transgenic mice were production from cryopreserved and in vitro fertilized eggs, respectively. All Tg mice produced from both eggs showed the expression of improved luciferase gene. These results indicated that efficiency of produced of Tg mice from cryopreserved eggs was comparable to that from in vitro fertilized eggs. Furthermore, it is suggested that microinjection of transgene into in vitro fertilized eggs cryopreserved by ultrarapid freezing is an easy and conveniently method for production of Tg mice.

• Applied Biological Chemistry
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• v.38 no.3
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• pp.224-231
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• 1995

To increase the expression of a foreign protein in transgenic plant, the benefits of 5'-untranslated leader sequences of tobacco mosaic virus (TMV) RNA or soybean glycinin gene, Gy2, fused to a protein coding sequence were exploited. pGA643-derived plasmid contains 355 promoter of cauliflower mosaic virus, protein coding sequence of maize 10 kDa zein (10kZ) and Gy2 terminator. The leader from Gy2 or TMV RNA was inserted between the promoter and the coding sequence in each construct. The recombinant DNAs were introduced into tobacco plants by Agrobacterium mediated leaf disc transformation method. Although the transgene without the leader had more transcripts than the others, mRNAs containing the leader were translated more efficiently. It might be due to difference in the length of 5'-untranslated sequence and context surrounding the AUG codon, but could be sequence specific rather. These results suggest that the leader sequences of Gy2 and TMV play important roles as an enhancer in translational control of foreign gene in transgenic tobacco plant.

• Korean journal of applied entomology
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• v.59 no.4
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• pp.341-348
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• 2020

The ATP-binding cassette (ABC) transporter superfamily represents the largest transmembrane protein that transports a variety of substrates across extra- and intra-cellular membranes. In insects, the ABC transporter proteins play crucial roles in insecticide resistance. To date, no studies have investigated the involvement of ABC transporter gene for cross-resistance to insecticide chemistries. Here, we studied such possible mechanisms against six conventional insecticides using transgenic Drosophila melanogaster strains carrying Mdr49 transcript variant A. For the 91-R and 91-C strains of Drosophila melanogaster, although they have a common origin, 91-R has been intensely selected with DDT for over 60 years, while 91-C has received no insecticide selection. Our transgenic analyses showed that overexpression of 91-R-MDR49 transcript variant A along with three amino acid variations can yield a relatively low degree of cross-resistance to carbofuran (2.0~6.7-fold) and permethrin (2.5~10.5-fold) but did not show cross-resistance to abamectin, imidacloprid, methoxychlor, and prothiofos as compared to the Gal4-driver control strain without transgene expression. These results indicate that the overexpression of Mdr49A in itself leads to a cross-resistance and three amino acid changes have additional effects on positive cross-resistance to carbofuran and permethrin.

• Korean Journal of Poultry Science
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• v.28 no.2
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• pp.135-142
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• 2001

A novel sterategy has been established to determine the origin of the Primordial Germ Cells (PGCs) in avian embryos directly and the developmental fate of the PGCs for the application to Poultry biotechnology. Cells were removed from 1) the centre of area pellucida, 2) the outer of area pellucida and 3) the area opaca of the stage X blastoderm (Eyal-Giladi & Kochav, 1976). When the cells were removed from the centre of area pellucida, the mean number of circulating PGCs in blood was significantly decreased in the embryo at stage 15 (Hamburger & Hamilton, 1951) as compared to intact embryos. When the cells were replenished with donor cells, no reduction in the PGCs number was observed. The removal of cells at the outer of area pellucida or at the area opaca had no effect on the number of PGCs. In case, another set of the manipulated embryos were cultured ex vivo to the hatching and reared to the sexual maturity, the absence of germ cells and degeneration of seminiferous tubules was observed in resulting chickens derived from the blastoderm in which the cells were removed from the centre of the area pellucida. It was concluded that the avian Primordial Germ cells are originated at the center of area pellucida. Developmental ability of the cells to differentiate into somatic cells and germ cells in chimeras were analyzed. Somatic chimerism was detected as black feather attributed from donor cells. Molecular identification by use of female - specific DNA was performed. It was confirmed that the donor cells could be differentiated into chimeric body and erythrocytes. Donor cells retained the ability to differentiate into germline in chimeric gonads. More than 70% of the generated chimeras transmitted donor derived gametes to their offspring indicating that the cells at the center of area pellucida had the high ability to differentiate into germ cells. A molecular technique to identify germline chimerism has been developed by use of gene scan analysis. Strain specific DNA fragments were amplified by the method. It would be greatly contributed for the detection of germline chimerism. Mixed- sex chimeras which contained both male and female cells were produced to investigate the developmental fate of male and female cells in ovary and testes. The sex combinations of donor and recipient of the resulting chimeras were following 4 pairs; (1) chimeras (ZZ/ZZ) produced by a male donor (ZZ) and a male recipient (ZZ), (2) chimeras (ZW/ZW) produced by a female donor (ZW) and a female recipient (ZW), (3) chimeras (ZZ/ZW) Produce by a male donor (ZZ) and a female recipient (ZW), (4) chimeras (ZW/ZZ) produced by a female donor (ZW) and a male recipient (ZZ). It was found that genetically male avian germ cells could differentiate into functional ova and that genetically female germ cells can differentiate into functional spermatozoa in the gonad of the mixed- sex chimeras. An ability for introduction of exogenous DNA into the PGCs from stage X blastoderms were analyzed. Two reporter genes, SV-$\beta$gal and RSV-GFP, were introduced into the PGCs. Expression of bacterial/gal was improved by complexing DNA with liposome detectedcc in 75% of embryos at 3 days embryos. At the embryos incubated for 1 day, expression of the GFP was observed all the embryos. At day 3 of incubation, GFP was detected in about 70% of the manipulated embryos. In case of GFP, expression of the transgene was detected in 30 %e of the manipulated embryos. These results suggested that the cells is one of the most promising vectors for transgenesis. The established strategy should be very powerfull for application to poultry biotechnology.

• The korean journal of orthodontics
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• v.33 no.6 s.101
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• pp.485-497
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• 2003

The form and function of the craniofacial structure critically depend on genetic information. With recent advances in the molecular technology, genes that are important for normal growth and morphogenesis of the craniofacial skeleton are being rapidly uncovered, shaping up modem craniofacial biology. One of them is fibroblast growth factor receptor 2 (FGFR2). Specific point mutations in the. FGFR2 gene have been linked to Apert syndrome, which is characterized by premature closure of cranial sutures and craniofacial anomalies as well as limb deformities. To study pathogenic mechanisms underlying craniosynostosis phenotype of Apert syndrome, we used a transgenic approach; an FGFR2 minigene construct containing an Apert mutation (a point mutation that substitute proline at the position 253 to arginine; P253R) was introduced into fertilized mouse germ cells by DNA microinjection. The injected cells were then allowed to develop into transgenic mice. We used a bone-specific promoter (a DNA fragment from the type I collagen gene) to confine the expression of mutant FGFR2 gene to the bone tissue, and asked whether expression of mutant FGFR2 in bone is sufficient to cause the craniosynostosis phenotype in mice. Initial characterization of these mice shows prematurely closed cranial sutures with facial deformities expected from Apert patients. We also demonstrate that the transgene produces mutant FGFR2 protein with increased functional activities. Having this useful mouse model, we now can ask questions regarding the role of FGFR2 in normal and abnormal development of cranial bones and sutures.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.227-233
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• 2017

Pig has been known to be one of the most feasible animals as a bioreactor to produce pharmaceuticals in milk and as a mediator in xenotransplantation research. Previously, we generated transgenic pigs for both purposes, which were expressing Factor 8, vWF, hTPA, and hEPO in milk, along with expression of MCP at GalT gene locus ($GalT^{-MCP/-MCP}$) as well as expressing MCP at GalT gene loci with CD73 expression ($GalT^{-MCP/+}/CD73$). In this study, we performed comparative analyses of sperm parameters between wild type male (WT) pig and those transgenic males to examine the effects of transgenes integrated into the pigs on motility, morphology, viability, and acrosome integrity of the spermatozoa. Our results showed that the rates of actively motile spermatozoa of WT, Factor 8, vWF, hTPA, hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs were 85.0%, 83.3%, 82.5%, 83.3%, 82.5%, 77.5%, and 78.7%, respectively. Whereas, the rates of morphologically normal spermatozoa of WT, Factor 8, vWF, hTPA, hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs were 90.0%, 80.0%, 80.0%, 83.3%, 85.0%, 91.8%, and 80.8%, respectively. In addition, the viability in spermatozoa of WT, Factor 8, vWF, hTPA, hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs were 93.9%, 82.4%, 89.9%, 83.9%, 87.4%, 92.8%, and 83.6%, respectively. The rates of spermatozoa with normal acrosome integrity in WT, Factor 8, vWF, hTPA, hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs were 98.1%, 98.6%, 98.6%, 98.7%, 98.1%, 99.5%, and 95.1%, respectively. There were no significant differences in motility, morphology, viability, and acrosome integrity of the spermatozoa among WT, Factor 8, vWF, hTPA, and hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs. These mean that neither random integration nor targeted integration of the transgene into chromosome of pig effect on characteristics of spermatozoa. Ultimately, the transgenic male pigs subjected in this study could apply to propagate their progenies for production of human therapeutic proteins and advancing the xenotransplantation research.

• Tuberculosis and Respiratory Diseases
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• v.46 no.2
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• pp.229-240
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• 1999

Background: The impact of the immune response on cancer gene therapy using viral vectors to deliver a "suicide gene" is currently unclear. A vigrous immune response targeted at viral proteins or transgene may enhance the efficacy of tumor destruction and even augment responses to tumor antigens. These responses may involve the release of cytokines and stimulation of tumor specific cytotoxic T-lymphocytes that enhance therapeutic efficacy. On the other hand, a vigorous rapid cellular immune response may destroy cells expressing the therapeutic gene and attenuate the response to therapy. Furthermore, development of neutralizing antibody responses may prevent readministration of virus, a potentially significant limitation. Evaluating the significance of these limitations in animal models and developing solutions are therefore of obvious importance. Methods: After retroviral transduction of mouse mesothelioma cell line(AB12) with Herpes Simplex Virus thymidine kinase (HSVtk) gene in vitro, subcutaneous flank tumors were established. To study the effect of intact immune system on efficacy of tumor erradication, the ability of the HSVtk/ganciclovir system to inhibit tumor growth was compared among normal Balb/c mice, immunodeficient Balb/c-nude and SCID mice, and Balb/c mice immunosuppressed with cyclosporin. Results: Ganciclovir treatment resulted in greater inhibition of tumor growth in Balb/c mice compared with immunodeficient Balb/c-nude mice and SCID mice(in immunodeficient mice, there were no growth inhibition by ganciclovir treatment). Ganciclovir treatment resulted in greater inhibition of tumor growth in noncyclosporin (CSA) treated Balb/c mice compared with CSA treated Balb/c mice. On day 8, mean ganciclovir-treated tumor volume were 65% of control tumor volume in Balb/c mice versus 77% control tumor volume in CSA-treated Balb/c mice. This effect was still evident during therapy (day 11 and 13). On day 13, non-CSA treated tumor volume was 35% of control tumor volume versus 60% of control tumor volume in CSA treated Balb/c mice. Duration of expression of HSVtk was not affected by the immunosuppression with CSA. Conclusion: These results indicate that the immune responses against retrovirally transduced cells enhance the efficacy of the HSVtk/ganciclovir system. These findings have important implications for clinical trials using currently available retrovirus vectors as well as for future vector design.

• Journal of Plant Biotechnology
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• v.34 no.4
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• pp.339-346
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• 2007

Pollen germination viability is an essential factor to produce seeds from pollination and fertilization, which are required to maintain plant generation. In this study, we tried to identify the effect of boric acid on pollen germination and tube grouch in non-transgenic and transgenic plants expressing monoclonal antibodies (anti-colorectal cancer mAb CO17-1A, anti-breast cancer mAb BR55, and anti-rabies virus mAb57). The pollen of non-transgenic plant was treated with different concentration of boric acid (0, 5, 10, 15, 20, $40{\mu}g/mL$) in germination buffer to investigate its effect on in vitro pollen germination. At $20{\mu}g/mL$ of boric acid, tile pollen germination rate was the highest (49.5%) compared to other concentrations. In general, the germination rate significantly increased 3-10 folds in boric acid ($20{\mu}g/mL$) treated group in non-transgenic and transgenic plants. Also, the pollen tube length increased in boric acid ($20{\mu}g/mL$) treated groups. In the treated group, the pollen tube length increased until 3 h boric acid treatment and decreased after the 3 h, indicating that the 3 h is the most appropriate incubation time period. Western blot analysis showed that the mAb transgene expression was more stable in leaf than pollen in transgenic plants. This study suggested that $20{\mu}g/mL$ of boric acid is ideal concentration to induce in vitro pollen germination of transgenic plants expressing therapeutic monoclonal antibodies, indicating stable pollination and fertilization in transgenic plants.

• Journal of Practical Agriculture & Fisheries Research
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• v.25 no.4
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• pp.138-147
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• 2024

Agrobacterium-mediated transformation (AMT) is a method that allows for the stable integration of DNA fragments into the plant genome. Transgenic plants generated through AMT typically exhibit a lower copy number of the transgene compared to those induced by particle bombardment. Furthermore, AMT offers a straightforward and efficient approach for generating transgenic plants. While the transformation efficiency of wheat is comparatively lower than that of other monocot plants such as Rice (Oryza sativa L.) and Maize (Zea mays L.), the cultivars 'Bobwhites' and 'Fielder' are commonly employed for wheat transformation. To date, there have been no reported instances of successful development of transgenic plants using Korean wheat varieties through AMT. This study aims to assess the transformation efficiency of 43 Korean wheat cultivars using the GUS assay, with the goal of identifying suitable Korean wheat cultivars for AMT. The pCAMBIA1301 vector, carrying the β-glucuronidase (GUS) gene, was incorporated into Agrobacterium strain EH105. Following the inoculation of Agrobacterium into immature embryos, GUS assays were conducted 'Saeol', 'Jopum', and 'Jonong' showed 100% (the number of embryos showing GUS spots/the number of embryos used for AMT) among 43 cultivars. In addition, cultivars with more than 70% were 'Saekeumgang', 'Jojung', 'Tapdong', 'Anbaek', 'Dabun', 'Sugang', 'Keumgang', 'Jeokjung', 'Seodun', 'Joeun', 'Dajung', and 'Baekjung'. It seems that the 15 cultivars above showed the possibility of using AMT. On the other hand, 'Yeonbaek', 'Goso', 'Baekgang', and 'Johan' showed less than 20% and GUS spots were not observed in 'Gru', 'Gobun', 'Milseong', and 'Shinmichal-1'. This study explores transient GUS expression in Korean wheat cultivars seven days after AMT. The observed initial high efficiency of transient transformation suggests the potential for subsequent stable transformation efficiency. Korean wheat cultivars demonstrating elevated transient transformation efficiency could serve as promising candidates for the development of stable transgenic wheat.