• International Journal of Industrial Entomology and Biomaterials
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• v.34 no.1
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• pp.11-15
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• 2017

We constructed the fibroin H-chain expression system to produce enhanced cyan fluorescent proteins (ECFP) in transgenic silkworm cocoon. Fluorescent cocoon could be made by fusing ECFP cDNA to the heavy chain gene and injecting it into a silkworm. The ECFP fusion protein, each with N- and C-terminal sequences of the fibroin H-chain, was designed to be secreted into the lumen of the posterior silk glands. The expression of the ECFP/H-chain fusion gene was regulated by the fibroin H-chain promoter. The use of the 3xP3-driven EGFP cDNA as a marker allowed us to rapidly distinguish transgenic silkworms. The EGFP fluorescence became visible in the ocelli and in the central and peripheral nervous system on the seventh day of embryonic development. A mixture of the donor and helper vector was micro-injected into 1,020 Kumokjam, bivoltin silkworm eggs. We obtained 6 broods. The cocoon was displayed strong blue fluorescence, proving that the fusion protein was present in the cocoon. Accordingly, we suggest that the ECFP fluorescence silk will enable the production of novel biomaterial based on the transgenic silk.

• Journal of Life Science
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• v.21 no.12
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• pp.1726-1731
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• 2011

Human Stem Cell Factor (hSCF) is a cytokine that binds to the c-Kit receptor and plays an important role in hematopoiesis, spermatogenesis, and melanogenesis. To produce the human Stem Cell Factor (hSCF) recombinant protein, we constructed a germline transgenic silkworm using the piggyback vector. The expression of the hSCF gene was driven by the Drosophila heat shock protein 70 (dHsp70) promoter. 3XP3 promotor-driven EGFP was used as a marker which allowed us to rapidly distinguish the transgenic silkworm. A mixture of the donor and helper vector was micro-injected into 1,020 eggs of bivoltin silkworms, Keomokjam. We obtained approximately 22 G1 broods that were EGFP-positive. The expression of the hSCF gene in the transgenic silkworm was analyzed by SDS-PAGE and immunoblotting. Also, analysis of insertion sites into the silkworm genome using inverse PCR showed that exogenous DNA was inserted into the transgenic silkworm genome. These results show that successfully constructed transgenic silkworm expresses the hSCF recombinant protein.

• Journal of Sericultural and Entomological Science
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• v.50 no.2
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• pp.87-92
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• 2012

We constructed the fibroin H-chain expression system to produce Discosoma sp. red fluorescent protein variant2 (DsRed2) in transgenic silkworm cocoon. Fluorescent cocoon could be made by fusing DsRed2 cDNA to the heavy chain gene and injecting it into a silkworm. The DsRed2 fusion protein, each with N- and C-terminal sequences of the fibroin H-chain, was designed to be secreted into the lumen of the posterior silk glands. The expression of the DsRed2/H-chain fusion gene was regulated by the fibroin H-chain promoter. The use of the 3xP3-driven EGFP cDNA as a marker allowed us to rapidly distinguish transgenic silkworms. The EGFP fluorescence became visible in the ocelli and in the central and peripheral nervous system on the seventh day of embryonic development. A mixture of the donor and helper vector was micro-injected into 1,020 Kumokjam, bivoltin silkworm eggs. We obtained 6 broods. The cocoon was displayed strong red fluorescence, proving that the fusion protein was present in the cocoon. Accordingly, we suggest that the DsRed2 fluorescence silk will enable the production of novel biomaterial based on the transgenic silk.

• Journal of Life Science
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• v.34 no.7
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• pp.493-499
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• 2024

Antimicrobial peptides are antimicrobial substances inherent in animals and plants, with strong antibacterial activity even in small amounts and with various other functions such as antiviral and antioxidant actions. Plants can be grown with just water and sunlight, allowing for their mass production at low costs. However, transforming a chloroplast into one that produces antimicrobial peptides, rather than growing plants, increases the amount of protein expression and minimizes contamination of the ecosystem because gene transfer by pollen does not occur. In that context, using transgenic plant chloroplasts to produce recombinant proteins increases protein degradation and reduces the solubility of proteins. To solve this problem, we fused SUMO, a fusion protein, with a recombinant protein. We also used a 6xHis tag to purify the fusion protein. The antimicrobial peptide stomoxyn is an antibacterial substance found in stable flies. Stomoxyn has an α-helix structure and is amphiphilic, which allows it to dissolve bacterial cell membranes. In this study, we constructed a transformation vector to express stomoxyn in both plant chloroplasts and Escherichia coli and used this vector to confirm the expression of stomoxyn in E. coli. The expression of the protein was then confirmed in E. coli using a transformation vector. The expressed stomoxyn was purified by nickel column and SUMOase treatment, and its antibacterial activity was confirmed using an agar diffusion assay. The EGFP gene was used to ensure that the transformed vector was inserted into the chloroplast.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.11
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• pp.1529-1540
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• 2011

Silencing of a specific gene using RNAi (RNA interference) is a valuable tool for functional analysis of a target gene. However, information on RNAi for analysis of gene function in farm animals is relatively nil. In the present study, we have validated the interfering effects of siRNA (small interfering RNA) using both quantitative and qualitative gene silencing in buffalo granulosa cells. Qualitative gene knockdown was validated using a fluorescent vector, enhanced green fluorescence protein (EGFP) and fluorescently labeled siRNA (Cy3) duplex. While quantitatively, siRNA targeted against the luciferase and CYP19 mRNA was used to validate the technique. CYP19 gene, a candidate fertility gene, was selected as a model to demonstrate the technique optimization. However, to sustain the expression of CYP19 gene in culture conditions using serum is difficult because granulosa cells have the tendency to luteinize in presence of serum. Therefore, serum free culture conditions were optimized for transfection and were found to be more suitable for the maintenance of CYP19 gene transcripts in comparison to culture conditions with serum. Decline in fluorescence intensity of green fluorescent protein (EGFP) was observed following co-transfection with plasmid generating siRNA targeted against EGFP gene. Quantitative decrease in luminescence was seen when co-transfected with siRNA against the luciferase gene. A significant suppressive effect on the mRNA levels of CYP19 gene at 100 nM siRNA concentration was observed. Also, measurement of estradiol levels using ELISA (enzyme-linked immunosorbent assay) showed a significant decline in comparison to control. In conclusion, the present study validated gene silencing using RNAi in cultured buffalo granulosa cells which can be used as an effective tool for functional analysis of target genes.

• Reproductive and Developmental Biology
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• v.32 no.2
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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.

• Journal of Microbiology and Biotechnology
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• v.14 no.6
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• pp.1333-1337
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• 2004

Oxidants such as hydrogen peroxide are powerful inducers of cell damage, ageing, and apoptosis. Since clusterin, a 75-80 kDa mammalian glycoprotein, is frequently found to be inducible in apoptotic cells and tissues, this study inquired into whether this would be a protective mechanism against further cell death. The aim was to find out whether overexpression of clusterin could protect cells from oxidant­induced stress and apoptosis. To clarify this issue, we generated and analyzed stable cell lines expressing fusion proteins of a rat clusterin with an enhanced green fluorescent protein (EGFP). When treated with varying concentrations of hydrogen peroxides, clusterin transfectants indeed showed increased resistance to apoptosis and exhibited a much higher survival rate than mock-transfected cells. On the other hand, neither intracellular re-distribution nor local concentration of clusterin-EGFP was observed, which leaves the question open about its anti-apoptotic mechanism. In conclusion, the overexpression of clusterin provides a means for protecting cells against oxidative stress and subsequent cell death.

• Parasites, Hosts and Diseases
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• v.53 no.3
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• pp.335-339
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• 2015

Cryptosporidium andersoni ATP-binding cassette (CaABC) is an important membrane protein involved in substrate transport across the membrane. In this research, the nucleotide binding domain (NBD) of CaABC gene was amplified by PCR, and the eukaryotic expression vector of pEGFP-C1-CaNBD was reconstructed. Then, the recombinant plasmid of pEGFP-C1-CaNBD was transformed into the mouse intestinal epithelial cells (IECs) to study the iron transportation function of CaABC. The results indicated that NBD region of CaABC gene can significantly elevate the transport efficiency of $Ca^{2+}$, $Mg^{2+}$, $K^+$, and $HCO_3{^-}$ in IECs (P<0.05). The significance of this study is to find the ATPase inhibitors for NBD region of CaABC gene and to inhibit ATP binding and nutrient transport of CaABC transporter. Thus, C. andersoni will be killed by inhibition of nutrient uptake. This will open up a new way for treatment of cryptosporidiosis.

• Journal of Microbiology and Biotechnology
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• v.21 no.4
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• pp.387-390
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• 2011

Nude mice (BALB/c) were grafted with human 293 cells and PERV (porcine endogenous retrovirus)-IRES-EGFP (a packageable retroviral vector plasmid containing an internal ribosome entry site-enhanced green fluorescent protein)-producing pig PK15 cells in order to determine whether the pig cells could transmit PERV-IRES-EGFP to mice and human 293 cells in vivo. None of the transplanted human 293 cell lines were infected by PERV, but PCR analysis identified PERV-B provirus integration into both the heart and salivary gland of the inoculated nude mice. Our data indicate that hearts and salivary glands can be used to identify PERV-B receptors.

• Biomedical Science Letters
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• v.11 no.2
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• pp.135-141
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• 2005

A Jopock (Jpk), a trans-acting factor associating with the position-specific regulatory element of murine Hoxa-7, has shown to have a toxicity to both prokaryotic and eukaryotic cells when overexpressed. Since Jpk protein harbors a transmembrane domain and a putative endoplasmic reticulum (ER)-retention signal at the N-terminus, a subcellular localization of the protein was analyzed after fusing it into the green fluorescent protein (GFP): Both N-term (Jpk-EGFP) and C-term tagged-Jpk (EGFP-Jpk) showed to be localized in the ER when analyzed under the fluorescence microscopy after staining the cells with ER- and MitoTracker. Since ER stress triggers the ER-stress mediated apoptosis to eliminate the damaged cells, we analyzed the expression pattern of Jpk under ER-stress condition. When MCF7 cells were treated with the ER-stress inducer such as DTT and EGTA, the expression of Jpk was upregulated at the transcriptional level like that of Grp78, a molecular chaperone well known to be overexpressed under ER-stress condition. In the presence of high concentration of ER-sterss inducer (10 mM), about 70 (DTT) to $95\%$ (EGTA) of cells died stronly expressing ($10\~12$ fold) Jpk. Whereas at the low concentration ($0.001\~1.0\;mM$) of the inducer, the expression of Jpk was increased about 2.5 (EGTA) to 5 fold (DTT), which is rather similar to those of ER chaperone protein Grp78. These results altogether indicate that the ER-stress upregulated the expression of Jpk and the excess stress induces the ER-stress induced apoptosis and the concomitant expression of Jpk.