• Journal of Life Science
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• v.26 no.8
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• pp.875-880
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• 2016

The plasma membrane plays a crucial role in relaying signals from the outside environment to the inside of the cells. In eukaryotic cells, the inner leaflets of the plasma membrane are composed mostly of phospholipids, including phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositides (PIs). In this study, we tried to analyze the morphological changes induced by EGFP-fused membrane binding proteins, which are targeted to the plasma membrane via specific phospholipids binding. As a result, we found that overexpression of EGFP-P4M-SidM, a specific PI4P binding protein, or EGFP alone, did not induce any morphological changes. On the other hand, overexpression of EGFP-PLCδ1(PH), which is a specific PI(4,5)P₂ binding protein, EGFP-AKT1(PH) which binds to PI(3,4,5)P₃, or EGFP-OSH2(PH)×2 which binds to PI4P and PI(4,5)P₂, could induce the filopodia and lamilapodia formation as well as cell shrinkage. Overexpression of Lact-C2-EGFP which is a specific PS-binding probe, EGFP fused Aplysia phosphodiesterase 4 (ApPDE4) long-form (L(N20)-EGFP) which is localized to the plasma membrane via hydrophobic interaction, or EGFP fused Aplysia PDE4 short-form (S(N-UCR1-2)-EGFP) which is localized to the plasma membrane via electrostatic interaction, could induce cell shrinkage, but not filopodia or lamilapodia formation. Taken together, our data support that the different phospholipid bindings in the plasma membrane could induce different characteristic morphological changes. Thus, we can analyze, characterize, and classify the cellular morphological changes induced by the various phospholipid binding proteins.

• Journal of Sericultural and Entomological Science
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• v.51 no.2
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• pp.153-158
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• 2013

To express green fluorescent protein in the cocoon of silkworm, we constructed the fibroin H-chain expression system to produce enhanced green fluorescent protein (EGFP) in the cocoon of transgenic silkworms. The EGFP 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 EGFP/H-chain fusion gene was regulated by the fibroin H-chain promoter. The use of the 3xP3-driven DsRed2 cDNA as a marker allowed us to rapidly distinguish transgenic silkworm. A mixture of the donor and helper vector was micro-injected into 1,200 eggs of bivoltin silkworms, Baegokjam. We obtained 8 broods. The cocoon displayed strong green fluorescence, proving that the fusion protein was present in the cocoon. Also, the presence of fusion proteins in cocoons was demonstrated by SDS-PAGE and immunoblotting. Accordingly, we suggest that the EGFP fluorescence silk will enable the production of the novel biomaterial based on the transgenic silk.

• Journal of Life Science
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• v.27 no.6
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• pp.617-622
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• 2017

In this study, we sought to develop an effective cloning system by which human cytochrome $b_5$ (cyt $b_5$) is introduced and expressed in zebrafish. First, the 414 bp human cyt $b_5$ gene was amplified from RNA extracts of HeLa cells using RT-PCR, and the amplicon was subsequently sequenced to confirm that it was intact. Next, cyt $b_5$ was cloned into the pEGFP-N3 vector, which also encodes a fluorescent gene. One-cell stage zebrafish embryos were microinjected with the recombinant vector containing the cyt $b_5$ gene. Fluorescence microscopy confirmed high expression of the fluorescent gene in the injected fry compared to the non-fluorescent control fry. Finally, we extracted RNA from the injected fry and performed RT-PCR to determine whether the human cyt $b_5$ gene is expressed in the transgenic zebrafish. Sequencing analysis further confirmed that the cloned human cyt $b_5$ gene was intact. The transgenic zebrafish model produced in this study will be a useful tool to study therapeutic approaches to cure various diseases related to the deficiency of functional human cyt $b_5$ as well as tools for cloning useful genes in fish.

• 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.

• 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.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.4
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• pp.492-499
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• 2009

A Silkie Bantam embryo fibroblast line (named SBF59 line) was successfully established by using direct explant culture and cryopreservation techniques. Cell morphology, viability, dynamic growth and contamination were tested and the karyotype and levels of isoenzymes of lactic dehydrogenase and malic dehydrogenase were analyzed. Four kinds of fluorescent protein extrogenes, including $pEGFP-N_3$, $pECFP-N_1$, $pEYFP-N_1$ and $pDsRed1-N_1$ were transfected into the cells. The results showed that the cells were healthy and possessed a fibrous structure without a change in morphology. The average viability of the cells was 96% before freezing and 90.5% after thawing. The growth curve appeared as typical "S" shape and the cell growth passed through a detention phase, a logarithmic phase and a platform phase; the estimated population doubling time (PDT) was 38.5 h; assays for the presence of bacteria, fungi, viruses and mycoplasmas were negative; the cell line showed no cross contamination when assessed by isoenzyme analysis; the chromosome number was 2n = 78 on more than 88% of occasions; four kinds of fluorescent protein extro-genes appeared to be expressed effectively with a high transfection efficiency between 18.3% and 42.3%. The cell line met the required quality control standard. It not only preserves the genetic resources of the important Silkie Bantam at the cellular level but also provides valuable materials for genomic, post-genomic, somatic cell cloning research and other applications.

• Journal of Embryo Transfer
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• v.29 no.1
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• pp.59-66
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• 2014

Transcription activator like effector nucleases (TALENs) are artificial restriction enzymes generated by fusing a TALE DNA binding domain to a DNA cleavage domain which remove and introduce specific genes to produce transgenic animals. To investigate the efficient laboratory techniques for the injection of TALEN mRNA, pEGFP-N1 commercial plasmid were microinjected into porcine parthenogenetic and in vitro fertilization (IVF). In Experiment 1, to investigate injection time, compared 4 different time durations (2 hr, 4 hrs, 6 hrs & 8 hrs) after post activation of parthenogenetic embryos and after 6 hrs of co-incubation with sperms in IVF embryos. There were significant difference (P<0.05) in development to the blastocysts (4.4, 8.9, 3.9, 0.6%), GFP expression in blastocysts (1.3, 5.7, 2.3, 0.0%) which injected after post activation of 4 hrs compared with other 3 groups. IVF embryos after 2 hrs and 4 hrs injected were expressed GFP significantly higher than rest of two groups (P<0.05). In Experiment 2, compared development of 2 different concentrations ($20ng/{\mu}l$ and $50ng/{\mu}l$) of EGFP injection. There were significant difference (P<0.05) between two treatments which has higher cleavage (58.8 vs 41.9%), blastocysts development rate (13.0 vs 11.1%) and GFP expressed blastocysts (5.7 vs 0.0%) in $20ng/{\mu}l$ than the $50ng/{\mu}l$ in parthenogenetic embryos. In IVF embryos, only $20ng/{\mu}l$ injected embryos were expressed GFP (4.2%) after 7 days of incubation and 77.3 vs 64.7% of cleavage, 26.4 vs 23.5% development to blastocysts. In Experiment 3, three different volumes (5, 10 and 20 pl) were microinjected into porcine embryos to determine the most appropriate volume. Out of 3 groups, significantly higher development rates of cleavage (68.3, 58.0, 29.3%), blastocysts (11.7, 12.7, 0.5%) and GFP expressed blastocysts (2.9, 7.8, 0.0%) were shown in the 10 pl group (P<0.05). In conclusion, these results imply that $20ng/{\mu}l$ concentration, 10 pl of volume and injection at 4 hrs after post activation for parthenogenetic and 2~4 hrs after IVF, $20ng/{\mu}l$ concentration and 10 pl volume for IVF embryos were more effective microinjection conditions.

• Journal of Sericultural and Entomological Science
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• v.52 no.2
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• pp.102-109
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• 2014

We constructed the fibroin H-chain expression system to produce enhanced yellow fluorescent proteins (EYFP) in the silk of transgenic silkworm. Fluorescent silk could be made by fusing EYFP cDNA to the heavy chain gene and injecting it into a silkworm. The EYFP 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 EYFP/H-chain fusion gene was regulated by the fibroin H-chain promoter. The yellow fluorescence proving that the fusion protein was present in the silk. Accordingly, we suggest that the EYFP fluorescence silk will enable the production of novel biomaterial based on the transgenic silk.

• Journal of Sericultural and Entomological Science
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• v.52 no.1
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• pp.25-32
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• 2014

We produced the transgenic silkworm that expressed the enhanced blue fluorescent protein (EBFP) in the cocoon of silkworms. The EBFP 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 EBFP/H-chain fusion gene was regulated by the fibroin H-chain promoter. The use of the $3{\times}P3$-driven DsRed2 cDNA as a marker allowed us to rapidly distinguish transgenic silkworm. A mixture of the donor and helper vector was micro-injected into 300 eggs of silkworms, Baegokjam. We obtained 5 broods. The cocoon displayed blue fluorescence, proving that the fusion protein was present in the cocoon. Also, the presence of fusion proteins in cocoons was demonstrated by SDS-PAGE and western blot analysis. Accordingly, we suggest that the EBFP fluorescence silk will enable the production of the silk-based biomaterials.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.3
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• pp.358-363
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• 2008

The intracytoplasmic sperm injection (ICSI) procedure has recently been utilized to produce transgenic animals and may serve as an alternative to the conventional pronuclear microinjection in species such as pigs whose ooplasm is opaque and pronuclei are often invisible. In this study, the effects of sperm membrane disruption and electrical activation of oocytes on in vitro development and expression of transgene green fluorescent protein (GFP) in ICSI embryos were tested to refine this recently developed procedure. Prior to ICSI, sperm heads were treated with Triton X-100+NaCl or Triton X-100+NaCl+NaOH, to disrupt membrane to be permeable to exogenous DNA, and incubated with linearized pEGFP-N1 vector. To induce activation of oocytes, a single DC pulse of 1.3 kV/cm was applied to oocytes for $30{\mu}sec$. After ICSI was performed with the aid of a micromanipulator, in vitro development of embryos and GFP expression were monitored. The chemical treatment to disrupt sperm membrane did not affect the developmental competence of embryos. 40 to 60% of oocytes were cleaved after injection of sperm heads with disrupted membrane, whereas 48.6% (34/70) were cleaved without chemical treatment. Regardless of electrical stimulation to induce activation, oocytes were cleaved after ICSI, reflecting that, despite sperm membrane disruption, the perinuclear soluble sperm factor known to mediate oocyte activation remained intact. After development to the 4-cell stage, 11.8 (2/17, Triton X-100+NaCl+NaOH) to 58.8% (10/17, Triton X-100+NaCl) of embryos expressed GFP. The expression of GFP beyond the stage of embryonic genome activation (4-cell stage in the pig) indicates that the exogenous DNA might have been integrated into the porcine genome. When sperm heads were co-incubated with exogenous DNA following the treatment of Triton X-100+NaCl, GFP expression was observed in high percentage (58.8%) of embryos, suggesting that transgenic pigs may efficiently be produced using ICSI.