To test the muscle cell specific gene expression, we examined the ability of human α-skeletal muscle actin (ACTA) promoter or human myoglobin (hMb) promoter to direct the expression of the GFP gene in both muscle and non-muscle cells, respectively. C2C12 cells, a mouse myoblast cell line, provide a powerful model to study skeletal muscle differentiation in vitro. We intended to use this cell line as a model for skeletal muscle-specific gene expression during myogenic differentiation from myoblast to myotubes. We compared marker gene expression profiles of proliferating and differentiated C2C12 cells using RT-PCR and fluorescent microscopy analysis. Also, we found that the expression of PCK1 gene under the control of ACTA promoter was proportionally increased as C2C12 differentiated into myotube form. PCK1 is involved in the regulation of gluconeogenesis. In previous research, transgenic mice with overexpressing PCK1 in skeletal muscle showed a greatly enhanced level of physical activity, which extends well into old age. This is due, in part, to an increased number of mitochondria and a high concentration of triglyceride in their skeletal muscles. These mice also had very little body fat, despite eating 60% more than controls. We also constructed a mesenchymal stem cell line and fetal fibroblast cell line for the experiments aiming to make transgenic animals in which the PCK1 gene is specifically expressed in muscle tissue. Accumulated knowledge of this approach could be applicable to a variety of related biological areas including transgenic animal research, gene function study, anti-aging study, etc. This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through Export Promotion Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (316002-5).

Tetranychus urticae and Myzus persicae are one of the most serious insect pests in many crops, vegetables, flowers, and fruit trees worldwide. Many insecticides have been developed to control green peach aphid and two spotted spider mite, but resistance to almost all insecticides has reduced their control effect. Particular groups of plant-beneficial microbials are not only root colonizers that provide plant disease suppression, but in addition are able to infect and kill insect larvae. Antimicrobial compounds produced by biocontrol microbes are effective weapons against a vast diversity of organisms such as fungi, nematodes, and viruses. In this study, we evaluated the effectiveness of mixtures plant extracts and improvement of culture process biocontrol microbials on insecticidal activity. Azadirachta indica and Derris elliptica mixed with micorbials, which are nutrient sources of mung bean extract and lecithin, were more effective than other the mixtures. Leaf spraying with the mixture of Pseudomonas fluorescens significantly showed the highest insecticidal power in vivo for 24 hours after treatment. The effect of spraying mixture was more than 50% at 2000 times dilution, and the spraying concentration of 90% or more showed a dilution of up to 500 times. Our results indicated that the nutrient sources of microbe act as a key antimicrobial metabolite in biocontrol of insect pests, and mixing with plant extracts can provide synergistic effects as an optimal usage of the biocontrol agents.

본 연구는 고농도 CO2 환경에 순응한 호접란 유묘의 CO2 교환 및 생장 반응을 조사하기 위해 실시하였다. 환경 조절이 가능한 식물생장상에서 6주령 호접란 ‘만천홍’을 27주 동안 야간에 각각 400 ± 100, 900 ± 100, 1500 ± 100, 2100 ± 100μmol CO2·mol-1 농도로 유지해주었다. 처리에 앞서 발달해 있었던 최상위 성숙엽으로 측정한 야간 중 CO2 흡수량은 900μmol CO2·mol-1 처리구에서 가장 높았고, 이어 2100, 1500, 400μmol CO2·mol-1 순으로 높았다. 그러나 처리 7주 이후 새로 발달한 성숙엽에서 측정한 결과, 2100μmol CO2·mol-1 처리구가 야간 중 가장 높은 CO2 흡수량을 보였으며, CO2의 흡수량은 처리 CO2 농도가 높아질수록 증가하는 경향을 보였다. 생장 반응에서는 최상위 성숙엽의 엽장, 엽폭 및 생체량은 900, 1500, 2100μmol CO2·mol-1 등 고농도 처리구에서 감소하였으나, 신엽 발달은 고농도 CO2 처리 하에서 촉진되었다. 이러한 결과들은 호접란 유묘의 높은 CO2 환경에 대한 적응이 CO2 흡수와 신엽 발달을 증진시킬 수 있다는 것을 보여준다. 그러나 900μmol CO2·mol-1 이상의 고농도에서는 잎의 생장과 생체량이 다소 줄어드는 경향이 있어 이에 대한 원인구명 등 추가적인 연구가 필요하다.

Human interferon alpha 2b (hIFNα-2b) is an important immune regulator widely used in clinic, for the treatment of chronic hepatitis, hairy cell leukemia, chronic myelogenous leukemia and multiple myeloma, etc. The clinically used hIFNα-2b is generally produced by E. Coli, which lacks the post-translational O-glycosylation of naturally synthesized protein, and has a short serum half-life. In this study, we report the successful generation of transgenic chickens that produce hIFNα-2b in the egg white using a feline immunodeficiency virus (FIV)-based lentiviral vector. In preliminary in vitro study, the hIFNα-2b gene under the control of CMV promoter expressed as much as 2,650 ng/㎖ in CEF-LNC-hIFNα-2bW cell. A FIV vector packaged with vesicular stomatitis virus G glycoprotein (VSV-G) was injected underneath the blastoderm of freshly laid chicken eggs (stage X) to produce a hIFNα -2b transgenic chicken. Out of 187 injected eggs, 55 chicks were hatched after 21 days of incubation, and 27 of the G0 hatched chicks expressed the vector-encoded hIFNα-2b gene. The expression of recombinant hIFNα-2b in transgenic chickens constitutes an important step towards low-cost and full biological activity production of this protein drug in bioreactor. This work was supported by the Bio-industry Technology Development Program, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea, and by a grant from the Next-Generation BioGreen 21 Program (No. PJ011178), Rural Development Administration, Republic of Korea.

In the present study, using a MoMLV-based retrovirus vector, we successfully generated a new transgenic chicken line expressing high levels of hEPO. A replication-defective Moloney murine leukemia virus (MoMLV)-based vectors packaged with vesicular stomatitis virus G glycoprotein (VSV-G) was injected beneath the blastoderm of non-incubated chicken embryos (stage X). One rooster was mated to wild-type hens to produce 748 G1 progeny. PCR analysis of blood samples from these progeny revealed that there were seven G1 transgenic offspring, corresponding to a 0.9% germline transmission rate. Subsequently, Southern blot analysis of the genomic DNA from three G1 transgenic chickens was carried out to verify the stable genomic integration and copy number of the transgene in the genome. Quantitative analyses of the blood samples taken from G1 transgenic chickens resulted in 4,150 ～ 10,823 IU/㎖ (34.6 ～ 90.2 ㎍/㎖) of hEPO in the blood. The biological activity of the recombinant hEPO in transgenic chicken serum was comparable to its commercially available counterpart. Red blood cell numbers were more than three-fold higher in the transgenic chickens compared to the non-transgenic chickens. Successful germline transmission of the transgene was also confirmed in G2 transgenic chicks produced from crossing G1 transgenic roosters with non-transgenic hens. We confirmed that 13 transgenic chicks of 45 G2 progeny, corresponding to a 28.9% germline transmission rate. These results will help establish a useful transgenic chicken model system for studies of embryonic development and for efficient production of transgenic chickens as bioreactors. This work was supported by the Bio-industry Technology Development Program, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea, and by a grant from the Next-Generation BioGreen 21 Program (No. PJ011178), Rural Development Administration, Republic of Korea.

Distillers dried grain (DDG) and makgeolli spent grain (MSG) are agricultural by-product to produce alcoholic beverage. However, they are known to contain enough nutrients. Mealworm is a promising insect resource for an animal feed ingredient as well as alternative human food. With low cost, DDG and MSG were investigated as a feed ingredient for rearing high quality mealworms. DDG and MSG were mixed with wheat bran and compared to control feed (only wheat bran) for its effects on larval survivorship, larval weight, duration for larval development, pupation rate, and pupal weight. Adding DDG on wheat bran showed positive results for larval weight, duration for larval developmental period, and pupation rate. However, adding MSG made longer duration for larval development, but it also improved larval weight, pupal weight with more than 90% pupation rate. We confirmed that adding 30～50% of DDG or MSG to conventional wheat bran have a strong potential to replace the conventional wheat bran insect feed for quality insect production.

Insect is an important player in the ecosystem as a prey for animals. Moreover, they are a valuable candidate food source for rearing animal. Tenebrio molitor (Coeloptera: Tenebrionidae) larvae are known as a good food source with high protein, unsaturated fatty acid, minerals. Therefore, it has strong potential to substitute the conventional meat consumption. To utilize T. molitor as a feed, the standard mass-rearing protocol is required. To make standard mass-rearing protocol, we tested different temperature(17.5, 20, 22.5, 25, 27.5 and 30°C) conditions for egg, larvae, pupae and adult T. molitor to identify the optimal rearing condition. Hatching was occurred within 15~32.5°C range. However, 17.5~27.5°C was required to get more than 70 % hatching rete. When the eggs were treated in 22.5~27.5°C, all eggs were hatched within 10 days. As larval development, shorter developmental period, higher pupation and eclosion rates were observed within 25~27.5°C temperature range. In addition, we compared the number of egg, oviposition duration and time required to start egg-laying. The minimum egg-laying(258.40±10.86) was observed at 17.5°C, but the maximum(749.10±7.45) was at 27.5°C. The maximum oviposition duration (137.00±12.73 day (mean±S.D.)) was achieved at 27.5°C, but the minimum (87.50±3.54 day (mean±S.D.)) was at 30°C. The time required to start egg-laying was less than 10 days at 17.5, 27.5, and 30°C. To consider all the factors, we concluded that the optimal temperature is 27.5 °C.

Hipparchia autonoe belongs to the family Nymphalidae (Lepidoptera) and is designated as an endangered insect and national monument in Korea. It only inhabits a very restricted area on Mt. Halla but is widely distributed in several Asian countries including Mongolia. A previous study conducted to understand the genetic relationship between Mt. Halla and Mongolian H. autonoe for conservation purposes suffered from a limited number of samples. Therefore, we sequenced the DNA barcode region of an additional 36 H. autonoe individuals, combined them with previous data from 19 individuals, and performed phylogenetic and population genetic analyses. Furthermore, the internal transcribed spacer 2 (ITS2) region was also sequenced from the 36 samples as a nuclear DNA marker. The existence of independent haplotypes, sequence types, and significant FST estimates (p < 0.05) between Mt. Halla and Mongolian populations indicated hampered gene flow between the populations. Nevertheless, an absence of a reciprocal monophyletic group in Mt. Halla and Mongolian populations by cytochrome oxidase subunit I gene- and ITS2-based phylogeny suggests that the genetic isolation of the Mt. Halla population from the Mongolian populations seemed not large enough to consider them independent genetic entities.

To identify subspecies and stocks of minke whale meats purchased from Korean markets during 2005-2007, we first obtained their complete sequences of mitochondrial DNA cytochrome b and control region sequences, and compared these sequences to the corresponding sequences of the common minke whale (Balaenoptera acutorostrata), obtained from GenBank. From analyses with partial cytochrome b sequences (383 bp) and non-coding, partial control region sequences (463 bp), Korean mink whale meats are identified as products from the North Pacific minke whale (B. a. scammoni). In addition, the sequences of the partial control region from these meats showed G at site no. 298 and G or A at site no. 463, and the meats appeared to originate from the J stock within this subspecies. Thus, because the J stock has been protected since 1986, implementation of strict regulation measures to reduce their accidental fisheries by catch seems urgent. In addition, B. a. scammoni is distinct from B. a. acutorostrata, with an average Jukes-Cantor distance of 2.21% in the complete control region sequence analysis (935 bp) and 1.31% in the complete cytochrome b gene sequence analysis; the current results support the current subspecies classification, although further sequencing analyses with nuclear genes are necessary.

Acteoside acts as an anti-oxidative activity and anti-apoptosis in the cells. But, it has been not studied on maturation and development of porcine oocytes. The aims of the present study were to examine the effects of acteoside on the morphological progress of meiosis, developmental competence, and ROS in porcine oocytes. Oocytes were matured in tissue culture medium-199, supplemented with acteoside at various concentrations: 0 (control), 10, 30 and 50 μM. The oocytes maturation rates of groups supplemented with acteoside were no significantly different (81.13, 85.96, 82.95 and 83.68%, respectively). Level of ROS was significantly decreased in acteoside treated group. Furthermore, the parthenogenetic blastocyst rate was significantly improved in 10 μM acteoside treated group compared with control group (44.83 vs. 27.75%). And we investigated effect of acteoside on the oocytes condition represented by cytoplasmic maturation by homogeneous distribution and formation of cytoplasmic organelles and regulation of apoptosis-related genes. In the results. during IVM, 10 μM acteoside treated oocytes showed that the mitochondria and lipid droplet were smaller and homogeneous distribution in cytoplasm compare with control oocytes. And reverse transcription polymerase chain reaction (RTPCR) of parthenogenetic blstocysts revealed that acteoside increased the anti-apoptotic genes (Mcl-1, Bcl-2 and Bcl-xL), whereas reduced the expression of pro-apoptotic genes (Bax and Bak). In conclusion, based on the results, the effect of acteoside on IVM was not attractive. However, in acteoside treated group, cytoplasmic maturation seemed to be improved with morphologically uniform distribution of cytoplasmic organelles. Furthermore, embryonic development in acteoside treated group was significantly highly increased than that of non-treated group. Our results represents that addition of acteoside to the IVM medium has a beneficial effect in physiology of porcine oocytes, providing a improved method for porcine oocytes in vitro. * This work was supported by a grant (Code# PJ008148) from BioGreen21 Program, Rural Development Administration, Republic of Korea.

The black-veined white, Aporia crataegi (Lepidoptera: Papilionoidea), is nearly extinct in South Korea, although substantial numbers of dried specimens are available. One of the common practices for such species is to launch re-introduction program after proper amount of genetic information are analyzed from donor and donee populations. In this study, we sequenced complete mitochondrial genome (mitogenome) of A. crataegi to design species-specific primers for subsequent population works and to further understand the mitogenome evolution in lepodiopteran Papilionoidea. The 15,140-bp long A. crataegi mitogenome that has typical sets of 37 genes is smallest among true butterfly species with overall slightly smaller size in genes and regions throughout the genome. Arrangement of the genome is identical to those of other lepidopteran mitogenomes, in which tRNA cluster located between the A+T-rich region and ND2 gene is translocated into tRNAMet, tRNAIle, and tRNAGln from ancestral arrangement, tRNAIle, and tRNAGln, tRNAMet. The A/T content of the genome at 81.3% is the highest in Pieridae, but lower than that of lycaenid species (81.7% ~ 82.7%) The high A/T content in the genome is also reflected in codon usage, accounting for 41.69% of A/T-composed codons (TTA, ATT, TTT, and ATA). Unlikely the diversified or modified usage of anticodon for tRNASer(AGN) the species of Pieridae including A. crataegi all unanimously have GCT that has been hypothesized as ancestral for Lepidoptera. A total of 111 bp of non-coding sequences are dispersed in 13 regions, ranging in size from 1–49 bp. Among them relatively longer ones (≥ 16 bp) all have relatively higher sequence identity to other regions of the genome, suggesting partial duplication of the sequences during A. crataegi evolution. As has been reported in some species of Lepidoptera, the A. crataegi A+T-region also has typically found conserved sequences (e.g., poly-T stretch, ATAGA motif, ATTTA element, microsatellite-like A/T sequence, and poly-A stretch) and one tRNA-like sequence, and this feature was commonly found in true butterfly species.