The necessity of conditional gene expression in pigs for transgenic models is raised. Thus, in this study, Cre-loxP conditional expression in porcine fetal fibroblasts was investigated and the transformed fibroblasts were reprogrammed in enucleated oocytes for further early embryonic development. Fetal fibroblasts from miniature pigs were used for transfection with pCALNL-DsRed including floxed neomycin resistant gene and selected with 750 ug/mL neomycin for two weeks. The transfected cells did not express DsRed under fluorescence microscope. After transient transfection of plasmid DNA expressing Cre, the fibroblasts began to express DsRed. The cells expressing Ds- Red were employed into somatic cell nuclear transfer (SCNT). A total of 121 oocytes were used for SCNT and 76 cloned embryos (62.8%)were cleaved. Six blastocysts were grown up after SCNT and expressed DsRed. Deletion of floxed neomycin resistant gene was confirmed by RT-PCR in cloned blastocysts. Taken together, this study demonstrated that Cre-loxP recombination in miniature pig fibroblasts were successfully worked and those sequential transformed cells were developed into pre-implantation stage via SCNT.

Freezing of bovine blastocysts has been proposed as a tool to improve the feasibility of cattle production by using embryo transfer technique. However, the low efficiency of frozen-thawed embryos survival and further development is a crucial problem. Thus, we examined the effect of artificial shrinkage before vitrification of bovine expanded, hatched and SCNT embryos on the survival rate, apoptosis index and further development after thawing. Expanded, hatched and SCNT embryos were vitrified after artificial shrinkage, which was performed by puncturing the blastocoele with a pulled pasteur pipet. Artificial shrinkage of the blastocyst was achieved after pushing a pulled pasteur pipet into the blastocoele cavity until it contracted. The shrunken and not shrunken embryos were exposed to cryoprotectant solution in 7.5% ethylene glycol-7.5% DMSOPBS with 20% FBS for 5 min. They were placed in a small volume of vitrification solution (15% ethylene glycol+15% DMSO+PBS+20% FBS+0.5 M sucrose) and plunged into liquid nitrogen on a cryotop. Then, after thawing, cryoprotectant was diluted in 1.0 M, 0.5 M, 0.25 M, and 0 M sucrose for 1, 3, 5, and 5 min. Under the optimal conditions, overall efficiency of the survival rate of bovine expanded, hatched, SCNT embryos in artificial shrinkage groups was higher compared with non-artificial shrinkage groups (p< 0.05). Especially, the numbers of TUNEL-positive nuclei in artificial shrinkage groups were significantly reduced than those of non-artificial shrinkage groups among frozen-thawed expanded, hatched, and SCNT blastocysts (p< 0.05). Our results showed that survival rates in cryopreserved expanded, hatched, SCNT embryos could be improved by reducing the fluid content. Therefore, we suggest that artificial shrinkage method is a effective pretreatment technique for the cryotop vitrification of expanded, hatched, SCNT bovine blastocysts.

<Objective> Injection of a linear transgene into male pronucleus has been widely used to produce Transgenic (Tg) mice. This approach however is inefficient and results in concatemerised transgene insertion and associated reduced protein expression from such insertions. The objective of this study is to develop active transgenesis method by using a piggyBac transposase plasmid DNA, and generate double transgene harboring transgenic mice. <Method> We examined the piggyBac transposase plasmid (pm- GENIE‐3) on its ability to produce transgenic animals with NaOH, HCl and FuGENE6 treated sperm followed by ICSI‐Tr, for its effectiveness in creating EGFP Tg mice, as judged by offspring epifluorescence. After these steps, we explored if the embryo development affects ICSI‐Tr efficiency by using substrate‐free media or aphidicolin. Moreover, we tested to determine if transgenesis is possible by directly injecting the DNA into the cytoplasm or into pronuclei. Finally, we attempted the introduction of two transgenes, such as EGFP and dsRED simultaneously in one transposon and the ability to generate double Tg mice by using NaOH treated sperm during ICSI‐Tr. <Results> The best results were obtained when sperm were treated with NaOH and co‐incubated with circular plasmid DNA of pmGENIE‐3. This resulted in Tg pups that could successfully express EGFP, with efficiencies of 37.9% of born animals being transgenic. Furthermore, the effectiveness of this method was proved by the production of Tg offspring from inbred strains of mice, such as C57BL/6, Balb/c and CD‐1 nude. While injection of DNA into the pronucleus or cytoplasm of one cell embryos, and delayed embryo development‐method were not as effective as ICSI‐Tr in producing Tg mice, they nevertheless proved successful. Finally, NaOH‐ICSI‐Tr successfully obtained Tg mice expressing both the EGFP and dsRED transgene. In conclusion, the current study developed an active form of NaOH‐ICSI‐Tr mediated transgenesis utilizing the piggyBac transposition machinery, and was successful in obtaining Tg mice which expressed simultaneously not only EGFP but also the dsRED transgene stably inserted in these animals.

The green peach aphid (Myzus persicae) is a cosmopolitan pest of agricultural and horticultural crops and causes serious economic damages. M. persica has rapidly developed resistance to a wide variety of insecticides, including pyrethroids. Target site insensitivity mechanism mediated by two mutations (L1014F and M918T) on the para-type voltage-sensitive sodium channel (vssc) is mainly responsible for pyrethroid resistance. To predict the vssc resistance allele frequency, quantitative sequencing (QS) protocol was established. Frequency prediction equations generated from the plots of signal ratios and amplification critical time showed a high correlation coefficient (r2>0.993), indicating its high accuracy in prediction. QS results revealed that the kdr-type L1014F mutation is only present in Pyeongchang strain. No field strains of M. persicae possessed the super-kdr type M918T mutation. However, a novel M918L mutation was found by genotyping approach. The allele frequencies of M918L and L1014F were 0% to 53% in populations examined, and the level of M918L mutation frequency was closely related with pyrethroid resistance. Therefore, QS-based detection of M918L mutation frequency should faciltate the monitoring of pyrethroid resistance in the field.

The green peach aphid (Myzus persicae) is a serious pest of agricultural and horticultural crops all over the world. M. persica has rapidly developed resistance to a wide variety of insecticides, including carbamates. The E4/FE4 carboxylesterase is known to be involved in carbamate resistance. To compare the E4/FE4 carboxylesterase gene copy number, as a genetic resistance marker, between seven field strains, quantitative real-time PCR (qPCR) was performed. In addition, quantitative sequencing (QS) was employed to predict the frequencies of acetylcholinesterase (AChE) mutations (A301S and S431F) that are associated with target site insensitivity. All M. persica strains examined possessed the S431F mutation in the heterozygous state except for a susceptible strain, implying the possibility of AChE duplication. In contrast, no A301S mutation was found. Frequency prediction equation was generated from the plots of signal ratios and amplification critical time, which showed a high correlation (r2>0.996). QS analysis of M. persicae populations revealed that the allele frequency of S431F ranged 4% to 63%. Taken together, the AChE resistance allele frequencies determined by QS and the E4/FE4 gene copy number by qPCR should facilitate the detection and monitoring of carbamate resistance in M. persicae in the field.

The screening of effective lethal genes was conducted via the systemic delivery of dsRNA for the RNAi-based management of Tetranychus urticae. Six candidate genes (COPI coatmer, T_COPI; ESCRT III_Snf7, T_SNF7; Ribosomal protein S4, T_RPS4; v-ATPase A subunit 2, T_V-ATPase; Aminopeptidase N, T_APN3; Acetylcholinesterase, T_AChE) and two reference genes (EGFP and T_AChEintron) were tested for the experiment. The permeated dsRNA to the leaf disc (ca. 30 mm diameter) was detected at 6 h after treatment, indicating that dsRNA could move through veins on the leaf. In the reference gene selection, the T_AChEintron was chosen for its low mortality compared with EGFP gene. In the evaluation of mortality, the T_COPI, T_V-ATPase and T_RPS4 exerted higher toxicities at 24 hour after treatment among six genes tested. Interestingly, T_APN3 showed toxicity after 72 hour. In summary, the dsRNA delivery via leaf disc was effective in screening lethal genes and some genes, such as COPI, V-ATPase and RPS4, can be applicable for establishing a RNAi-based control system against T. urticae.

The resistance levels against carbamates (CB) and organophosphates (OP) were determined through bioassay and quantitative sequencing (QS) methods in 16 field populations of Nilaparvata lugens. The resistance levels to CB and OP by bioassay were 1.3~47.5-fold and 1.4~14.4-fold higher than a susceptible strain, respectively. The QS protocol was established to determine the allele frequencies of eight point mutations on acetylcholinesterase putatively associated CB and OP resistance. The allele frequencies of four mutations in local populations (G119A, F/S330Y, F331H and I332L) ranged from ca. 0.0~51.7%, 1.0~44.3%, 8.5~57.3% and 7.12~56.6%, respectively. The average prediction limits were –9.6±5.1~7.7±2.9%. The F330Y, F331H and I332L were tightly linked each other, suggesting these mutations may occur simultaneously. In the correlation analysis, G119A was not well correlated with both insecticides (r2= less 0.25), whereas F/S330Y, F331H and I332L showed better correlation with the resistance levels of carbamate (r2=0.590) than organophosphate (r2=0.235). This finding indicates that F/S330Y, F331H and I332L mutation frequencies are suitable for detecting carbamate resistance in N. lugens. QS will be applicable for the rapid monitoring of resistance levels to CB insecticides in N. lugens.

A carbofuran-resistant strain (CAS) showed ca. 41.1- and 15.1-fold resistance compared to a susceptible strain (SUS) and a non-selected field strain (FM), respectively. Enhanced activities of carboxylesterase and P450 were found as ca. 3- and 1.6-fold higher in CAS strain, suggesting these enzymes play a minor role in carbofuran resistance. Interestingly, the insensitivity of acetylcholinesterase (AChE) to carbofuran was revealed to be ca. 5.5- and 3.7-fold higher in CAS strain compared to, indicating that AChE insensitivity mechanism is associated with carbofuran resistance. In the western blot analysis, two kinds of AChEs were found and type-1 AChE (Nlace1) was identified as the major AChE in N. lugens. The open reading frame of Nlace1 is composed of 2,106 bp (ca. 78 Kd) and revealed 52.5% and 24.3% identity compared with Nephotettix cincticeps and Drosophila melanogaster, respectively. In the screening of point mutations, four amino acid substitutions (G119A, F/S330Y, F331I and H332L) were identified in the CAS strain that likely contribute to the AChE insensitivity. The allele frequencies of these mutations increased in the survived populations following the selection by LC50 of carbofuran, confirming that they are in fact associated with reduced sensitivity to carbofuran in N. lugens. These point mutation can be useful for the monitoring of resistance levels in conjunction with QS methods.

We identified and characterized the full-length cDNA sequences encoding two acetylcholinesterases (ClAChE1 and ClAChE2) and a salivary gland-specific cholinesterase (ClSChE) from the common bed bug, Cimex lectularius. All three cholinesterase genes (Clac1, Clace2 and Clsce) have conserved motifs, including a catalytic triad, a choline binding site and an acyl pocket. Phylogenetic analysis showed that ClAChE1 belongs to the insect AChE1 clade, whereas ClAChE2 belongs to the insect AChE2 clade. ClSChE was grouped into the clade containing all AChE1s, suggesting its paralogous relationship to ClAChE1. Transcription levels of Clace1 were higher than those of Clace2 in all tissues examined, including the central nervous system (CNS). In contrast, the Clsce transcript was not detected in the CNS but specifically found in the salivary gland in much higher levels (>3000 fold) than those of Clace1 and Clace2. Western blot analysis using anti-ClAChE antibodies in conjunction with activity staining revealed that ClAChE1 is more active than ClAChE2 whereas ClSChE has little enzyme activity. Three-dimensional structure modeling suggested that ClAChEs and ClSChE shared structural similarities, but had some differences in the residues forming the acyl pocket and oxyanion hole. The current findings should provide valuable insights into the evolution and functional diversification of insect cholinesterase.

Capsaicin β-glucoside was isolated from the feces of Helicoverpa armigera, H. assulta and H. zea that fed on capsaicin-supplemented artificial diet. The chemical structure was identified by NMR spectroscopic analysis as well as by enzymatic hydrolysis. The excretion rates of the glucoside were different among the three species; those in the two generalists, H. armigera and H. zea, were higher than in a specialist, H. assulta. UDP-glycosyltransferases (UGT) enzyme activities measured from the whole larval homogenate of the three species with capsaicin and UDP-glucose as substrates were also higher in the two generalists. Compared among five different larval tissues (labial glands, testes from male larvae, midgut, the Malpighian tubules, and fatbody) from the three species, the formation of the capsaicin glucoside by one or more UGT is high in the fat body of all the three species as expected, as well as in H. assulta Malpighian tubules. Optimization of the enzyme assay method is also described in detail. Although the lower excretion rate of the unaltered capsaicin in H. assulta indicates higher metabolic capacity toward capsaicin than in the other two generalists, the glucosylation per se seems to be insufficient to explain the decrease of capsaicin in the specialist, suggesting H. assulta might have another important mechanism to deal with capsaicin more specifically.

Autophagy is a process of intracellular bulk protein degradation, in which the accumulated proteins and cytoplasmic organelles are degraded. It plays important roles in cellular homeostasis, apoptosis, and development, but its role during early embryo development remains contentious. Therefore, in the present study, we investigated the effects of 3-methyladenine (3-MA) on early embryonic development in pigs. we also investigated several indicators of developmental potential, including mitochondrial distribution, genes expressions (autophagy-, apoptosis- related genes), apoptosis and ER-stress, which are affected by 3-MA. After in vitro maturation and fertilization, presumptive pig embryos were cultured in PZM-3 medium supplemented with 3-MA for 2 days at 39℃, 5% CO2 in air. Developmental competence to the blastocyst stage in the presence of 3-MA was gradually decreased according to increasing concentration. Thus, all further experiments were performed using 2 mM 3-MA. Blastocysts that developed in the 3-MA treated group decreased LC3-II intensity and expressions of autophagy related genes than those of the untreated control, resulting in down-regulates the autophagy. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 3-MA treated group compared with control (6.0±1.0 vs 3.3±0.6, p<0.05). Also, the expression of the pro-apoptotic gene Bax increased in 3-MA treated group, whereas expression of the anti-apoptotic gene Bcl-XL decreased. Mito Tracker Green FM staining showed that blastocysts derived from the 3-MA treated group had lower mitochondrial integrity than that of the untreated control, resulting in decrease the embryonic qualities of preimplantation porcine blastocysts. Then, the expression of the spliced form of pXBP-1 product (pXBP-1s) increased in 3-MA treated group, resulting increase of ERstress. Taken together, these results indicate that inhibition of autophagy by 3-MA is closely associated with apoptosis and ER-stress during preimplantation periods of porcine embryos.

본 논문에서는 산업 현장에서 비전기술을 이용한 객체의 검사나 분류에 사용되는 빠르고 효율적인 최소 사각형 근사화 방법을 대해서 소개한다. 주어진 객체의 최소 사각형 근사를 위해서 객체의 전체 외곽선을 사용하지 않고 n 개로 샘플링된 에지 점들에 대해서만 각도 히스토그램을 생성하고 이를 이용하여 객체의 장축과 단축을 계산한다. 최종 근사 사각형을 찾기 위해 이전 단계에서 얻어진 장축과 단축을 중심으로 하는 4개의 모서리점을 갖는 사각형을 찾는다. 이후에, 잡음에 의한 오차를 줄이기 위해 첫 번째 사각형의 각도를-1∼+1로 변형하여 추가적으로 2개의 후보 사각형을 생성한 후, 3개의 추정된 사각형과 원본 영상의 외곽선의 매칭 정도를 측정하여 오류가 최소화 되는 사각형을 최종 사각형 영역으로 선택한다. 제안된 방법을 몇 개의 실험데이터에 대해 실험한 결과 기존의 방법에 비해 빠르고 효율적인 최소 사각형 근사가 가능하였다.

A 20' X 20' region around 30 Doradus in the Large Magellanic Cloud (LMC) is observed and analyzed in the near-infrared. We obtain polarimetry data in the J, H, and Ks bands using the SIRIUS polarimeter SIRPOL at the Infrared Survey Facility 1.4 m telescope. We measure the Stokes parameters of 2562 point-like sources to derive the degree of polarization and the polarization position angles. We discuss the statistics of the groups classified by color-magnitude diagram and proper motions of the sources, in order to separate the Galactic foreground sources from those present in the LMC. We notice that groups classified by the proper motion data show a tendency towards different polarimetric properties.

The forest of Korea had been severely degraded since early 1900s until 1950s. Korean Government has successfully accomplished the reforestation works since 1960s. However, some plantations showed poor survival and growth caused by ignoring site characteristics in selecting plantation species and lack of tending works such as thinning. The natural regeneration of indigenous species, such as Quercus species and Pinus densiflora Siebold & Zucc., were examined in the plantations of Pinus koraiensis Siebold & Zucc. and Larix kaempferi Fortune ex Gordon. Quercus species regenerated mainly by sprouting while P. densiflora regenerated naturally from a few mother trees that remained in the plantations. P. koraiensis showed poor survival (IVI ≤ 25%) and suppressed growth (height ≤ 3 m and DBH ≤ 3 cm at 20 year-old) by Quercus species or P. densiflora in the plantation areas, however had high survival (IVI ≥70%) and growth (8 m height and 14.1 cm DBH at 20 year-old) in areas where silvicultural practices were conducted. L. kaempferi showed good survival (IVI ≥40%) and growth (17.2 m height and 16.3 cm DBH at 30 year-old) mostly in valley areas, while it was nearly dead (IVI ≤ 10%) in ridge or ridge-slope areas and was replaced by indigenous species such as Quercus species (IVI ≥ 25～55%) or P. densiflora (IVI ≥ 18～50%).

목적: International Association of Contact Lens Educators(IACLE)가 실시하는 STE결과를 분석하여 한국에서의 콘택트렌즈 교육 실태를 알아보고자 한다. 방법: 2010년 IACLE에서 주관한 STE에 참여한 12개 대학 안경광학과 학생들의 시험성적을 분석하여 대학 및 국가간 비교를 실시하였다. 결과: 12개 대학의 평균은 100점 만점을 기준으로 하여 54.01점이었으며 평균 합격률은 62.75%였다. 문항을 주제별로 7개 부류로 나누어 분석한 결과 평균 점수는 각각 전안부 해부 및 생리 48.73점, 콘택트렌즈 디자인 및 관리 46.14점, 콘택트렌즈 피팅원리 49.08점, 환자 검사 및 콘택트렌즈 처방 73.37점, 콘택트렌즈 관리 및 유지 66.12점, 콘택트렌즈에 대한 안구 반응 55.45점, 콘택트렌즈 착용으로 인한 부작용 52.54점으로 나타났다. 교육기간에 따라 2,3년제 대학과 4년제 대학을 비교해 본 결과 2,3년제 대학의 평균 점수가 53.35점, 4년제 대학의 점수가 54.94점으로 큰 차이를 보이지 않았다. 국가간 비교에서는 중국, 인도, 프랑스, 독일에 비해 한국 학생들의 점수가 다소 낮은 것으로 확인 되었다. 결론: 평균점수와 합격률에 있어 학교 및 과목 사이에 편차가 확인 되었다.

Ovulation synchronization (ovsynch) has proved to increase the number of insemination in cattle by overcoming the problems of heat detection. The aim of this study was to do ovsynch in water buffaloes where heat detection is a major reproductive problem and to determine the conception rates after timed artificial insemination (TAI). Twenty cyclic buffaloes at 60 days postpartum were selected by examining 24 unobserved estrus buffaloes based on milk progesterone assay (progesterone concentration 1.0 ng/ml) from the Mymensingh district of Bangladesh. Ovsynch treatment regimen was started irrespective of the stage of estrous cycle. Gonadorelin (500 ) was injected intramuscularly at Day 0 followed by Alfaprostol (8 mg) at Day 7. A second injection of Gonadorelin was given at Day 9 and TAI was done with frozen semen from Mediterranean buffalo bulls at 16~20 hours of the second Gonadorelin injection. Milk progesterone ELISA at Day 10~12 post AI confirmed ovulation in 16 out of 20 (80%) buffaloes (progesterone concentration 1.0 ng/ml). High progesterone concentration ( 1.0 ng/ml) at Day 10~12 and Day 22~24 of AI showed pregnancy in six out of 20 (30%) buffaloes. Pregnancy was further confirmed by ultrasonography at Day 40 in these six buffaloes. In conclusion, ovsynch followed by TAI could be applied in cyclic buffaloes for overcoming the estrus detection problems; however, more studies are needed to increase the conception rate.