This study was to analyse the usability of morphological evaluation of vitrified-thawed oocyte before somatic cell nuclear transfer (SCNT) using Oosight imaging system to show spindle. For the vitrification, in vitro matured bovine MII oocytes were treated by two-step freezing medium without (control group) or with 5 ug/ml cytochalasin-b (CCB group). In Exp. 1, after thawing, recovered oocytes in each treatment group were assessed by live image using Oosight imaging system or/and cytoskeletal protein image using immunostaining. In Exp. 2, in each treatment group the in vitro developmental potential of frozen-thawed bovine oocytes post evaluation using Oosight imaging system and then SCNT was investigated. The SCNT embryos were cultured in CR1aa medium supplemented with 10% FBS, 1 mM EGF and 1 mM IGF at 38.5 C in 5% O2 and 5% CO2 in air for 8 days. In Exp.1, the rates of in vitro survival, morphological good grade and spindle normality of CCB treatment group (91.1%, 54.2% and 55.5%) were better than those of control group (86.1%, 48.5% and 48.5%). After SCNT using vitrified-thawed oocyte, the rates of fusion, reconstructed embryos and blastocyst development were also high in CCB treatment group (66.6%, 36.4% and 3.0%) than control group (60.0%, 27.3% and 0%). These results demonstrated that the identification of morphological spindle image of the vitrified-thawed bovine oocytes using Oosight imaging system helps to predict the SCNT embryo quality.
The aim of this study was to investigate the correlation among bone mineral density(BMD), body composition and body circumference on 20's college women in Hwaseong. A total of 86 subjects were measured with BMD and body composition and body circumference. To evaluate the correlation between BMD and body composition, bone density and body weight, body mass index(BMI), lean body mass, muscle mass, fat mass and body fat mass were compared. The results of this study, weight was considered the strong correlation with BMD than the height and BMI seems to be greater significance rather than the lumbar spine and femur BMD. In addition, the relationship between body composition and BMD, lean body mass, muscle mass, body fat mass were the most relevant factors and BMD. The relationship between BMD and body circumference that have been difficult because of not enough previous studies but somewhat the study showed that association.
This study was carried out to identify how a self-stretching exercise program affects pain for each body area, pain relief and job satisfaction for care workers. 20 of 40 care workers with musculoskeletal symptom were randomly selected and participated a self-stretching exercise program consisting of 15 motions. The intervention was done five times or more per weeks for 8 weeks and 1 session lasted within 15 minutes. 'Musculoskeletal symptom survey table' of the Korea Occupational Safety and Health Agency(KOSHA) and JDI(Job Descriptive Index) was used for pain on the musculoskeletal symptom and job satisfaction. Survey were done twice before and after the program. The result of this study showed that self-stretching exercise program group(SSPG) relieved from pain significantly in the shoulders(p<.01) and lumbar(p<.05), comparing to the non selfstretching exercise program group(NSPG). Although no significant difference on variations in the JDI appeared in SSPG, the significant reduction appeared from the colleague relationship and organization in NSPG(p<.05). SSPG showed the significant increase on variations in JDI from the job and organization comparing to NSPG. Especially, the improvement on satisfaction for the organization was shown(p<.05). Accordingly, the self-stretching exercise program for care workers can be said to positively affect the overall pain relief and increase on the JDI.
Urokinas type plasminogen activator (uPA) has been used as a therapeutic agent for treating human diseases such as thrombosis. Attempts to transgenically overexpress the uPA in animal bioreactors have been hampered due to side effects associated with this functional protein hormone on homeostasis. Recently, chicken has been emerged as a potential candidate for use as bioreactor to produce proteins of pharmaceutical importance. Since this species has low homology uPA sequence with mammals, we hypothesized that chicken could be used as a potential bioreactor for production of human uPA. In this study, using replication‐defective Murine Leukemia Virus (MLV)‐based retrovirus vectors encapsidated with Vesicular Stomatitis Virus G Glycoprotein (VSV‐G), we attempted to make transgenic chicken expressing human uPA (huPA). The recombinant retrovirus was injected beneath the blastoderm of non‐incubated chicken embryos (stage X, at laying). After 21 days of incubation (at hatching), all of the 38 living chicks that assayed, were found to express the vector‐encoded huPA gene in various organs and tissues, which was under the control of the Rous Sarcoma Virus (RSV) or Cytomegalovirus (CMV) promoter. Using specific primer set for huPA, PCR and RTPCR analyses of gDNA isolated from these samples demonstrated these chickens were transgenic for huPA. Furthermore, successful germ line transmission of huPA transgene was confirmed and next generation whole body huPA transgenic chickens were also produced. We also assayed huPA protein titer in blood (17.1 IU/ml) and eggs (4.4 IU/ml) of whole body huPA transgenic chicken. Thus, our results demonstrated that chicken could be used as bioreactors to produce huPA.
Somatic cell nuclear transfer (SCNT) is an efficient technique which has been successfully applied to developmental biology, and resulted in the production of offspring from various species. It offers many opportunities in basic and medical research as well as endangered species preservation. On the other hand, embryonic stem (ES) cells are useful research tools for genetic engineering and developing disease models. In previous study, we established bovine IVF embryo derived ES cell line which can be grow indefinitely as undifferentiated cell state. In this study, we compared the effect of two different age cells (bovine ES cell; JNU-ibES-05 or adult ear fibroblast cell) on in vitro developmental potential of bovine SCNT embryo. To produce SCNT embryos, the ES cells or somatic cells were dissociated and transferred into enucleated MⅡ oocytes, and cleaved reconstructed embryos were cultured in CR1aa medium containing 10% FBS, 1 ug/ml epidermal growth factor (EGF) and 1 ug/ml insulin growth factor (IGF) for 8 days. In the result, blastocyst development rate was similar between ES cell treatment group and somatic cell treatment group, 27.7% (10/36) and 28.9% (11/ 38), respectively. However, there was particular difference in development speed from day 5 post SCNT, blastocyst expanding was 1 day faster in ES cell group than in somatic cell group. This difference was analyzed by semi-quantitative RT-PCR using pluripotency, growth and cell cycle gene markers. These results demonstrated that SCNT embryo using ES cell as a donor cell has better growth potential than somatic cell, and it will be a useful tool for a transgenic animal production.
본 연구에서는 azoxymethane (AOM)과 dextran sodium sulfate (DSS)로 유도된 대장 발암과정에 대한 셀레늄의 방어 효과를 조사하였다. 셀레늄 결핍(0.02 ppm Se), 정상(0.1 ppm Se), 과다(0.5 ppm Se)사료를 12주간 식이로 급여하여 혈액검사와 대장암 발생의 초기단계인 aberrant crypt foci (ACF)수를 측정했으며, 암 발생율을 조사하였다. ICP-AES 를 사용하여 간의 셀레늄 농도를 측정하였으며, 또한 셀레늄포함 항산화효소인 glutathione peroxidase (GPx) 활성을 알아보았다. 또한 TUNEL assay와 PCNA, β-catenin에 대한 면역조직 염색을 수행하였다. ACF 수 및 종양 발생률에 있어서, 셀레늄과다사료를 급여한 군이 정상셀레늄사료를 급여한 군보다 낮았으며, 셀레늄결핍사료를 급여한 군은 오히려 ACF 수 및 종양 발생률이 높았다. GPx 활성은 셀레늄의 섭취가 과다한 군에서 높게 나타났으며, 이 때, TUNEL 에서 apoptotic positive cell이 증가하는 것을 확인했다. 또 한 셀레늄의 섭취가 과다한 군에서 PCNA와 β-catenin의 발현이 감소됨을 볼 수 있었다. 본 마우스 모델실험에서 셀레늄은 여러 기전에 의해 대장암 발생을 억제할 수 있을 것으로 사료된다.
The porcine reproductive and respiratory syndrome virus (PRRSV) has three major structural proteins which designated as GP4, GP5, and M. They have been considered very important to arouse the humoral and cellular immune responses against PRRSV infection and proposed to be the excellent candidate proteins in the design of PRRS bioengineering vaccine. However, the PRRSV structural proteins are produced in low levels in the infected cells because it forms insoluble protein and possesses several transmembrane regions. To overcome this problem, we fused the GP4, GP5, and M with SUMO (Small ubiquitin-related modifier), and expressed the fused gene in Bm5 cells and silkworm larvae. Expression of the proteins were analyzed by 12% SDS-PAGE and western blotting using 6xHis tag and porcine anti-PRRSV antibodies. In results, SUMO fused proteins were expressed at a high level in Bm5 cells. The levels of protein using the silkworm larvae is higher than that using Bm5 cells. The fused protein was purified by Ni-NTA affinity chromatography. This study demonstrated that SUMO, when fused with PRRSV structural proteins, was able to promote its soluble expression. This may be a better method to produce PRRSV structural proteins for vaccine development.
Apolipophorin-III (apoLp-III) is a hemolymph protein whose function is to facilitate lipid transport in an aqueous medium in insects. Recently, apolipophorin-III in Galleria mellonella and Hyphantria cunea was shown to play an unexpected role in insect immune activation. We show here a novel possible function/role of the apoLp-III in insects. To investigate the genes which have a relationship with apoLp-III in fall webworm larvae, we reduced endogenous Hc apoLp-III mRNA levels in larvae via RNA interference (RNAi). The RNAi-mediated Hc apoLp-III reduction resulted in the reduction of antioxidants, like MnSOD, catalase, and glutathione S transferase as well as immune proteins. In particular, expression of MnSOD commonly decreased in fat body, midgut, and hemocytes following the knockdown of Hc apoLp-III, which induced an elevated level of superoxide anion in Hyphantria cunea larvae. The observed effect of Hc apoLp-III RNAi suggests that Hc apoLp-III is related to the action/expression of antioxidants, especially MnSOD.
A new insect member of the STAT family of transcription factors (HcSTAT) has been cloned from the lepidopteran, Hyphantria cunea. The domain involved in DNA interaction and the SH2 domain are well conserved. The gene is transcribed at a low level during all stages of development, and transcribed in hemocyte, fat body, midgut, epidermis, and Malpighian tubule. Especially, hemocyte and Malpighian tubule showed transcriptional activation of HcSTAT upon Gram-negative and -positive bacteria challenge. Gram-negative and -positive bacteria challenge specifically results in nuclear translocation of HcSTAT protein and induction of DNA-binding activity that recognizes a STAT target site in H. cunea hemocyte. In vivo treatment with sodium orthovanadatetranslocates HcSTAT to the nucleus in hemocyte cells.
The Classical Swine Fever Virus (CSFV) is a member of the Pestivirus genus of the Flaviviridae. The genome of CSFV is a positive single-stranded RNA molecule 12.3 kb and contains a single large open reading frame (ORF). The polyprotein composed of eight nonstructural and four structural proteins (nucleocapsid protein C and three envelope glycoprotein E0, E1 and E2). E2, the most immunogenic of the CSFV glycoproteins, induces a protective immune response in swine. To determine the characteristics of the CSFV, LOM strain, we investigated the nucleotide sequence of the glycoprotein E0, E1 and E2. Comparison of the LOM with the other strains revealed nucleotide sequence identity ranging from 97 to 98%. Expression of the glycoprotein E2 was identified by SDS-PAGE and Western blot analysis using anti-CSFV E2 monoclonal antibodies in Sf21 cells. The expression levels of glycoprotein E2 were observed from day 3 and 5 days maximum. In addition, its expression efficiency by media and cell line was investigated. The result showed that High-Five cells and Grace’s insect media for Sf21 were the best conditions for the expression of the glycoprotein E2.
Apolipophorin-Ⅲ (apoLp-Ⅲ) is a hemolymph protein whose function is to facilitate lipid transport in an aqueous medium in insect. Recently, apolipophorin-Ⅲ in Galleria mellonella and Hyphantria cunea was shown to play an unexpected role in insect immune activation. We show here a novel possible function/role of apoLp-Ⅲ in insects. To investigate the genes which have a relationship with apoLp-Ⅲ in fall webworm larvae, we reduction of endogenous Hc apoLp-Ⅲ mRNA levels in larvae via RNA interference (RNAi). The RNAi-mediated Hc apoLp-Ⅲ reduction resulted in the reduction of antioxidants, like MnSOD, catalase, and glutathione S transferase as well as immune proteins. In particular, expression of MnSOD commonly decreased in fat body, midgut, and hemocytes following the knockdown of Hc apoLp-Ⅲ, which induced an elevated level of superoxide anion in H. cunea larvae. The observed effect of Hc apoLp-Ⅲ RNAi suggests that Hc apoLp-Ⅲ is related to the action/expression of antioxidants.
Innate immunity responses are triggered by the immune challenge and therefore involve signaling processes. The cellular response is initiated by hemocytes and mainly involves phagocytosis and encapsulation of intruders by these cells. To address whether Hc-STAT is activated upon bacterial challenge, we examined the subcellular location of STAT protein in hemocyte by immunostaining. A new insect member of the STAT family of transcription factors (Hc-STAT) has been cloned from the lepidopteran, Hyphantria cunea. The domain involved in DNA interaction and the SH2 domain are well conserved. The gene is transcribed at a low level during all stages of development, and the protein is present in hemocytes, fat body, midgut, epidermis, and Malphigian tuble (Mt). Especially, hemocytes and Mt showed transcriptional activation of Hc-STAT upon Gram (-) bacteria and fungal challenge. Gram (-) bacteria and fungal challenge specifically results in nuclear translocation of Hc-STAT protein and induction of DNA-binding activity that recognizes a STAT target site in H. cunea hemocyte. In vitro treatment with pervanadate translocates Hc-STAT to the nucleus in hemocyte cells. Here we report the first evidence for the involvement hemocyte JAK/STAT pathway upon microbial infection in lepidopteran insect.
Embryonic stem (ES) cells are known to have an infinite proliferation and pluripotency that are associated with complex processes. The objective of this study was to examine expression of genes differentially regulated during differentiation of human ES cells by suppression subtractive hybridization (SSH). Human ES cells were induced to differentiate into neural precursor cells via embryoid body. Neural precursor cells were isolated physically based on morphological criteria. Immunocytochemical analysis showed expression of pax6 in neural precursor cells, confirming that the isolated cells were neural precursor cells. Undifferentiated human ES cells and neural precursor cells were subject to the SSH. TPX2 (Targeting Protein for Xklp2 (Xenopus centrosomal kinesin-like protein 2)) was identified, cloned and analyzed during differentiation of human ES cells into neural lineages. Expression of TPX2 was gradually down-regulated in embryoid bodies and neural precursor cells relative to undifferentiated ES cells. Targeting Protein for Xklp2 has been shown to be involved in cell division by interaction with microtubule development in cancer cells. Taken together, result of this study suggests that TPX2 may be involved in proliferation and differentiation of human ES cells.
Somatic cells nuclear transfer (SCNT) is a useful tool in studies of developmental biology and animal cloning. However, SCNT experiments only are allowed to skilled technical experts. In this experiment, laser-assisted zona pellucida piercing tool (LASER) was applied in murine SCNT. LASER minimized the use of piezo-driven micromanipulator (PIEZO), reducing chances of problems caused by PIEZO pulses. LASER reduced time that took to pierce zona pellucida in removal of nucleus from oocyte and somatic cell injection, which might have taken longer time with PIEZO. Time and difficulties that took researcher of equivalent skilled for their experiments were decreased with LASER, and this might affect the improvement of embryonic development. (LASER, 6.2% versus PIEZO, 2.9%; P<0.05). Thus, these data support that the use of LASER can be used for zona pellucida piercing in murine SCNT program as an alternative to PIEZO.
The present study investigated the effects of follicle stimulating hormone (FSH) and human chorionic gonadotrophin (hCG) on the nuclear maturation of canine oocytes. Oocytes were recovered from mongrel female ovaries in various reproductive states; follicular, luteal or anestrous stage. Oocytes were cultured in serum-free tissue culture medium (TCM)-199 supplemented with various concentrations of FSH (Exp. 1: 0, 0.5, 1.0 or 10 IU) or hCG (Exp. 2: 0, 0.5, 1.0 or 10 IU) or both (Exp. 3: 1 IU FSH + 1 IU hCG) for 72 hr to determine the effective concentration of these hormones, and to examine their combined effect. After maturation culture, oocytes were denuded in PBS containing 0.1% (w/v) hyaluronidase by gentle pipetting. The denuded oocytes were stained with 1.9 μM. Hoechst 33342 in glycerol and the nuclear state of oocytes was evaluated under UV light. More (p<0.05) oocytes matured to MII stage when follicular stage oocytes were supplemented with 1 IU FSH (6.2%) compared with the control, 0.1 or 10.0 IU FSH (0 to 1.2%). Significantly higher (p<0.05) maturation rate to MII stage was observed in follicular stage oocytes supplemented with 1.0 IU hCG (7.2%) compared with the control or other hCG supplemented groups (0 to 1.5%). However, the combination of FSH and hCG did not improve the nuclear maturation rate of canine oocyte (2.4 %) compared with FSH (6.2%) and hCG alone (7.2%). In conclusion, FSH or hCG alone significantly increased the maturation of canine oocytes to MII stage.