RNAi (RNA interference) is a tool for silencing of target genes through sequence-specific manner. Spodoptera exigua belongs to Noctuidae family of Lepidoptera and is serious threat to crops of economic importance. One of S. exigua chymotrypsin gene (SeCHY2) was cloned into the L4440 vector to produce sequence specific dsRNAs (double-stranded RNAs). Recombinant L4440 vectors were transformed into Escherichia coli strain HT115 (DE3). Oral delivery of bacterially expressed dsRNA gave significant larval mortality. Quantitative real-time PCR results showed that expression level of target SeCHY2 gene in the larval gut tissue was significantly down-regulated. Pretreatment with an ultra-sonication and heating to disrupt bacterial cell wall/membrane significantly increased the insecticidal activity of the transformed bacteria

Malignant gliomas and glioblastomas are the most common type of primary brain tumors. The treatment of malignant glioma involves surgery, radiation, and chemotherapy. These therapies have not been successful in curing malignant glioma and typically associated with dismal prognosis. Therefore, we can investigate thymidine kinase activity and cytotoxic effect after transfer suicide gene in U-251 glioma cells. We assessed expression patterns of green fluorescence protein(GFP) after infected with adenovirus in U-251 cells. After infection of HSV-tk in U-251 cells, we observed thymidine kinase activity with [3H]-penciclovir and cytotoxic effect by treated with ganciclovir. We could observe that expression level of GFP was increased according to infected concentrations in U-251 cells. GFP was not expressed in 1moi and 10moi, and slightly expressed in 30moi. Expression level of GFP was largely increased in 50moi and almost cells expressed GFP in 100moi. GFP expression has shown clear image in 100moi compared with other concentrations. We also investigated thymidine kinase activity using [3H]-penciclovir after infection of suicide gene HSV-tk into U-251 cells. Thymidine kinase activity increased in 10moi concentration compared with empty adenovirus infection. We could find that thymidine kinase activity was elevated proportional to HSV-tk infection amount in 30moi and 50moi. For evaluation of cellular cytotoxic effect of HSV-tk, we treated ganciclovir to U-251 cells and assessed cytotoxicity by using MTT assay. We could identifiy that cytotoicity appeared in very low concentration of HSV-tk compared with cancer cells originated with other organs. Cytotoxic effect was shown about 15% of U-251 cells of total cells in 5moi. By infection 10moi of HSV-tk, cytotoxic effect was intensively increased and about 60% of U-251 cells became extinct. About 70% cells exhibited cytotoxic effect in 30moi and more than 80% cells also appeared cytotoxic effect by infection of HSV-tk in 50moi, 100moi, and 200moi. Therefore, we could confirm to gene expression in U-251 cells was increased proportional to infected gene concentrations. Also we could find that thymidine kinase activity elevated with according to infected concentration and cellular cytotoxic effect was shown in very low concentration and higher cytotoxic effect also appeared by infection of suicide gene HSV-tk into U-251 glioma cells. These results suggest that gene therapy with suicide gene will be successful in curing brain tumors containing malignant glioma and glioblastoma.

The objective of this study was to investigate the proteome composition in pretermand term‐derived human umbilical cord. Umbilical cord samples were collected from 6 preterm infants with gestational age less than 36 weeks or 4 full terms together with medical information during prenatal period. Several biomarkers are routinely used clinically for predicting preterm labor; however, these factors are either nonspecific or detected too late. Protein profiles were performed on samples from both preterm‐ and termderived human umbilical cord by using Two‐dimensional gel electrophoresis (2‐DE). Approximately 200 different proteins were identified between preterm‐ and term‐delivered umbilical cords. Among them, differentially expressed 34 proteins were identified in 48 protein spots. In the preterm‐derived human umbilical cords, 15 proteins were present at higher levels (2.0‐ to 9.28‐fold increases) and 19 were present at lower levels (2.0‐ to 11.8‐ fold decreases or not detectable) compared to the control term‐derived umbilical cords. Proteomics approaches such as 2‐DE could greatly facilitate the discovery of new and better biomarkers. The high sensitivity and specificity achieved by the combined use of the selected biomarkers show great potential for the early detection of Adverse pregnancy outcomes such as pre‐eclampsia, small for gestational age infants, preterm delivery and placental abruption are associated with higher mortality. Increased amount of HIF‐1 α, GAPDH and HSP27 were observed in preterm‐derived umbilical cords was due to hypoxia‐ dependant and oxidative stress‐independent manner. Moreover, we isolated HUVEC (Human umbilical vascular endothelial cells) from preterm‐ and term‐derived umbilical cords and examined LDH activity. The results of the current study may provide important insights into the molecular mechanisms underlying umbilical cord development and also these data will contribute to a better understanding of the composition of preterm‐ and term ‐ derived human umbilical cord and aid the discovery of novel biomarkers for the prenatal diagnosis of fetal abnormalities

For investigate intracellular function and role of genes in the biological processes, various gene delivery methods into cell have been developed. Many studies performed to construct optimum conditions of gene delivery into cells and tissues. In this study, we examined efficiency of gene delivery-complexed with cationic lipid vector in human cancer cell lines. GFP plasmids were complexed with cationic lipid and transfected into human cancer cell lines at different concentrations. And then, expression of GFP was analysed with fluorescent microscope and FACS. To determine efficiency of gene delivery, we investigated GFP expression level in various cancer cell lines. GFP expression cells were not shown in hepatocellular carcinoma cell line HepG2 and lung carcimona cell line A549 after 24hr transfection, while, GFP expression cells were observed at 500ng concentration after 48hr transfection. In colorectal carcinoma cell line HCT116, GFP expression cells were observed at 100ng and 500ng concentrations after 24hr transfection and slightly increased at 48hr. After transfection into ovary adenocarcinoma cell line SKOV3, we could found that many cells expressed GFP at 500ng concentration after 24hr and highly elevated GFP expression cells after 48hr. For further evaluate gene expression level, we confirmed GFP expression level by using FACS analysis after 48hr transfection. As a result, HepG2 was expressed GFP in very low level at 10ng, 100ng, and 500ng concentrations. We also identified that GFP was expressed low level at 10ng and 100ng in HCT116 and A549, but highly increased at 500ng concentration to 14.19% and 16.57%, respectively. In case of SKOV3, GFP expression was highly elevated to 13.14% at 100ng and 58.10% at 500ng compared with 10ng transfection. By Comparing efficiency of gene expression among cancer cell lines, GFP expression was similar with cell lines at 10ng transfection, but significantly differed from cell lines at 500ng higher concentration. Additionally, GFP expression level of SKOV3 was showed about 10 fold higher than HepG2, and about 4 fold higher than HCT116 and A549 at 500ng. These results demonstrated that efficiency of gene delivery-complexed with cationic lipid vector was the highest in SKOV3, while HepG2 was showed the lowest efficiency. Taken together, we could determined that efficiency of gene delivery into cells differed from each human cancer cell lines. Our study suggest that cellular properties should be considered in gene delivery-complexed with cationic lipid vector to improve cellular expression efficiency of gene.

조혈 줄기 세포에의 효과적인 유전자 전달은 유전자 치료의 새로운 가능성을 제시할 수 있다. 레트로바이러스를 이용한 유전자 전달 기술은 많은 기초 연구와 임상 시도가 이루어진 대표적인 바이러스이다. 그러나 현재 사용되고 있는 in vitro에서의 조혈 줄기 세포에의 유전자 도입은 조혈 줄기 세포의 분화 유도, 자기 복제 능력과homing 능력의 저하 등 많은 문제점이 있다. 본 연구는 이러한 문제점을 극복하기 위한 방법으로서 마우스의 대퇴골에 직접 레트로바이러스를 이식하는 IBM (Intra-Bone Marrow) 방법을 이용하여 조혈 줄기 세포에의 효과적인 유전자 도입을 시도하였다. IBM 이식 2주 후 마우스의 각 조직을 분석한 결과, 골수뿐 아니라 림파절, 비장, 간장 세포 등에서 유전자가 안정적으로 발현하는 것을 관찰하였다. 또한, 6.4+-2.7%의 골수조직 존재 조혈줄기/전구세포에서 도입된 유전자가 안정적으로 발현하고 있는 사실을 확인하였다. 본 연구의 결과를 바탕으로 IBM 이식 방법을 이용한 생체 조직 내 레트로바이러스의 유전자 도입은 조혈 줄기 세포를 이용한 유전자 치료에 매우 효과적인 방법이라는 사실을 시사해주고 있다.

Gene delivery is one of the keen interests in animal industry as well as research on gene functions. Some of the in vivo gene delivery techniques have been successively used in various tissues for the gene therapy and transgenesis. Despite intensive efforts, it still remains to overcome problems of limited local and regional administration and low transgene expression. To improve the efficiency of gene delivery, a new procedure was tested. We injected exogenous DNA containing LacZ into the female or male gonads and then pulsed electric field. Electroporated gonads showed positive X-gal staining in many seminiferous tubules of the porcine fetal gonads. Exogenously introduced LacZ genes were also expressed in female porcine gonad. In addition, we demonstrated efficient gene delivery in gonad of adult mouse. Furthermore, we succeed to generate genetically modified germline cells showing GFP and positive X-gal signals. The results suggest that the newly developed gene delivery is an effective way of in vivo transfection in mammals. The developed gene delivery procedure should be useful in producing transgenic animals when combined with primary cell culture and nuclear transplantation.