• Journal of Plant Biotechnology
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• 제5권4호
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• pp.215-219
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• 2003

Optimal shoot regeneration and transformation conditions of root type chicory (Cichorium intybus L. var. sativus cv Cesare) were studied. Leaf explants were co-cultured with Agrobacterium tumefaciens, which contained NPTII as a selectable marker and a rice homeotic gene, OsMADS1, that encodes a MADS-domain-containing transcription factor. After one day of co-cultivation, explants were transferred to selection media consisting of MS basal medium supplemented with 0.5 mg/L BAP, 0.1 mg/L IAA, 70 mg/L kanamycin, and 250 mg/L cefotaxime. PCR and Southern blot analyses revealed stable integration of the OsMADS1 gene in the chicory genome. Four-teen original transgenic plants ($T_o$ plants) were acclimatized in the greenhouse and examined for their morphological characters. Most of the transgenic plants showed altered morphologies, such as short, bushy, and early-flowering phenotypes with reduced apical dominance. Additionally, half of the transgenic plants exhibited altered leaf shapes, and 4 out of 14 plants were sterile. These phenotypes were inherited by the next generation. Northern blot analysis confirmed expression of the OsMADS1 gene in both floral and vegetative organs.

• The Plant Pathology Journal
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• 제25권2호
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• pp.179-183
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• 2009

To facilitate the genetic manipulation of Cladosporium phlei, a causal agent of leaf spot disease in timothy (Phleum pretense), protoplast-mediated transformation of C. phlei has been developed and the resulting transformants were characterized in this study. Hygromycin B resistance was applied as a dominant selection marker due to the sensitivity of C. phlei to this antibiotic. The transformation efficiency ranged from approximately 20-100 transformants per experiment. Southern blot analysis of stable transformants revealed that transformation occurred by way of stable integration of the vector DNA into the fungal chromosome. PCR analysis and plasmid rescuing of randomly selected transformants suggested that integration of tandem repeat copies of vector DNA was common. In addition, multiple integrations of the transforming vector at different chromosomal sites were also observed. The establishment of a transformation method for C. phlei facilitates strain improvement of this fungus and can be applied as an initial step in the molecular analysis of pigment production in this fungus.

• Asian Pacific Journal of Cancer Prevention
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• 제16권15호
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• pp.6663-6668
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• 2015

Background: Treatment failure in leukemia is due to either pharmacokinetic resistance or cell resistance to drugs. Materials and Methods: Gene expression of multiple drug resistance protein (MDR-1), multidrug resistance-related protein (MRP) and low resistance protein (LRP) was assessed in 45 pediatric ALL cases and 7 healthy controls by real time PCR. The expression was scored as negative, weak, moderate and strong. Results: The male female ratio of cases was 2.75:1 and the mean age was 5.2 years. Some 26/45 (58%) were in standard risk, 17/45(38%) intermediate and 2/45 (4%) in high risk categorie, 42/45 (93%) being B-ALL and recurrent translocations being noted in 5/45 (11.0%). Rapid early response (RER) at day 14 was seen in 37/45 (82.3%) and slow early response (SER) in 8/45 (17.7%) cases. Positive expression of MDR-1, LRP and MRP was noted in 14/45 (31%), 15/45 (33%) and 27/45 (60%) cases and strong expression in 3/14 (21%), 11/27 (40.7%) and 8/15 (53.3%) cases respectively. Dual or more gene positivity was noted in 17/45 (38%) cases. 46.5 % (7/15) of LRP positive cases at day 14 were in RER as compared to 100% (30/30) of LRP negative cases (p<0.05). All 8 (100%) LRP positive cases in SER had strong LRP expression (p=<0.05). Moreover, only 53.3% of LRP positive cases were in haematological remission at day 30 as compared to 100% of LRP negative cases (p=<0.05). Conclusions: Our study indicated that increased LRP expression at diagnosis in pediatric ALL predicts poor response to early treatment and hence can be used as a prognostic marker. However, larger prospective studies with longer follow up are needed, to understand the clinical relevance of drug resistance proteins.

• Asian Pacific Journal of Cancer Prevention
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• 제13권12호
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• pp.6475-6479
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• 2012

Background: Recent studies have demonstrated an association between insulin growth factor (IGF) and insulin growth factor binding protein-3 (IGFBP-III) serum levels and increased risk for various cancers. However, little information is available on clinical implications of the IGF system in Indian patients with cervical cancer. This study explored associations by analyzing their expression profiles in cervical cancer cases. Materials and Methods: Totals of 50 patients with advanced cervical cancer and 40 healthy controls were enrolled. Human papillomavirus (HPV) and cervical biopsy sample were obtained from all participating women. Circulatory levels were estimated by ELISA and the tissue expression was assessed using RT-PCR and Western blotting. Results: Levels of IGF-I and II showed significant increase whereas IGFBP-III showed significant decline in all patients as compared to controls. Spearman correlation analysis between IGFs and HPV status showed significant correlations. Conclusions: We demonstrated elevated circulating levels and tissue expression of IGF-I and IGF-II in advancer cancer cervix patients, as compared with controls, with a converse trend being apparent for IGFBP-III. In future, associations of the IGF system and clinical outcome of cervical cancer patients in post treatment samples might point to significance in disease mapping as a prognostic marker after validation with a larger patient series.

• Asian Pacific Journal of Cancer Prevention
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• 제13권4호
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• pp.1297-1303
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• 2012

Aims: Primary hepatocellular carcinoma (HCC) is a common malignancy often related to hepatitis viral infection. Smad4 is known to mediate the TGF-${\beta}$ pathway to suppress tumorigenesis. However, the function of Smad4 in HCC is still controversial. In this study we compared levels of Smad4 in HCC tissues with or without hepatitis virus infection and adjacent normal-appearing liver. Methods: Samples from HCC patients were analyzed for Smad4 protein and mRNA expression by immunohistochemistry (IHC), RT-PCR and Western blotting. Results: We found that tumor tissues expressed less Smad4 mRNA and protein than the adjacent tissues. Most HCC tumor tissues were negative for Smad4 in IHC staining, while the majority of adjacent tissues were positively stained. Interestingly, protein levels were higher in HCC tissues with viral hepatitis than those without virus infection. Suppression of expression appeared closely related to HCC, so that Smad4 appears to function as a tumor suppressor gene (TSG). Conclusion: Patients with hepatitis viral infection, at higher risk for HCC, exhibited increased Smad4 protein expression suggesting hepatitis virus may modulate Smad4 expression, which is functionally distinct from its putative role as a TSG. Smad4 expression may thus be an applicable marker for diagnosis and/or a target to develop therapeutic agents for HCC.

• Asian Pacific Journal of Cancer Prevention
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• 제15권17호
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• pp.7091-7095
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• 2014

Background: The study aimed to evaluate the incidence of CpG island promoter methylation of BMP6, a member of the transforming growth factor beta family, in tissue samples from colorectal cancers (CRC) and look for its association with BMP6 expression and clinicopathological correlation. Materials and Methods: Methylation specific PCR for the BMP6 promoter region was performed with 85 frozen tissue samples of CRC and 45 of normal colon. Methylation status of MLH1 was also determined by the same method. Expression of BMP6 was evaluated by immunohistochemistry (IHC), using Allred's scoring system. The methylation status was analyzed against clinical and pathological parameters in CRC. Results: The study revealed BMP6 hypermethylation in 34 of 85 tumor specimens (40%), and 15 out of 45 normal tissue samples from CRC (33%). The incidence of hypermethylation was inversely correlated with IHC score. Allred's scores of 7 or more were correlated with lower frequency of BMP6 hypermethylation (29% compared to 50% in the remaining, p-value 0.049). However, there was no association between hypermethylation status and any clinicopathological parameters. The methylation status of BMP6 was not correlated with that of MLH1, a key methylation determinant in CRC. On survival analysis, there was no significant difference in progress-free survival (PFS) between the cases with and without hypermethylation (2-year PFS 74% and 76%, respectively). Conclusions: CpG island methylation of BMP6 is found in high frequency in CRC and this epigenetic event is associated with suppressed protein expression in the tumor tissue. However, the marker is not associated with tumor progression of the disease.

• 농업과학연구
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• 제43권2호
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• pp.205-214
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• 2016

A drought-tolerant transgenic rice (Agb0103) was developed using a pepper methionine sulfoxide reductase (CaMsrB2) under the control of rice Rab21 promoter with a selection marker, the phosphinothricin acetyltransferase (PAT) gene. Commercialization of genetically modified (GM) crops will require the evaluation of risks associated with the release of GM crops. With the potential problems associated to GM crops safety testing, the investigation of their effects on non-target organisms is necessary for environmental risk research. This study was carried out to assess acute toxicity of a GM crop using the water flea (Daphnia magna) for non-target organism risk evaluation. The effect of acute toxicity on Daphnia magna of Agb0103 rice and a non-GM rice, Ilmibyeo, were investigated at different concentrations (0, 625, 1,250, 2,500, 5,000, and 10,000 mg/L). The Agb0103 rice used for the test was confirmed to express the CaMsrB2/PAT gene by the PCR and ELISA. Daphnia magna feeding tests showed no significant differences in cumulative immobility or abnormal response with either Agb0103 rice or non-GM rice. The 48hr-EC50 values showed no difference between Agb0103 rice (2243 mg/L) and non-GM rice (2694 mg/L). These results suggest that there is no significant difference in toxicity to Daphnia magna between Agb0103 rice and its non-GM counterpart.

• 한국작물학회지
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• 제49권4호
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• pp.358-362
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• 2004

In order to develop molecular markers related to stay green phenotype, AFLP analysis was conducted using near-isogenic lines for either stay green or non stay green trait. Both lines have characteristics of multi-ear and tillers (MET). Two out of 64 primer combinations of selective amplification identified three reproducible polymorphic fragments in MET corn with stay green. Both of E+AGC/M+CAC and E+AAG/M+CAA primer combinations produced two and one specific polymorphic fragments linked to stay green trait, respectively. For the conversion of AFLPs to sequence tag sites (STSs), primers were designed form both end sequences of each two polymorphic fragments. One fragment, which was amplified with E+AAG/M+CAA primer combinations, possessed 298 bp long and showed a $91\%$ homology with maize retrotransposon Cinful-l. One out of two polymorphic fragments produced with E+AGC/M+CAC primer combination had 236 bp long and matched a $96\%$ homology with an intron region of 22kDa alpha zein gene cluster in Zea mays. One out of two PCR fragments amplified with MET2 primer set in the stay green MET was not produced in the non-stay green MET. The developed AFLP and STS marker could be used as an efficient tool for selection of the stay green trait in the MET inbred.

• Journal of Gastric Cancer
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• 제3권4호
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• pp.201-205
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• 2003

Purpose: Telomerase activity is generally absent in primary cell cultures and normal tissues. Telomerase is known to be induced upon immortalization or malignant transformation of human cells. Telomerase activity can be increased in immature lymphocytes and activated lymphocytes, but it is not detected in the peripheral blood of normal persons. The authors analyzed peripheral blood telomerase from patients of gastric cancer to evaluate the possibility of using it for diagnosis and as a prognostic factor. Materials and Methods: We obtained blood samples from 11 inflammatory patients and 64 gastric cancer patients. The telomerase activity was measured using the [PCR-ELISA] method. The results were correlated with the T, N, M stage, cell differentiation, vascular, neural, and lymphatic invasion, tumor size, and tumor location. Results: In the 11 inflammatory patients, telomerase activity was not detected while in the gastric cancer patients, a positive rate of $28.1\%$ was noted. The peripheral telomerase activity was not related with tumor size, tumor site, lymphatic and vascular invasion, stage, or histologic differentiation. Conclusion: The peripheral blood telomerase activity for patients of gastric cancer can be utilized as a marker for the diagnosis of not only advanced gastric cancer, but also relatively early stage gastric cancer, but not as a prognostic factor.

• Asian-Australasian Journal of Animal Sciences
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• 제15권9호
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• pp.1227-1231
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• 2002

A powerful tool for chicken transgenesis could be established by employing a germline chimera production through primordial germ cell transplantation. This study was conducted to examine whether foreign gene-transfected gonadal primordial germ cells (gPGCs) have a migration activity into the gonad after transfer to recipient embryos. In Experiment 1, gPGCs of Korean Ogol Chicken were retrieved from 5.5-day-old embryos and subsequently transferred to the dorsal aorta of 2.5-day-old White Leghorn embryos after being labeled with PKH26 fluorescent dye. To confirm migration activity after transplantation, recipient embryos were sacrificed and examined on 3 days after transfer. Sex determination was concomitantly undertaken to examine whether sex of recipient embryos could affect the migration activity of gPGCs. All of embryonic gonads examined showed positive signals with PKH26 fluorescence and W-chromosome specific band by polymerase chain reaction (PCR) was detected in male embryos when gPGCs with ZW chromosome were transferred to recipient embryos. In Experiment 2, retrieved gPGCs were transfected with LacZ gene-containing cytomegalovirus promoter ($pCMV{\beta}$) by electroporation and subsequently transferred to recipient embryos. LacZ gene expression was identified in the gonads of 6 or 10-day-old recipient embryos and hatched-chicks. A total of 20 embryos and 12 hatched-chicks were examined and 11 of them (10 embryos and one hatched chicken; 11/32=34.4%) expressed $\beta$-galactosidase, a marker substance of LacZ gene. The results of this study demonstrated that foreign gene-transfected gPGCs can migrate and settle down into the gonad after being transferred into the blood vessel of the recipient embryos. This established technique will contribute to developing a peer biotechnology for transgenic chicken.