Radioiodine i s a routine therapy for differentiated thyroid cancers.Non-thyroid cancers can intake radioiodine after transfection of the human sodium iodide symporter (hNIS) gene.The human telomerase reverse transcri...Radioiodine i s a routine therapy for differentiated thyroid cancers.Non-thyroid cancers can intake radioiodine after transfection of the human sodium iodide symporter (hNIS) gene.The human telomerase reverse transcriptase (hTERT) promoter,an excellent tumor-specific promoter,has potential value for targeted gene therapy of glioma.We used the hTERT promoter to drive the expression of the hNIS and human thyroid peroxidase (hTPO) gene as a primary step for testing the effects of radioiodine therapy on malignant glioma.The U87 and U251 cells were co-transfected with two adenoviral vectors,in which the hNIS gene had been coupled to the hTERT promoter and the hTPO gene had been coupled to the CMV promoter,respectively.Then,we performed Western blot,125I intake and efflux assays,and clonogenic assay with cancer cells.We also did 99mTc tumor imaging of nude mice models.After co-transfection with Ad-hTERT-hNIS and Ad-CMV-hTPO,glioma cells showed the 125I intake almost 1.5 times higher than cells transfected with Ad-hTERT-hNIS alone.Western blots revealed bands of approximately 70 kDa and 110 kDa,consistent with the hNIS and hTPO proteins.In clonogenic assay,approximately 90% of cotransfected cells were killed,compared to 50% of control cells after incubated with 37 MBq of 131I.These results demonstrated that radioiodine therapy was effective in treating malignant glioma cell lines following induction of tumor-specific iodide intake by the hTERT promoter-directed hNIS expression in vitro.Cotransfected hNIS and hTPO genes can result in increased intake and longer retention of radioiodine.Nude mice harboring xenografts transfected with Ad-hTERT-NIS can take 99mTc scans.展开更多
[Objective] To clone ovarian-specific promoter-1 （OSP-1） fragment in rats and detect the tissue-specific expression of OSP-1 at cell level. [Method] According to the known OSP-1 sequence in rats, the specific pdmers...[Objective] To clone ovarian-specific promoter-1 （OSP-1） fragment in rats and detect the tissue-specific expression of OSP-1 at cell level. [Method] According to the known OSP-1 sequence in rats, the specific pdmers were designed. The OSP-1 fragment was amplified by PCR and compared with published OSP-1 sequence. After eukaryotic expression vector pOSP-1-EGFP-N1 was constructed by cloning OSP-1 promoter into the pEGFP-N1 vector without CMV, the recombinant plasmid was transfected into buffalo granulocytes, mammary epithelial cells and fetal fibroblast cells under mediation of liposome LipofectamineTM 2000. The green fluxorescent protein （GFP） expression was observed with the fluorescence microscope after transfection for 12, 24 and 48 h. [Result] The amplified OSP-1 fragment was 480 bp, and the homology to published rat OSP-1 sequence was 96%. The sequence analysis showed that this fragment contained a core promoter cis-element similar with TATA box and ChAT box, and multiple C/EBP beta transcription factor binding sites. The EGFP-expressing positive granulccytes were firstly observed at 12 h after transfection and they were increased. All the EGFP-expressing and non-expressing cells from granulocytes were large and round. After transfection for 48 h, the EGFP-expressing positive granulocytes were decreased. The EGFP was not expressed in buffalo mammary epithelial cell and fetal fibroblast cell. [ Conclusion] EGFP exDression controlled bv OSP-1 promoter can be found in buffao granulocvtes.展开更多
The promoter is a cis-acting element in regulating gene expression. A promoterless plasmid containing UidA gene was transformed into tritordeum by barmbadment. Histochemical analysis of various tissues in transgenic t...The promoter is a cis-acting element in regulating gene expression. A promoterless plasmid containing UidA gene was transformed into tritordeum by barmbadment. Histochemical analysis of various tissues in transgenic tritordeum was carried to examine tissue-specific expression of GUS(beta-glucuronidase) activity. The pollen-specific promoter was trapped and identified successfully in a transformant line. PCR(polymerase chain reaction) method was used to isolate this pollen-specific promoter. By sequencing and analyzing the amplified fragment from PCR, a part of UidA gene and a flanking sequence were obtained. Some essential elements of plant promoters were found in the sequence. To determine the function of it, the cloned fragment was fused with UidA gene, then cloned and transformed into Triticum durum. The transgenic plant transformed by this vector showed GUS expression only in pollen. Therefore a pollen-specific promoter was isolated successfully.展开更多
Dear Editor, The structure of eukaryotic promoter is modular, consisting of different sub-domains （Benfey et al., 1990）. Cross-talk among these sub-domains and effective combinatorial interac- tions between the spe...Dear Editor, The structure of eukaryotic promoter is modular, consisting of different sub-domains （Benfey et al., 1990）. Cross-talk among these sub-domains and effective combinatorial interac- tions between the specific cis-element/s with respective trans- factor/s usually control the strength and tissue specificity of the promoter. Further, specificity/inducibility of promoter can be modified by altering its genetic architecture through ＇cis-rearrangement＇ （Rushton et al., 2002） and ＇swapping of sub-domains＇ （Bhullar et al., 2003）. The fundamental motiva- tion behind developing such modified promoters lies in thebelief that the swapping/shuffling of the upstream activation sequences carrying a specific set of cis-elements that bind to a particular trans-factor from one promoter to the other con- taininq a TATA seauence that miaht result in a novel chimericregu!atory module,展开更多
基金supported by a grant from Tianjin Basic Research and Leading Edge Science Project of China (No. 08JCZDJC23900)
文摘Radioiodine i s a routine therapy for differentiated thyroid cancers.Non-thyroid cancers can intake radioiodine after transfection of the human sodium iodide symporter (hNIS) gene.The human telomerase reverse transcriptase (hTERT) promoter,an excellent tumor-specific promoter,has potential value for targeted gene therapy of glioma.We used the hTERT promoter to drive the expression of the hNIS and human thyroid peroxidase (hTPO) gene as a primary step for testing the effects of radioiodine therapy on malignant glioma.The U87 and U251 cells were co-transfected with two adenoviral vectors,in which the hNIS gene had been coupled to the hTERT promoter and the hTPO gene had been coupled to the CMV promoter,respectively.Then,we performed Western blot,125I intake and efflux assays,and clonogenic assay with cancer cells.We also did 99mTc tumor imaging of nude mice models.After co-transfection with Ad-hTERT-hNIS and Ad-CMV-hTPO,glioma cells showed the 125I intake almost 1.5 times higher than cells transfected with Ad-hTERT-hNIS alone.Western blots revealed bands of approximately 70 kDa and 110 kDa,consistent with the hNIS and hTPO proteins.In clonogenic assay,approximately 90% of cotransfected cells were killed,compared to 50% of control cells after incubated with 37 MBq of 131I.These results demonstrated that radioiodine therapy was effective in treating malignant glioma cell lines following induction of tumor-specific iodide intake by the hTERT promoter-directed hNIS expression in vitro.Cotransfected hNIS and hTPO genes can result in increased intake and longer retention of radioiodine.Nude mice harboring xenografts transfected with Ad-hTERT-NIS can take 99mTc scans.
基金supported by the Major Projects of Genetically Modified Organism (GMO) New Varieties Breeding of Ministry of Agriculture ( 2008ZX08007-003)
文摘[Objective] To clone ovarian-specific promoter-1 （OSP-1） fragment in rats and detect the tissue-specific expression of OSP-1 at cell level. [Method] According to the known OSP-1 sequence in rats, the specific pdmers were designed. The OSP-1 fragment was amplified by PCR and compared with published OSP-1 sequence. After eukaryotic expression vector pOSP-1-EGFP-N1 was constructed by cloning OSP-1 promoter into the pEGFP-N1 vector without CMV, the recombinant plasmid was transfected into buffalo granulocytes, mammary epithelial cells and fetal fibroblast cells under mediation of liposome LipofectamineTM 2000. The green fluxorescent protein （GFP） expression was observed with the fluorescence microscope after transfection for 12, 24 and 48 h. [Result] The amplified OSP-1 fragment was 480 bp, and the homology to published rat OSP-1 sequence was 96%. The sequence analysis showed that this fragment contained a core promoter cis-element similar with TATA box and ChAT box, and multiple C/EBP beta transcription factor binding sites. The EGFP-expressing positive granulccytes were firstly observed at 12 h after transfection and they were increased. All the EGFP-expressing and non-expressing cells from granulocytes were large and round. After transfection for 48 h, the EGFP-expressing positive granulocytes were decreased. The EGFP was not expressed in buffalo mammary epithelial cell and fetal fibroblast cell. [ Conclusion] EGFP exDression controlled bv OSP-1 promoter can be found in buffao granulocvtes.
基金NSFC foundation,Guangdong Province and China Education Ministry joint production-education-research funding Program ( No. 2009B090300198)the Fundamental Research Funds for the Central Universities,HUST( No. M2009060)PhD dissertation Foundation of Huazhong University of Science & Technology
文摘The promoter is a cis-acting element in regulating gene expression. A promoterless plasmid containing UidA gene was transformed into tritordeum by barmbadment. Histochemical analysis of various tissues in transgenic tritordeum was carried to examine tissue-specific expression of GUS(beta-glucuronidase) activity. The pollen-specific promoter was trapped and identified successfully in a transformant line. PCR(polymerase chain reaction) method was used to isolate this pollen-specific promoter. By sequencing and analyzing the amplified fragment from PCR, a part of UidA gene and a flanking sequence were obtained. Some essential elements of plant promoters were found in the sequence. To determine the function of it, the cloned fragment was fused with UidA gene, then cloned and transformed into Triticum durum. The transgenic plant transformed by this vector showed GUS expression only in pollen. Therefore a pollen-specific promoter was isolated successfully.
文摘Dear Editor, The structure of eukaryotic promoter is modular, consisting of different sub-domains （Benfey et al., 1990）. Cross-talk among these sub-domains and effective combinatorial interac- tions between the specific cis-element/s with respective trans- factor/s usually control the strength and tissue specificity of the promoter. Further, specificity/inducibility of promoter can be modified by altering its genetic architecture through ＇cis-rearrangement＇ （Rushton et al., 2002） and ＇swapping of sub-domains＇ （Bhullar et al., 2003）. The fundamental motiva- tion behind developing such modified promoters lies in thebelief that the swapping/shuffling of the upstream activation sequences carrying a specific set of cis-elements that bind to a particular trans-factor from one promoter to the other con- taininq a TATA seauence that miaht result in a novel chimericregu!atory module,
