Connected a segment of CMV enhancer to the front of MyoG gene promoter and then constructed the corresponding dual luciferase expression vector pGL3-CMV-MyoGpro. We set four eukaryotic expression vectors including pGL...Connected a segment of CMV enhancer to the front of MyoG gene promoter and then constructed the corresponding dual luciferase expression vector pGL3-CMV-MyoGpro. We set four eukaryotic expression vectors including pGL3-CMV, pGL3MyoGpro, pGL3-CMV-MyoGpro, and pGL3-Basic which contained CMV promoter, MyoG promoter, CMV-MyoG synthesis promoter, and a promoterless negative control, respectively. Then the four vectors and internal control Renilla luciferase report gene vector phRL-TK were transfected into bovine skeletal muscle satellite cells, mouse C2C12 cells and bovine fetal fibroblast cells to detect the promoter activity with dual luciferase report system. The results showed that CMV enhancer could significantly improve the transcription activity of bovine MyoG gene promoter in muscle satellite cells and mouse C2C12 cells, and it had certain specificity. This study provided experimental materials for increasing the high expression of exogenous gene in bovine muscle cells, and also laid the molecular theoretical basis for obtaining the high specific promoter of bovine muscle and the transgenic beef cattle.展开更多
Objective To screen the 5’ regulatory region of the aldose reductase (AR) gene for genetic variabilities causing changes in protein expression and affecting the promoter function. Methods The screenings were carri...Objective To screen the 5’ regulatory region of the aldose reductase (AR) gene for genetic variabilities causing changes in protein expression and affecting the promoter function. Methods The screenings were carried out by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). All SSCP variants were submitted for DNA sequencing and inserted into the plasmid chloromycetin acetyl transferase (CAT) enhancer vector. The constructs were used to transfect Hela cells,and CAT assays were performed to assess promoter activity. Gel mobility shift and footprinting assays were also performed to determine the interaction between the DNA and nuclear proteins. Results Two polymorphisms, C(-106)T and C(-12)G, were identified in the regulatory region in 123 Chinese control subjects and 145 patients with type 2 diabetes mellitus. The frequencies of genotypes WT/WT, WT/C(-12)G and WT/C(-106)T were not significantly different between the subjects and patients. In the patients with and without retinopathy, frequencies of WT/C(-106)T were 31.5% and 17.5% (P【0.05) respectively, and the frequencies of WT/C(-12)G were 10.5% and 2.5% (P】0.05) respectively. The total frequency of WT/C(-12)G and WT/C(-106)T in patients with retinopathy was 41.8%, significantly higher than that (20.0%) in patients without retinopathy (P【0.025). The relative transcription activities of the wild-type, the C(-12)G and the C(-106)T were 15.7%, 31.0% and 32.2%, respectively. The results of DNA-protein interaction assays showed that these variations did not change the binding site of DNA with trans-acting factors. Conclusion The polymorphisms C(-12)G and C(-106)T strongly associated with diabetic retinopathy in the Chinese population have been identified in the regulatory region of the aldose reductase gene.展开更多
基金Supported by the Major Special Projects of New Product Training of Transgenic Organisms(zx080072008-2008)
文摘Connected a segment of CMV enhancer to the front of MyoG gene promoter and then constructed the corresponding dual luciferase expression vector pGL3-CMV-MyoGpro. We set four eukaryotic expression vectors including pGL3-CMV, pGL3MyoGpro, pGL3-CMV-MyoGpro, and pGL3-Basic which contained CMV promoter, MyoG promoter, CMV-MyoG synthesis promoter, and a promoterless negative control, respectively. Then the four vectors and internal control Renilla luciferase report gene vector phRL-TK were transfected into bovine skeletal muscle satellite cells, mouse C2C12 cells and bovine fetal fibroblast cells to detect the promoter activity with dual luciferase report system. The results showed that CMV enhancer could significantly improve the transcription activity of bovine MyoG gene promoter in muscle satellite cells and mouse C2C12 cells, and it had certain specificity. This study provided experimental materials for increasing the high expression of exogenous gene in bovine muscle cells, and also laid the molecular theoretical basis for obtaining the high specific promoter of bovine muscle and the transgenic beef cattle.
基金grantsfromtheNationalNaturalScienceFoundationofChina (No 396 70 35 2 )
文摘Objective To screen the 5’ regulatory region of the aldose reductase (AR) gene for genetic variabilities causing changes in protein expression and affecting the promoter function. Methods The screenings were carried out by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). All SSCP variants were submitted for DNA sequencing and inserted into the plasmid chloromycetin acetyl transferase (CAT) enhancer vector. The constructs were used to transfect Hela cells,and CAT assays were performed to assess promoter activity. Gel mobility shift and footprinting assays were also performed to determine the interaction between the DNA and nuclear proteins. Results Two polymorphisms, C(-106)T and C(-12)G, were identified in the regulatory region in 123 Chinese control subjects and 145 patients with type 2 diabetes mellitus. The frequencies of genotypes WT/WT, WT/C(-12)G and WT/C(-106)T were not significantly different between the subjects and patients. In the patients with and without retinopathy, frequencies of WT/C(-106)T were 31.5% and 17.5% (P【0.05) respectively, and the frequencies of WT/C(-12)G were 10.5% and 2.5% (P】0.05) respectively. The total frequency of WT/C(-12)G and WT/C(-106)T in patients with retinopathy was 41.8%, significantly higher than that (20.0%) in patients without retinopathy (P【0.025). The relative transcription activities of the wild-type, the C(-12)G and the C(-106)T were 15.7%, 31.0% and 32.2%, respectively. The results of DNA-protein interaction assays showed that these variations did not change the binding site of DNA with trans-acting factors. Conclusion The polymorphisms C(-12)G and C(-106)T strongly associated with diabetic retinopathy in the Chinese population have been identified in the regulatory region of the aldose reductase gene.
