ABSTRACT
Objective To construct the luciferase report vector carrying a disintegrin and metalloprotease 10(adam10) gene promoter,to screen its stable expression cell line and to analyze its activity.Methods The genome DNA of human neuroblastoma SH-SY5Y cells was extracted as the template.The adam10 gene promoter was amplified by PCR and was cloned into luciferase reporter vector pGL4.17.The adam10 gene promoter luciferase reporter vector pGL4.17-adam10 was constructed and transfected in to SH-SY5Y cells(pGL4.17 vector without promotoer as the negative control and pGL4.17 vector with CMV promoter as the positive control).Then the stable expression cell line was screened by G418 and its fluorescence activity was detect after treating with 1 tμmol/L retinoic acid(RA) for 4 d.Results About 438 bp adam10 gene promoter was successfully amplified by PCR.The pGL4.17-adam10 vector was correct by pCR and double enzyme digestion identification.The cell line stably expressing adam10 gene promoter was obtained after transfecting SH-SY5Y cells by this vector and screening by G418,which had stronger transcriptional activity by detection;1 μmol/L RA could induce high efficiency expression of adam10 gene promoter.Conclusion Human adam10 gene promoter luciferase vector is successfully constructed.adam10 gene promoter can be stably expressed in SH-SY5Y cells,which provides a basis for deeply studying adam10 gene expression regulation,polymorphism analysis and high-throughput drug screening.
ABSTRACT
Objective To construct the luciferase report vector carrying a disintegrin and metalloprotease 10(adam10) gene promoter,to screen its stable expression cell line and to analyze its activity.Methods The genome DNA of human neuroblastoma SH-SY5Y cells was extracted as the template.The adam10 gene promoter was amplified by PCR and was cloned into luciferase reporter vector pGL4.17.The adam10 gene promoter luciferase reporter vector pGL4.17-adam10 was constructed and transfected in to SH-SY5Y cells(pGL4.17 vector without promotoer as the negative control and pGL4.17 vector with CMV promoter as the positive control).Then the stable expression cell line was screened by G418 and its fluorescence activity was detect after treating with 1 tμmol/L retinoic acid(RA) for 4 d.Results About 438 bp adam10 gene promoter was successfully amplified by PCR.The pGL4.17-adam10 vector was correct by pCR and double enzyme digestion identification.The cell line stably expressing adam10 gene promoter was obtained after transfecting SH-SY5Y cells by this vector and screening by G418,which had stronger transcriptional activity by detection;1 μmol/L RA could induce high efficiency expression of adam10 gene promoter.Conclusion Human adam10 gene promoter luciferase vector is successfully constructed.adam10 gene promoter can be stably expressed in SH-SY5Y cells,which provides a basis for deeply studying adam10 gene expression regulation,polymorphism analysis and high-throughput drug screening.