Objective To construct effective RNA-interference plasmids targeting mouse HIF-la gene and testify their effects and specificities in interfering HIF-1α expression. Methods Three RNA-interference plasmids targeting m...Objective To construct effective RNA-interference plasmids targeting mouse HIF-la gene and testify their effects and specificities in interfering HIF-1α expression. Methods Three RNA-interference plasmids targeting mouse HIF- 1α gene, pBS/U6/HIF-1α-siRNAI-Ⅲ, were constructed and identified using double digestion method in the present study. RT-PCR, immunostaining and western blotting were employed to detect the expression alterations of HIF-1α in 293T cells following transfections of the three plasmids, respectively. The interference effect of pBS/U6/HIF1αi-II in SH-SY5Y cell line was further investigated. Results All the three RNA-interference plasmids, especially pBS/U6/HIF1αi-Ⅱ, showed significant inhibition in HIF-1α expression in 293T cell line. pBS/U6/HIF1αi-Ⅱ could also inhibit HIF-1α expression in SH-SY5Y cell line, in a dosedependent way. Conclusion Plasmid pBS/U6/HIF1αi-Ⅱ constructed in our study can effectively and specifically inhibit HIF- 1α expression, and its role in neural tube development and dysfunction will be further investigated. Construct of pBS/U6/ HIF1αi-Ⅱ plasmid will provide a useful tool to study the role of HIF-1 pathway in embryogenesis, oncogenesis and i schemia development.展开更多
基金supported by the Innovative Foundation of the Third Military Medical University (N0.2007XG42)
文摘Objective To construct effective RNA-interference plasmids targeting mouse HIF-la gene and testify their effects and specificities in interfering HIF-1α expression. Methods Three RNA-interference plasmids targeting mouse HIF- 1α gene, pBS/U6/HIF-1α-siRNAI-Ⅲ, were constructed and identified using double digestion method in the present study. RT-PCR, immunostaining and western blotting were employed to detect the expression alterations of HIF-1α in 293T cells following transfections of the three plasmids, respectively. The interference effect of pBS/U6/HIF1αi-II in SH-SY5Y cell line was further investigated. Results All the three RNA-interference plasmids, especially pBS/U6/HIF1αi-Ⅱ, showed significant inhibition in HIF-1α expression in 293T cell line. pBS/U6/HIF1αi-Ⅱ could also inhibit HIF-1α expression in SH-SY5Y cell line, in a dosedependent way. Conclusion Plasmid pBS/U6/HIF1αi-Ⅱ constructed in our study can effectively and specifically inhibit HIF- 1α expression, and its role in neural tube development and dysfunction will be further investigated. Construct of pBS/U6/ HIF1αi-Ⅱ plasmid will provide a useful tool to study the role of HIF-1 pathway in embryogenesis, oncogenesis and i schemia development.
