摘要
Technology for monitoring in vivo microRNA (miRNA) activity is extremely important for elucidating miRNA biology.However,in vivo studies of miRNA have been hampered by the lack of a convenient approach to reliably reflect real-time functional changes in miRNAs.Sensors for miRNA were developed by adding miRNA target sequences to the 3'-untranslated region of Gaussia princeps luciferase (Gluc) mRNA.These sensors were then evaluated in vitro and in vivo by measuring Gluc activity in cell supernatants and in peripheral blood.Sensors driven by the CMV promoter were effective for monitoring miR-122 in living cells,but not for the long-term monitoring of miR-122 or miR-142 in mouse liver because of CMV-promoter silencing.Replacing the CMV promoter with a CAG promoter rendered these sensors effective for the long-term monitoring of relevant liver miRNA activities.We subsequently used the CAG-promoter-based sensor for the long-term monitoring of endogenous liver miR-122,miR142 and miR-34a activities,as well as for exogenous miR-34a activity.Our study demonstrates that real-time in vivo activities of miRNAs can be continuously and conveniently detected in mouse liver using the sensors that we have developed.
Technology for monitoring in vivo microRNA （miRNA） activity is extremely important for elucidating miRNA biology. However, in vivo studies of miRNA have been hampered by the lack of a convenient approach to reliably reflect real-time functional changes in miRNAs. Sensors for miRNA were developed by adding miRNA target sequences to the 3＇-untranslated region of Gaussia princeps luciferase （Gluc） mRNA. These sensors were then evaluated in vitro and in vivo by measuring Gluc activity in cell supernatants and in peripheral blood. Sensors driven by the CMV promoter were effective for monitoring miR-122 in living cells, but not for the long-term monitoring of miR-122 or miR-142 in mouse liver because of CMV-promoter silencing. Replacing the CMV promoter with a CAG promoter rendered these sensors effective for the long-term monitoring of relevant liver miRNA activities. We subsequently used the CAG-promoter-based sensor for the long-term monitoring of endogenous liver miR-122, miR142 and miR-34a activities, as well as for exogenous miR-34a activity Our study demonstrates that real-time in vivo activities of miRNAs can be continuously and conveniently detected in mouse liver using the sensors that we have developed.
基金
supported by the China Special Key Program on Infectious Diseases (Grant Nos.2008ZX10002-023 and 2008ZX10001-012)
the Research Program from the State Key Laboratory of Molecular Virology and Genetic Engineering (Grant No.2008-S-0003)
