Gaussia Luciferase Reporter Assay for Assessment of Gene Delivery Systems in Vivo

Objective To develop an alternative method for assessment of gene delivery systems in vivo.Methods Mouse primary spleen lymphocytes were genetically modified in vitro by a retroviral vector harboring a Gaussia luciferase(Gluc) expression cassette.After implantation of these cells into recipient mice,the expression of Gluc was detected in whole blood or plasma collected.Results As little as 10 μL whole blood drawn from the recipient mice could guarantee prompt reading of Gluc activity with a luminometer.And the reading was found in good correlation with the number of genetically modified spleen lymphocytes implanted to the mice.Conclusions Gluc may be useful as an in vivo reporter for gene therapy researches,and Gluc blood assay could provide an alternative method for assessment of gene delivery systems in vivo.

Long-term ex vivo monitoring of in vivo microRNA activity in liver using a secreted luciferase sensor

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.

A cell-based high-throughput approach to identify inhibitors of influenza A virus

Influenza is one of the most common infections threatening public health worldwide and is caused by the influenza virus.Rapid emergence of drug resistance has led to an urgent need to develop new anti-influenza inhibitors.In this study we established a 293T cell line that constitutively synthesizes a virus-based negative strand RNA,which expresses Gaussia luciferase upon influenza A virus infection.Using this cell line,an assay was developed and optimized to search for inhibitors of influenza virus replication.Biochemical studies and statistical analyses presented herein demonstrate the sensitivity and reproducibility of the assay in a high-throughput format(Z 0 factor value40.8).A pilot screening provides further evidence for validation of the assay.Taken together,this work provides a simple,convenient,and reliable HTS assay to identify compounds with anti-influenza activity.

An RNA polymerase I-driven human respiratory syncytial virus minigenome as a tool for quantifying virus titers and screening antiviral drug

Human respiratory syncytial virus（RSV） is an important pediatric pathogen of lower respiratory tract worldwide. No vaccines and antiviral drugs are available. Herein the use of an RNA polymerase I-driven RSV minigenome for analyzing RSV replication and screening anti-RSV drugs was investigated. The RNA polymerase I（Pol I） was used to transcribe RSV minigenome from the constructed plasmid, designated p HM-RSV-Gluc, of minigenome c DNA which comprised trailer region, gene start sequence（GS）, reverse complementary copy of Gaussia luciferase（Gluc） gene, gene end sequence（GE）, and leader region in the direction of 5＇–3＇end and was flanked by promoter and terminator of Pol I. The expression of Gluc was confirmed in p HM-RSV-Gluc transfected HEp-2 cells following RSV infection and had the characteristics of dose-dependent, which provided a rapid, sensitive, and quantitative method for quantifying virus titers and screening antiviral drugs.