![]() , and luciferase activity was evaluated using a CCD camera (Xenogen) after intraperitoneal injection of 3 mg of luciferin substrate (Promega) Hydrodynamic injections were performed as described with 5mg of luciferase expressing plasmid and 20mg of an unrelated carrier plasmid Eight week-old Balb/C mice were used in all experiments following the guidelines from the institutional ethical committee. ![]() In a Berthold Luminometer (Lumat LB 9507). Renilla and firefly luciferase activities were measured using the Dual Luciferase® System (Promega) as previously described Cells were transfected with calcium phosphate HeLa and HuH7 cells were obtained from the ATCC and treated as recommended The same plasmids have been introduced in mice by hydrodynamic injection, and luciferase activity has been evaluated with a CCD camera at different times after plasmid administration. We have cloned the regulatory sequences in a plasmid that contains luciferase sequences and evaluated luciferase expression in cultured cells to ensure functionality. The aim of our work was to evaluate the efficacy of regulatory sequences in a mouse animal model. These systems often find exogenous genes as unwanted and activate machineries developed to inhibit their expression such as the immune system or molecular mechanisms that silence gene expression such as promoter methylation. Expression of exogenous genes in animals is hampered by natural systems developed to avoid expression of toxic factors. When functionality is assayed in animal models, many of these methods behave differently and do not yield high expression levels for long periods of time. However, most of the tools described to increase expression of exogenous genes have been evaluated in cultured cells. These methods function either by inducing replication of episomal DNA or integration of exogenous DNA into the cell genome. Interestingly, methods that allow replication of exogenous DNA upon cell division also have been described. ![]() These include enhancers and promoters, which increase transcription introns and mRNA stabilization and transport sequences, which favour processing and accumulation of mRNAs and translation enhancers, which increase translation efficiency.Īdditionally, several protocols have been described to improve introduction of exogenous DNA in the nucleus of the cell of interest and increase nuclear DNA stability. To obtain high expression, several sequences have been described. High expression levels over long periods of time are required for many gene therapy applications.
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