Team:Heidelberg/Project/Mouse Infection
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==Methods== | ==Methods== | ||
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+ | In order to enable in vivo analysis of our gene therapy approach using AAV tropism as well as miRNA binding sites as trigger for expression the following constructs have been subcloned into the AAV context in order to allow for virus production: (1) positive control, (2) off-targeting construct, (3) synthetic tuning construct and (4) on-targeting construct. All but of one virus were packaged in the adeno-associated virus (AAV) rep and cap gene with Adenovirus 5 (Ad5) as a helper plasmid. Accordingly one virus was packaged into a shuffled cap gene from our homology based capsid shuffling attempt (VERLINKUNG). (1) The positive control consists of the sv40promoter driving a firefly luciferase (luc2) gene, thereby leading a no target-specific expression of the firefly protein in all mice tissues. The positive control was also packaged as a transgene into our shuffled capsid which after selection pressure was already able to positively transduce Huh7 and HepG2 cells in cell culture (VERLINKUNG). (2) The off-targeting construct is composed of an sv40 promoter driving a firefly luciferase (luc2) with binding sites against miR-122 behind it. In order to achieve the highest expression in all mice cells but the liver cells, a perfect binding site of miR-122 was used for in vivo study. (3) The synthetic tuning construct consists of two viruses injected at the same time in the mice. The one virus packaged the expression construct of shRNA haat driven by the H1 promoter. The second virus packaged the following transgene: sv40 promoter driving luc2 with binding sites for shRNA haat behind it. In order to ensure a synthetic tuning effect a perfect binding site and one with an introduced bulge at position 9-12 respectively were used for in vivo experiments. Those two binding sites should lead to a knockdown in the first case and a repression of luciferase expression in the latter in comparison to the positive control. (4) The on-targeting construct consist of again two independent viruses which were then co-infected into mice. One of these viruses packages the Tet Repressor (TetR) driven by an sv40 promoter. The expression of TetR is under the control of miR-122 as four binding sites of this miRNA are cloned into the 3’UTR of the gene. The second virus is composed of an sv40 promoter driving the tet operater (TetO2) which monitors the expression of luc2. | ||
==References== | ==References== |
Revision as of 06:30, 25 October 2010
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