Team:Heidelberg/Project/Mouse Infection
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==Methods== | ==Methods== | ||
===Contructs=== | ===Contructs=== | ||
+ | The <i>in vivo</i> analysis should enlighten our gene therapy approach using AAV tropism as well as miRNA binding sites as trigger for expression. The following constructs have been subcloned separately into the AAV context to accomplish those tasks: <br/> | ||
+ | # positive control, | ||
+ | # off-targeting construct, | ||
+ | # synthetic tuning construct and | ||
+ | # on-targeting construct. | ||
+ | <br/> | ||
+ | All but one virus were packaged by the AAV rep and cap gene with Adenovirus 5 (Ad5) as a helper plasmid. Accordingly, one virus construct was packaged into a shuffled cap gene from our [https://2010.igem.org/Team:Heidelberg/Project/Capsid_Shuffling/Homology_Based homology based capsid shuffling] attempt. | ||
+ | <br/> | ||
+ | # The positive control (see sidebar, fig. 1) consisted of the SV40 promoter driving a firefly luciferase (luc2) gene, thereby leading to an unspecific expression of the luciferase protein in all mice tissues. In addition to packaging this construct into a wild type AAV virus, the positive control was also packaged as a transgene into our [https://2010.igem.org/Team:Heidelberg/Project/Capsid_Shuffling/Homology_Based shuffled capsid] which after random selection <!--selection pressure--> was already able to [https://2010.igem.org/Team:Heidelberg/Notebook/Homology_Based/October#16/10/2010 positively transduce Huh7 and HepG2 cells] <i>in vitro</i>. | ||
+ | # The off-targeting construct (see sidebar, fig. 2) was composed of an SV40 promoter driving a firefly luciferase (luc2) gene with binding sites against miR-122 behind it. In order to achieve the highest expression in all mice cells but the liver cells - a single perfect binding site of miR-122 was used for <i>in vivo</i> study. | ||
+ | # The synthetic tuning construct (see sidebar, fig. 3) consisted 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 ("tuning" construct, see sidebar, fig.3). The second virus packaged the following transgene: SV40 promoter driving luc2 with shRNA haat binding site behind it ("tuned" construct, see sidebar, fig. 4). In order to ensure a synthetic tuning effect, a perfect binding site and one with a bulge that was introduced at position 9-12 were used for <i>in vivo</i> experiments, respectively. Those two binding sites should lead to a significant knockdown in the first case and a slight repression of luciferase expression in the latter as compared to the positive control. | ||
+ | # The on-targeting construct consisted of two independent viruses which were co-infected into mice, as well. One of these viruses packaged the Tet Repressor (TetR) driven by an SV40 promoter ("repressor" construct, see sidebar, fig. 5). The expression of TetR is under the control of miR-122 as four binding sites of this miRNA were cloned into the 3’UTR of the gene. The second virus was composed of an SV40 promoter driving the Tet operator (TetO<sub>2</sub>) which monitors the expression of luc2 ("operator" construct, see sidebar, fig. 6). With this setup, luc2 expression should be inhibited by the TetR in all mice tissues except for liver cells, where TetR is down-regulated by miRNA 122. | ||
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===Production of recombinant virus=== | ===Production of recombinant virus=== | ||
The viruses were produced in HEK 293-T cells and purified on an iodixanol gradient according to [https://2010.igem.org/Team:Heidelberg/Notebook/Methods#Virus_Production the virus production protocol]. | The viruses were produced in HEK 293-T cells and purified on an iodixanol gradient according to [https://2010.igem.org/Team:Heidelberg/Notebook/Methods#Virus_Production the virus production protocol]. |
Revision as of 03:22, 28 October 2010
![](https://static.igem.org/mediawiki/2010/9/92/Bg_plasmid.png)
in vivo studyAbstractGene therapy offers a great tool for treatment of various diseases. Nevertheless it is only useful if it allows for specific, fine-tuning of the gene of interest (GOI). IntroductionGene therapy offers a great tool for treatment of various diseases such as failure, muscular dystrophies, and cystic fibrosis (Zincarelli, Soltys et al. 2008). ResultsConstructsThe in vivo analysis should enlighten our gene therapy approach using AAV tropism as well as miRNA binding sites as trigger for expression. The following constructs have been subcloned separately into the AAV context to accomplish those tasks:
Mice injectionThe tail vein injection was chosen so as to assess AAV serotype tissue tropism; the luciferase transgene was used for visualizing the relative vector distribution in all the animals in a real-time manner. This allows for in vivo imaging and time lapse experiments. Bioluminescence imagingDiscussionMethodsContructsThe in vivo analysis should enlighten our gene therapy approach using AAV tropism as well as miRNA binding sites as trigger for expression. The following constructs have been subcloned separately into the AAV context to accomplish those tasks:
Production of recombinant virusThe viruses were produced in HEK 293-T cells and purified on an iodixanol gradient according to the virus production protocol. Before infection, the titer of the viruses was quantified using quantitative realtime PCR. Procedure involving animalsThe mouse experiments were conducted in accordance with the animal facility of the German Cancer Research Center in Heidelberg. Female NMRI mice were obtained from a collaboration with Dr. Oliver Müller. At 8-10 weeks of age, the animals were injected in the tail vein (TV), with ~ 1x1011 particles of AAV-SV40-luciferase in 200µl of 1x phosphate-buffered saline. The mice are transferred to a holding device which restrains the mouse while allowing access to the tail vein. The tails were warmed before the injections and injections were carried out using 27 gauge needles. All the mice recoverd from the injection quickly without loss of mobility or interruption of grooming activity [http://2010.igem.org/Team:Heidelberg/Project/Mouse_Infection#References (Zincarelli et al., 2008)]. in vivo animal imagingMice were anesthesized in an isofluran chamber. The mice were injected intraperitoneally with 200µl of a 30 mg/ml concentration of D-luciferin. This injection starts the luminescence of luc2. Mice were measured for one to seven minutes post injection under the in vivo bioluminometer. ReferencesGrimm, D. (2002). "Production methods for gene transfer vectors based on adeno-associated virus serotypes." Methods 28(2): 146-157.
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