Team:Heidelberg/Project/Mouse Infection
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==Abstract== | ==Abstract== | ||
- | Gene therapy offers a great tool for treatment of various diseases. Nevertheless | + | Gene therapy offers a great tool for treatment of various human diseases and has shown tremendous promise and success in many recent clinical trials. Nevertheless, two most critical hurdles that remain are 1) the development of delivery vectors that allow for the highly cell- and tissue-specific expression of a gene of interest (GOI), as well as 2) the implementation of novel systems that permit the tightly controlled and fine-tuned expression of this gene in human patients. To fill in these two essential gaps, the iGEM Team 2010 of Heidelberg is introducing molecular evolution of viral gene transfer vectors as well as mi(cro)RNA-based gene regulation technology into the realm of synthetic biology. By combining these two powerful new methodologies, we have succeeded in engineering synthetic viral miRNA vectors that we could demonstrate to provide potent, specific and regulated gene expression in cultured mouse and human hepatocytes. Most importantly, we also document the successful use of our novel approach to achieve tightly controlled and liver-specific expression of a vector-encoded recombinant DNA in intact livers of adult mice. Our work and data thus pave the way for the future further expansion and utilization of our unique miRNA/vector kits in numerous biotechnological and especially biomedical applications. |
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==Introduction== | ==Introduction== |
Revision as of 03:43, 28 October 2010
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in vivo studyAbstractGene therapy offers a great tool for treatment of various human diseases and has shown tremendous promise and success in many recent clinical trials. Nevertheless, two most critical hurdles that remain are 1) the development of delivery vectors that allow for the highly cell- and tissue-specific expression of a gene of interest (GOI), as well as 2) the implementation of novel systems that permit the tightly controlled and fine-tuned expression of this gene in human patients. To fill in these two essential gaps, the iGEM Team 2010 of Heidelberg is introducing molecular evolution of viral gene transfer vectors as well as mi(cro)RNA-based gene regulation technology into the realm of synthetic biology. By combining these two powerful new methodologies, we have succeeded in engineering synthetic viral miRNA vectors that we could demonstrate to provide potent, specific and regulated gene expression in cultured mouse and human hepatocytes. Most importantly, we also document the successful use of our novel approach to achieve tightly controlled and liver-specific expression of a vector-encoded recombinant DNA in intact livers of adult mice. Our work and data thus pave the way for the future further expansion and utilization of our unique miRNA/vector kits in numerous biotechnological and especially biomedical applications. IntroductionGene therapy offers a great tool for treatment of various diseases such as failure, muscular dystrophies, and cystic fibrosis (Zincarelli, Soltys et al. 2008). ResultsMice 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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