305. Generation of Light-Producing, Somatic-Transgenic Mice Using Lentivirus and Adeno-Associated Virus Vectors
Authors: Rajvinder Karda, Dany P. Perocheau, Suzanne M.K. Buckley, Juliette M. K.M. Delhove, Michael Hughes, Ahad A. Rahim, Mark R. Johnson, Joanne Ng, Natalie Sufi, Tristan R. Mckay, Simon N. Waddington
Journal: Molecular Therapy (2016)

Germ line light producing transgenic mice, where luciferase expression is controlled by a surrogate promoter or by a minimal promoter downstream of tandem, synthetic, transcription factor binding elements, are used to provide an in vivo readout of disease processes. However, as every cell within the organism contains the luciferase reporter gene, it is therefore not specific to individual organs. We have developed a novel technology for the generation of light emitting somatic transgenic animals using lentiviral vectors. This allows signalling pathways in diseased organs to be monitored specifically, continually and in a non-invasive manner [1]. In this study, we aimed to deliver NFkB driving a luciferase reporter constructs to the nervous system of neonatal mice to generate somatic-transgenic mice using both lentivirus and adeno-associated viral (AAV) vectors. Lentivirus vector pseudotyped with VSV-G viral envelope glycoproteins or AAV8 serotyped vector carrying an NFkB response element was injected intracranially or intravenously to outbred CD1 neonatal (P1) mice and luciferase expression was monitored continually by whole body bioluminescence imaging of conscious mice. The ability to image conscious mice holds advantages when studying neuropathology. After weaning, pathological activation of NFKB was induced by intraperitoneal injection of lipopolysaccharide (LPS). Following intracranial injection, the VSV-G lentivirus NFkB biosensor showed transduction of the brain and spinal cord. Luciferase expression was upregulated 24 hours after administration of LPS. Immunohistochemistry revealed only modest spread throughout the brain. Conversely, intracranial injection of AAV8 NFkB biosensor showed a much wider spread and increased luciferase expression. Finally we administered AAV8 NFkB biosensor intravenously at P1. Whereas previous studies show AAV8 serotype transduces many systemic tissues [2] through this route; we observed luciferase expression predominantly in the brain and spine (see figure). Using a standard Gateway® cloning system we have established a library of more than 25 lentivirus biosensors where some have been tested in vitro and in vivo. We plan on incorporating these response elements into the AAV backbone described above. Therefore, enabling the generation of somatic-transgenic mice which have a wider spread of AAV biosensor. This will complement existing germ line transgenic, light producing technology by maximising the use, and reducing the numbers, of animals used in biomedical research. 1. Buckley, SMK et al. 2015. In vivo bioimaging with tissue-specific transcription factor activated luciferase reporters. Scientific Reports. 2. Inagaki, K et al. 2008. Frequency and spectrum of genomic integration of recombinant AAV serotype 8 vector in neonatal mouse liver. Journal of virology.