Generation of light-producing somatic-transgenic mice using adeno-associated virus vectors

Abstract

We have previously designed a library of lentiviral vectors to generate somatic-transgenic rodents to monitor signalling pathways in diseased organs using whole-body bioluminescence imaging, in conscious, freely moving rodents. We have now expanded this technology to adeno-associated viral vectors. We first explored bio-distribution by assessing GFP expression after neonatal intravenous delivery of AAV8. We observed widespread gene expression in, central and peripheral nervous system, liver, kidney and skeletal muscle. next, we selected a constitutive SffV promoter and nfκB binding sequence for bioluminescence and biosensor evaluation. An intravenous injection of AAV8 containing firefly luciferase and eGFP under transcriptional control of either element resulted in strong and persistent widespread luciferase expression. A single dose of LPS-induced a 10-fold increase in luciferase expression in AAV8-NFκB mice and immunohistochemistry revealed Gfp expression in cells of astrocytic and neuronal morphology. Importantly, whole-body bioluminescence persisted up to 240 days. We have validated a novel biosensor technology in an AAV system by using an nfκB response element and revealed its potential to monitor signalling pathway in a non-invasive manner in a model of LPS-induced inflammation. This technology complements existing germline-transgenic models and may be applicable to other rodent disease models.Germline light-producing transgenic mice where luciferase expression is controlled by an endogenous promoter, 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 physiological and pathological processes 1,2 . One of the advantages of this technology is that every cell will contain a copy of the luciferase transgene and therefore provide a whole-body transgene expression profile under the control of a specific promoter of choice. However, producing germline transgenics requires frequent backcrossing and therefore becomes a time-consuming and costly process, using many rodents.We have previously developed a novel technology which allows the generation of light-producing somatic transgenic rodents, using lentiviral vectors as a proof-of-concept system 3 and have validated this technology both in vitro 4,5 and in vivo 1,6 . We have also demonstrated that signalling pathways in diseased organs can be monitored specifically, continually and in a non-invasive manner 1,2 . Exploiting the immune tolerance of neonatal mice

Spinal cord pathology in a Dravet Syndrome mouse model

“…40µm sections were cut using a Micron HM 430 freezing microtome (Thermo Fisher Scientific). To analyse CD68 & GFAP immunoreactivity free-Floating Immunohistochemistry was performed following previously described protocol (32, 33). Tissue images were captured using a stereoscopic fluorescence microscope (DM4000; Leica).…”

A brief review of reporter gene imaging in oncolytic virotherapy and gene therapy

“…With the exception of GFP, protein shapes are artistic interpretations. Permissions for imaging panels: (A and B) From Karda et al., 121 modified via cropping of the original figure and reproduced under CCA 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ ). (C) From Hutzen et al.…”

Recent Advances in In Vivo Somatic Cell Gene Modification in Newborn Pups

“…In contrast to the germline manipulation, a new strategy called "in vivo somatic cell genome editing" [5] or "somatic-Tg mice" [6] is emerging as an alternative to the abovementioned germline manipulation, which generates somatic gene modification in the fetal stage through in utero injection of nucleic acids, or in the adult stage through tail vein injection or local administration of nucleic acids. This approach does not require manipulation of early embryos or strain maintenance, and it can test the results of genome editing in a short period.…”

“…These data demonstrate that systemic rAAV2/9 has a high affinity for peripheral neural tissue and is useful for future therapeutic development and basic studies on the ENS. Karda et al [6] generated somatic Tg rodents to monitor signaling pathways in organs using whole-body bioluminescence imaging, through neonatal i.v. delivery of rAAV8 carrying EGFP cDNA.…”

“…Importantly, whole-body bioluminescence persisted for up to 8 months. According to Karda et al [6], this technology can complement existing germline Tg models, and it may be applicable to other rodent disease models.…”