A minimally invasive, lentiviral based method for the rapid and sustained genetic manipulation of renal tubules.

Athena Matakidou,David J Adams,David A Tuveson,Judit Espana-Agusti

doi:10.1038/srep11061

Abstract

The accelerated discovery of disease-related genes emerging from genomic studies has strained the capacity of traditional genetically engineered mouse models (GEMMs) to provide in-vivo validation. Direct, somatic, genetic engineering approaches allow for accelerated and flexible genetic manipulation and represent an attractive alternative to GEMMs. In this study we investigated the feasibility, safety and efficiency of a minimally invasive, lentiviral based approach for the sustained in-vivo modification of renal tubular epithelial cells. Using ultrasound guidance, reporter vectors were directly injected into the mouse renal parenchyma. We observed transgene expression confined to the renal cortex (specifically proximal and distal tubules) and sustained beyond 2 months post injection. Furthermore, we demonstrate the ability of this methodology to induce long-term, in-vivo knockdown of candidate genes either through somatic recombination of floxed alleles or by direct delivery of specific shRNA sequences. This study demonstrates that ultrasound-guided injection of lentiviral vectors provides a safe and efficient method for the genetic manipulation of renal tubules, representing a quick and versatile alternative to GEMMs for the functional characterisation of disease-related genes.

Highlights

Luminescence was successfully detected from the left flank of ELS injected mice but not from litter-mates injected with a control lentiviral vector (Fig. 1c)
To evaluate the extent of renal lentiviral transduction achieved by our approach, we examined multiple sections of both left and right kidneys of injected mice
We demonstrate that US-guided intraparenchymal delivery of lentiviral vectors can induce sustained genetic modification of renal tubular epithelial cells bypassing the need to engineer novel genetically engineered mouse models (GEMMs) and to breed multiple animals for the generation of compound mutants

Summary

Introduction

To establish lentiviral vector integration and transgene expression in-vivo bioluminescence was assessed at 7 days post US-guided intraparenchymal injection. Immunohistochemical analysis of injected kidneys at 7, 15 and 60 days post infection revealed sustained Strawberry expression limited to the renal cortex and corticomedullary junction (Fig. 1d), supporting stable lentiviral integration and transgene expression. To evaluate the extent of renal lentiviral transduction achieved by our approach, we examined multiple sections of both left and right kidneys of injected mice.

Results

Conclusion