Methods enabling kidney-specific gene transfer in adult mice are needed to develop new therapies for kidney disease. We attempted kidney-specific gene transfer following hydrodynamic tail vein injection using the kidney-specific podocin and gamma-glutamyl transferase promoters, but found expression primarily in the liver. In order to achieve kidney-specific transgene expression, we tested direct hydrodynamic injection of a DNA solution into the renal pelvis and found that luciferase expression was strong in the kidney and absent from extra-renal tissues. We observed heterogeneous, low-level transfection of the collecting duct, proximal tubule, distal tubule, interstitial cells, and rarely glomerular cells following injection. To assess renal injury, we performed the renal pelvis injections on uninephrectomised mice and found that their blood urea nitrogen was elevated at two days post-transfer but resolved within two weeks. Although luciferase expression quickly decreased following renal pelvis injection, the use of the piggyBac transposon system improved long-term expression. Immunosuppression with cyclophosphamide stabilised luciferase expression, suggesting immune clearance of the transfected cells occurs in immunocompetent animals. Injection of a transposon expressing erythropoietin raised the haematocrit, indicating that the developed injection technique can elicit a biologic effect in vivo. Hydrodynamic renal pelvis injection enables transposon mediated-kidney specific gene transfer in adult mice.
All nephrons in the mammalian kidney arise from a transient nephron progenitor population that is lost close to the time of birth. The generation of new nephron progenitors and their maintenance in culture are central to the success of kidney regenerative strategies. Using a lentiviral screening approach, we previously generated a human induced nephron progenitor-like state in vitro using a pool of six transcription factors. Here, we sought to develop a more efficient approach for direct reprogramming of human cells that could be applied in vivo. PiggyBac transposons are a non-viral integrating gene delivery system that is suitable for in vivo use and allows for simultaneous
Hydrodynamic injection creates a local, high-pressure environment to transfect various tissues with plasmid DNA and other substances. Hydrodynamic tail vein injection, for example, is a well-established method by which the liver can be transfected. This manuscript describes an application of hydrodynamic principles by injection of the mouse kidney directly with plasmid DNA for kidney-specific gene expression. Mice are anesthetized and the kidney is exposed by a flank incision followed by a fast injection of a plasmid DNA-containing solution directly into the renal pelvis. The needle is kept in place for ten sseconds and the incision site is sutured. The following day, live animal imaging, Western blot, or immunohistochemistry may be used to assay gene expression, or other assays suited to the transgene of choice are used for detection of the protein of interest. Published methods to prolong gene expression include piggyBac transposon-mediated transgene integration and cyclophosphamide treatment to inhibit the immune response to the transgene.
SummaryCystinuria Type A is a relatively common genetic kidney disease occurring in 1 in 7,000 people worldwide that results from mutation of the cystine transporter rBAT encoded by Slc3a1. We used CRISPR/Cas9 technology to engineer cystinuria Type A mice via genome editing of the C57BL/6NHsd background. These mice are an improvement on currently available models as they are on a coisogenic genetic background and have a single defined mutation. In order to use albinism to track Cas9 activity, we co‐injected gRNAs targeting Slc3a1 and tyrosinase (Tyr) with Cas9 expressing plasmid DNA into mouse embryos. Two different Slc3a1 mutational alleles were derived, with homozygous mice of both demonstrating elevated urinary cystine levels, cystine crystals, and bladder stones. We used whole genome sequencing to evaluate for potential off‐target editing. No off‐target indels were observed for the top 10 predicted off‐targets for Slc3a1 or Tyr. Therefore, we used CRISPR/Cas9 to generate coisogenic albino cystinuria Type A mice that could be used for in vivo imaging, further study, or developing new treatments of cystinuria.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.