1FM Kirby Center of Molecular Ophthalmology, Scheie Eye Institute, USA
2Department of Medicine, Medical University of South Carolina, USA
33Department of Medicine, Ralph H Johnson VAMC, USA
*Corresponding author: Joshua H Lipschutz, Medical University of South Carolina, Clinical Science Building 829, 96 Jonathan Lucas Street, Charleston, SC 29425, USA, Tel: 843792-7659; Fax: 843 792-8399; E-mail: Lipschut@musc.edu
Submission: January 23, 2018; Published: February 05, 2018
Volume1 Issue4 February 2018
Background/Aims: Degeneration of primary cilia has been implicated in many diseases, including retinal degeneration and polycystic kidney disease. This study aims to demonstrate gene transfer to adult murine ciliated kidney tubule cells, in vivo, facilitated by φC31 integrase and electroporation.
Methods: Cohorts of 6-10 week old adult C57BL/6 mice were used. After adequate anesthesia via inhaled isoflurane, mice were prepped and draped for abdominal surgery. A midline laparotomy was performed, and the left kidney was isolated. Connective and adipose tissue was bluntly dissected away from the ureter and renal pelvis. A 10μL retrograde intra-ureteral microinjection was performed utilizing a heat-pulled glass capillary tube. The capillary tube was filled with two plasmids, one containing the marker gene, enhanced green fluorescent protein with the attachment site B sequence (pCMV.eGFP.attB), and the other encoding for the integrase enzyme (pCMV.Int), and electroporation was performed. Three days later, the mice were sacrificed, and kidneys were collected, placed in 4% paraformaldehyde, cryoprotected, sectioned, and evaluated using fluorescence microscopy.
Results: Green fluorescence protein expression was detected in renal papilla and renal tubule cells. No gross inflammation was appreciated.
Conclusion: φC integrase can facilitate gene transfer to the adult murine kidney in vivo. Further optimization will be needed to increase efficiency of transduction, document stability of transgene expression, and evaluate potential toxicities.
Keywords: Radical salvage prostatectomy; Pelvic lymphadenectomy; Prostatic radiation therapy
Abbreviations:: PKD: Polycystic Kidney Disease; AKI: Acute Kidney Injury; ESRD: End-Stage Renal Disease