Department of Veterans Affairs, Nashville, TN 37212 USA.
Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232 USA.
Sci Rep. 2017 Mar 20;7:44904. doi: 10.1038/srep44904.
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.
方法使肾脏特异性基因转移在成年小鼠需要开发新的治疗肾脏疾病。我们试图肾脏特异性基因转移后，尾静脉高压注射使用肾脏特异性足细胞和γ-谷氨酰转移酶启动子，但发现主要在肝脏表达。为了达到肾脏特异性转基因表达，我们测试了直接注射到肾盂 DNA 溶液和发现，表达的荧光素酶在肾脏和不存在肾外组织强。我们观察到不同的，低水平的转染集合管、近端肾小管、远端肾小管、间质细胞和肾小球细胞很少注射后。为了评估肾损伤，我们进行了肾盂注射在单侧肾切除小鼠，发现他们的血尿素氮升高两天后转移，但在两周内解决。虽然荧光素酶表达很快减少肾盂注射后，使用 piggyBac 转座子系统改善了长期表达。环磷酰胺免疫抑制稳定的荧光素酶表达，表明免疫清除转染细胞发生在免疫活性动物。注射一个转座子表达促红细胞生成素提高了红细胞比容，表明开发的注射技术可以引起体内的生物学效应。流体动力学肾盂注射使转座子介导的成年小鼠肾脏特异性基因转移。
