Targeting gene expression to specific cells of kidney tubules in vivo, using adenoviral promoter fragments.
Targeting gene expression to specific cells of kidney tubules in vivo, using adenoviral promoter fragments.
复制标题
DOI：
10.1371/journal.pone.0168638
复制
发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Uchida S
中科院分区：
文献类型：
--
作者：
Watanabe S;Ogasawara T;Tamura Y;Saito T;Ikeda T;Suzuki N;Shimosawa T;Shibata S;Chung UI;Nangaku M;Uchida S
尽管通过病毒转移进行细胞特异性基因表达的技术已经发展，但许多挑战（例如，病毒载体设计、将基因转导到特定靶细胞中）仍然存在。我们研究了一种新的，简单的方法，使用腺病毒转移到靶向特定细胞的肾小管表达外源蛋白。我们选择了编码近端小管中钠依赖性磷酸盐转运蛋白2a（NPT 2a）、汉勒氏粗升支（TALH）中钠-钾-2-氯协同转运蛋白（NKCC 2）和集合管中水通道蛋白2（AQP 2）的基因。将这三个基因的启动子连接到GFP编码片段上，然后将最终的构建体掺入腺病毒载体中，然后将其用于产生基因操纵的病毒。在冲洗循环血液后，将病毒直接注射到大鼠的肾动脉中，并使其在肾小管细胞中位点特异性表达，然后将大鼠安乐死，以获得用于免疫组织化学的肾组织。检查用腺病毒衍生的EGFP和内源蛋白的双重染色以验证原位表达，即“腺病毒驱动的NPT 2a-EGFP和内源NHE 3蛋白”、“腺病毒驱动的NKCC 2-EGFP和内源NKCC 2蛋白”和“腺病毒驱动的AQP 2-EGFP和内源AQP 2蛋白”。由于缺乏发现良好的工作抗NPT 2a抗体，使用了针对也在近端小管中特异性表达的不同蛋白质（钠-氢交换器3或NHE 3）的抗体。肾脏结构保存良好，其他器官组织未显示EGFP染色。我们的基因转移方法比使用基因工程动物更容易，并且它具有允许在出生后进行基因转移的优点。这是第一种成功地将基因表达靶向肾小管中特定细胞的方法。本研究为安全有效的基因治疗肾小管疾病迈出了第一步。
Although techniques for cell-specific gene expression via viral transfer have advanced, many challenges (e.g., viral vector design, transduction of genes into specific target cells) still remain. We investigated a novel, simple methodology for using adenovirus transfer to target specific cells of the kidney tubules for the expression of exogenous proteins. We selected genes encoding sodium-dependent phosphate transporter type 2a (NPT2a) in the proximal tubule, sodium-potassium-2-chloride cotransporter (NKCC2) in the thick ascending limb of Henle (TALH), and aquaporin 2 (AQP2) in the collecting duct. The promoters of the three genes were linked to a GFP-coding fragment, the final constructs were then incorporated into an adenovirus vector, and this was then used to generate gene-manipulated viruses. After flushing circulating blood, viruses were directly injected into the renal arteries of rats and were allowed to site-specifically expression in tubule cells, and rats were then euthanized to obtain kidney tissues for immunohistochemistry. Double staining with adenovirus-derived EGFP and endogenous proteins were examined to verify orthotopic expression, i.e. “adenovirus driven NPT2a-EGFP and endogenous NHE3 protein”, “adenovirus driven NKCC2-EGFP and endogenous NKCC2 protein” and “adenovirus driven AQP2-EGFP and endogenous AQP2 protein”. Owing to a lack of finding good working anti-NPT2a antibody, an antibody against a different protein (sodium-hydrogen exchanger 3 or NHE3) that is also specifically expressed in the proximal tubule was used. Kidney structures were well-preserved, and other organ tissues did not show EGFP staining. Our gene transfer method is easier than using genetically engineered animals, and it confers the advantage of allowing the manipulation of gene transfer after birth. This is the first method to successfully target gene expression to specific cells in the kidney tubules. This study may serve as the first step for safe and effective gene therapy in the kidney tubule diseases.