In vivo gene delivery and visualization of corneal stromal cells using an adenoviral vector and keratocyte-specific promoter
In vivo gene delivery and visualization of corneal stromal cells using an adenoviral vector and keratocyte-specific promoter
复制标题
DOI：
10.1167/iovs.03-1224
复制
发表时间：
2004-07-01
影响因子：
4.4
通讯作者：
Perez, VL
中科院分区：
文献类型：
--
作者：
Carlson, EC;Liu, CY;Perez, VL
目的.本研究旨在确定角膜基质细胞内注射腺病毒构建体是否可以有效地在体内转染角膜基质细胞，并确定组织特异性启动子是否可以用于在角膜基质细胞中表达外源基因。将具有巨细胞病毒（pCMV）驱动的增强型绿色荧光蛋白（EGFP）报告基因的腺病毒构建体注射到小鼠角膜基质中。通过体内立体显微镜定量体内表达并分析样品，通过共聚焦三维（3-D）重建测定离体表达。使用3.2-kb角膜蛋白聚糖启动子驱动组织特异性报告基因在体内表达。在角膜基质中注射腺-EGFP后11小时，在体内首次检测到EGFP表达，持续时间约3周。离体整体角膜共焦分析与3-D重建允许可视化的EGFP表达在角膜基质细胞，以准确地评估细胞的结构和分布在角膜基质。裸pCMV-EGFP质粒DNA不表达腺病毒-EGFP水平的报告基因。3.2kb的角膜蛋白聚糖启动子能够驱动EGFP在角膜中的组织特异性表达。角膜基质内注射腺病毒包装的DNA构建体是在角膜基质中递送和表达基因的快速和有效的方式。基质内注射还能够将组织特异性启动子构建体递送至角膜基质用于基因表达。此外，三维重建提供了一个强大的工具，增强可视化的角膜基质环境和细胞生物学。
PURPOSE. This study was conducted to determine whether intrastromal injection of adenoviral construct could be used to transfect corneal stroma cells effectively in vivo and to determine whether a tissue-specific promoter could be used to express exogenous genes in keratocytes.METHODS. An adenoviral construct with a cytomegalovirus (pCMV)-driven enhanced green fluorescent protein (EGFP) reporter gene was injected into the stroma of murine corneas. In vivo expression was quantitated and samples were analyzed by in vivo stereomicroscopy, and ex vivo expression was determined by confocal three dimensional (3-D) reconstruction. The 3.2-kb keratocan promoter was used to drive tissue-specific reporter gene expression in vivo.RESULTS. EGFP expression was first detected in vivo 11 hours after injection of adeno-EGFP in the corneal stroma, with a duration of approximately 3 weeks. Ex vivo wholemount cornea confocal analysis with 3-D reconstruction allowed visualization of EGFP expression in corneal stroma cells, to accurately assess cellular architecture and distribution in the corneal stroma. Naked pCMV-EGFP plasmid DNA did not express the reporter gene to the levels of the adeno-EGFP. The 3.2-kb keratocan promoter was capable of driving EGFP tissue-specific expression in the cornea.CONCLUSIONS. Intrastromal injection of adenovirus packaged DNA constructs is a rapid and efficient way to deliver and express genes in the corneal stroma. Intrastromal injection is also capable of delivering tissue-specific promoter constructs to the corneal stroma for gene expression. Furthermore, 3-D reconstruction provides a powerful tool for enhanced visualization of the corneal stroma environment and cellular biology.