In vivo gene delivery and visualization of corneal stromal cells using an adenoviral vector and keratocyte-specific promoter.
OBJECTIVE
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.
Carlson
Eric C
EC
Ophthalmic Research, Cole Eye Institute, The Cleveland Clinic Foundation, Ohio 44195, USA.
Liu
Chia-Yang
CY
Yang
Xiaoping
X
Gregory
Meredith
M
Ksander
Bruce
B
Drazba
Judith
J
Perez
Victor L
VL
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D003319:Corneal Stroma / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D021621:Imaging, Three-Dimensional; D007267:Injections; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D011401:Promoter Regions, Genetic; D011509:Proteoglycans / Q000235:genetics*; D014162:Transfection / Q000379:methods*
C099280:Kera protein, mouse; D008164:Luminescent Proteins; D011509:Proteoglycans; D049452:Green Fluorescent Proteins