Specific targeting of gene expression to a subset of human trabecular meshwork cells using the chitinase 3-like 1 promoter.
OBJECTIVE
To compare the gene expression profile of trabecular meshwork (TM) and Schlemm's canal (SC) primary cultures and to identify promoters for targeting gene expression to specific cells in the outflow pathway.


METHODS
The differential gene expression profile of four human TM and three SC primary cultures was analyzed by gene microarrays (Affymetrix, Santa Clara, CA) and confirmed by quantitative real-time PCR. Based on the results, a recombinant adenovirus was constructed with the expression of the reporter gene LacZ driven by the 5' promoter region of the chitinase 3-like 1 (Ch3L1) gene (AdCh3L1-LacZ). The expression of the Ch3L1 promoter was analyzed in human TM and SC cells and in human perfused anterior segments infected with AdCh3L1-LacZ.


RESULTS
gamma-Sarcoglycan, fibulin-2, and collagen XV were identified as the genes more highly expressed in SC than in TM cells. Ch3L1 showed the highest levels of differential expression in TM versus SC cells. Expression analysis of the Ch3L1 promoter demonstrated specific expression in a subset of the TM cells in cell culture and in perfused anterior segments.


CONCLUSIONS
Comparative analysis of gene expression between SC and TM primary cultures identified several genes with promoters potentially capable of targeting gene expression to specific cells within the outflow pathway. Results with the Ch3L1 promoter indicated that two different cell subtypes may be present in the TM. This study provides a new potential tool to investigate the role of these different cell types in both normal and pathophysiological function of the outflow pathway, with implications for possible future glaucoma gene therapy.
Liton
Paloma B
PB
Department of Ophthalmology, Duke University, Durham, North Carolina 27710, USA.
Liu
Xialin
X
Stamer
W Daniel
WD
Challa
Pratap
P
Epstein
David L
DL
Gonzalez
Pedro
P
D000256:Adenoviridae / Q000235:genetics; D054392:Adipokines; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D000071451:Chitinase-3-Like Protein 1; D003094:Collagen / Q000235:genetics / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D020869:Gene Expression Profiling; D018390:Gene Targeting; D006023:Glycoproteins / Q000235:genetics*; D006801:Humans; D037102:Lectins; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D049031:Sarcoglycans / Q000235:genetics / Q000378:metabolism; D014129:Trabecular Meshwork / Q000166:cytology / Q000378:metabolism*; D001616:beta-Galactosidase / Q000378:metabolism
D054392:Adipokines; C084533:CHI3L1 protein, human; C085811:COL15A1 protein, human; D002135:Calcium-Binding Proteins; D000071451:Chitinase-3-Like Protein 1; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; D037102:Lectins; D049031:Sarcoglycans; C084612:fibulin 2; D003094:Collagen; D001616:beta-Galactosidase