Microarray Analysis of Gene Expression in Human Donor Corneas

Jun, Albert S.; Liu, Sammy H.; Koo, Ellen; Diana, V.; Stark, Walter J.; Gottsch, John D.

doi:10.1001/archopht.119.11.1629

Cited by 30 publications

(15 citation statements)

References 35 publications

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“…2,3 A previous work described the use of complementary DNA (cDNA) microarray technology to provide a gene expression profile of the human cornea, characterizing some 1200 genes. 4 Microarrays, however, are limited by the finite number of oligonucleotide sequences localized on a chip and are not able to identify the expression of novel genes.…”

mentioning

confidence: 99%

Gene Expression in Donor Corneal Endothelium

et al. 2003

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confidence: 99%

Gene Expression in Donor Corneal Endothelium

et al. 2003

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“…Corneas were fixed for 30 min in phosphate-buffered saline (PBS) containing 1% glutaraldehyde, then rinsed thrice in cold PBS containing 2 m M of MgCl 2 . Corneas were then incubated for 30 min at 37 ° C in a solution containing 0.2% X-Gal (5-bromo-4-chloro-3-inodyl-␤ -galactosidase, Sigma), 10 6 , which gave a blue coloration to the transfected ECs. After rinsing in distilled water, corneas were placed on a backlit table and photographed at low magnification.…”

Section: Transfection With ␤ -Galactosidase: Measurement Of Transfectmentioning

confidence: 99%

“…Overexpression of certain genes of interest and the inactivation of others may allow determination of the role and interactions of certain proteins in the corneal endothelium. This is all the more important as insight into the genes expressed most specifically in the ECs of healthy subjects and in patients with Fuchs' dystrophy was provided several years ago [6][7][8][9][10] . In the long term, the prospect of local gene therapy for management of early stages of Fuchs' dystrophy and for modulation of accelerated EC loss during corneal preservation and post-graft rejection is equally attractive.…”

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confidence: 99%

Ex vivo Gene Electrotransfer to the Endothelium of Organ Cultured Human Corneas

Hé¹,

Pipparelli²,

Manissolle³

et al. 2009

Ophthalmic Res

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Aims: To describe an innovative device that allows gene electrotransfer to human corneal endothelial cells (EC) during storage in organ culture. Methods: Customized electrodes without endothelial contact were developed. Two plasmids containing the cytomegalovirus promoter and reporter genes [enhanced green fluorescent protein (eGFP) or beta-galactosidase (β-gal)] were electroporated in 2 series of human corneas with eight 1-Hz 100-ms pulses of 125 mA square current. Controls were exposed to naked DNA without electric pulses. eGFP-transduced corneas were used to determine the transgene expression kinetics, whereas β-gal measured transfection efficiency using image analysis tools. Overall, endothelial toxicity was determined by: (1) cytotoxicity tests using triple staining with Hoechst 33342, ethidium homodimer III, and calcein AM, 3 h and 3 and 14 days after electroporation on the series of 15 eGFP-transfected paired corneas; (2) anti-ZO-1 staining to assess tight junctions’ integrity. Results: All electroporated corneas carried transfected ECs, whereas the controls carried none. eGFP expression was observed 3 h after electrotransfer, and was then present from days 1 to 28. Transfection efficiency determined on 63 corneas transfected with β-gal ranged from 0.1 to 54% of the transfected ECs (mean ± SD: 7 ± 11%, median: 2.9%) with significant reproducibility for paired corneas from the same donor. Electroporation produced low early EC death. Anti ZO-1 staining revealed no dramatic change in EC mosaic continuity, neither 1 and 3 nor 28 days after electroporation. Conclusions: Gene electrotransfer to the endothelium of organ-cultured human corneas with custom-designed electrodes allows rapid and easy EC transfection. However, further optimization is required to ensure reproducible results.

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“…In an effort to create a preliminary database of human corneal gene expression Jun et al (2001) performed a microarray study using a cDNA array constructed from human donor corneas. The study showed a wide range of genes being expressed including, genes for six types of collagen subunit were found to be expressed (e.g.…”

Section: Introductionmentioning

confidence: 99%

Limbal stem cells, a review of their identification and culture for clinical use

O’Sullivan

Clynes

2007

Cytotechnology

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The surface of the eye is covered by two distinct epithelial populations, the conjunctival and corneal epithelia. The stem cell population for the corneal epithelia has been found to be located at the area known as the limbus. This is a narrow ring of tissue at the transitional zone between the cornea and conjunctiva. This stem cell population is responsible for generating transient amplifying cells which are responsible for renewing the cornea epithelia. There are currently no definitive markers for the stem cell population in the limbus. Instead using morphological features, such as small cells with a high nucleus-to-cytoplasm ratio, in conjunction with the presence of certain markers e.g. DNP63a and the absence of others, e.g. the cytokeratin pair 3 & 12, are taken as being indicative of the stem cell population. Damage can occur to the corneal epithelium due to a number of causes including, Steven-Johnson syndrome, and chemical or thermal burns. This results in invasion of the cornea by the conjunctival epithelium resulting in impaired vision. In 1997 Pellegrini et al. (Lancet 349, 990) successfully used cells sheets from cultured limbal cells to successfully treat patients with corneal damage. Since then several other groups, have successfully treated patients, using similar methods.

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Microarray Analysis of Gene Expression in Human Donor Corneas

Cited by 30 publications

References 35 publications

Gene Expression in Donor Corneal Endothelium

Gene Expression in Donor Corneal Endothelium

Ex vivo Gene Electrotransfer to the Endothelium of Organ Cultured Human Corneas

Limbal stem cells, a review of their identification and culture for clinical use

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