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T. T. McMahon, H. Ying, X. Shen, B. Y. J. T. Yue; CRB1 Gene Expression in the Human Cornea. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4653.
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To investigate whether CRB1, or the Crumbs homolog 1 gene is expressed in the human cornea. An association of the CRB1 genotype variant of Leber Congenital Amaurosis and keratoconus, a bilateral corneal thinning disorder, has recently been described. 1 CRB1 is believed to be responsible for photoreceptor orientation and polarity in the developing retina. The expression of CRB1 in the human cornea however has not been described.
Expression of the CRB1 gene was examined by reverse transcription-PCR (RT-PCR) and immunohistochemical staining. Total RNA extracted from normal human cornea and retina (positive control) using the QIAGEN RNAeasy mini Kit was reverse transcribed into cDNA via the Fermentas First Strand cDNA Synthesis Kit. Human CRB1-specific primer sets (forward, 5’- AACAACACCAGGTGCCTCTC -3’; and reverse, 5’-GGCATGTAGCCTCGTTCTTG -3’) were used in PCR. The predicted product size for the human CRB1 gene was 516 bp. For immunohistochemistry, normal human corneal and retinal paraffin sections were prepared. Following deparaffinization and antigen retrieval, tissue sections were blocked in blocking buffer and incubated with goat anti-CRB1 antibody (1:100) overnight at 4 C. The slides were further incubated with biotin conjugated rabbit anti-goat secondary antibody (1:500) and the immunoreactive products were visualized using the VECTASTAIN ABC kit.
: By PCR, a 516-bp product was obtained from both normal human retinal and corneal cDNA samples but not from the water negative control. Sequence analyses confirmed the identity of this PCR product to be the human CRB1 gene. Immunohistochemical staining using anti-CRB1 yielded positive brown deposits in the human cornea. The staining intensity observed was much weaker than that in the retina. In the cornea, the most prominent staining was seen in the epithelial layer. Nuclear staining of CRB1 was distinctively noted in basal epithelial cells.
Both CRB1 transcript and protein are detected in the human cornea. These results support the conclusion that the CRB1 gene is expressed in the adult cornea.
1. McMahon TT, Kim LS, Fishman GA, et al. CRB1 gene mutations are associated with keratoconus in patients with leber congenital amaurosis. Invest Ophthalmol Vis Sci 2009;50(7):3185-7.
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