Purchase this article with an account.
M.A. Akinci, M.T. Budak, J.M. Wolosin; Identification of Limbal genes by Differential Display . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2031.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: Progress in the study of the limbo-corneal epithelial (LCE) proliferation, survival and differentiation is presently hindered by the dearth of information on enzymes and proteins selectively expressed in the limbal (and stem cell)-domains. Accordingly, the purpose of this study was to identify genes differentially expressed in the limbus. Methods: Limbal (Li) and corneal (Co) biopsies from Dutch Belted rabbits, were explanted for 88 hr in permeable membrane inserts placed in plates containing a feeder layer of 3T3 cells. Pigmentation allowed accurate dissection of Li tissue. After ~72 hr, suprabasal cells shed off, leaving basal cells monolayers. These cells displayed known characteristics of the respective tissue cells, including clonogenic capacity (approx. 1:16 cells for Li; nil for Co) and K3 and Cx43 expression (Li, neg./low.; Co, pos.). Total RNA was prepared in parallel from both cell types. Equal RNA amounts were reverse transcribed and subjected to PCR using 110 distinct primer pair combinations. Reactions included 33P-dCTP. Products were resolved in ultrathin polyacrylamide gels and visualized by autoradiography. Bands for which the Li:Co density ratio was > 5 were considered to be differentially displayed (DD) Li amplicons (A). They were cut out of gels and subcloned using TOPO-TA. For each Li DDA four clones were sequenced. When > 2 clones were identical the sequence was considered a putative Li-specific gene. Gene identity was determined by BLAST against the mammalian genomic database. The definitive status of each such gene was confirmed by real time quantitative (RTQ) PCR using RNAs generated from fresh Li and Co epithelia. Primers were designed using the Oligo4 software. Results: The PCR reactions generated about 10,000 amplicons, 147 DDAs and 53 putative Li genes. Twenty-seven of those later sequences were homologues of known mammalian proteins (E value < 10-15). RTQPCR confirmed the selective expression of 17 of these genes including: inhibitor of apoptosis p2 (IAP2, a survivin-type protein; Li to Co expression ratio, 62:1); Pim3 (an anti-apoptotic ser/threo kinase; 55:1); Celsr-1/flamingo (developmental cadherin; 22:1); Smarf1 (involved in control of chromatin remodeling /binds P53 and Rb and effects tumor suppression; 23:1); SIRP-1α(hematopoietic precursor membrane glycoprotein CD172; 150:1). Conclusions: Differential display allows identification of relevant signal transduction and membrane proteins that could be critical for the control of limbal phenotype, limbal cell survival and can yield markers for stem cell isolation.
This PDF is available to Subscribers Only