Development of Gene Microarray Chips for Rabbit and Dog Ocular Tissues

Abstract

Abstract: : Purpose: Microarray gene chips allow a broad analysis of changes in gene expression patterns during biological processes or disease states. Rabbit and dog are common animal models for ophthalmic research but microarray gene chips have not been available for these species. Methods: Samples of normal tissues from anterior segment (cornea/conjunctiva) and posterior segment were separated and harvested from NZW rabbit and beagle dog eyes and processed through the NEIBank pipeline. Five cDNA libraries (rabbit lens library failed) were constructed in pCMVSport6 and almost 4000 clones from each library were sequenced from both 5' and 3' ends at the NIH Intramural Sequencing Center. These sequences and other rabbit or dog ophthalmic genes from GenBank were assembled and further filtered by UF/ICBR to derive two sets of non–redundant sequences for dog and rabbit respectively, which were annotated with sequence orientation, potential CDS, gene identity and GeneOntology terms. These non–redundant sequences were then sent to Agilent Technologies for probe design. Custom arrays were manufactured with Agilent SureHyb technology in which 60 base oligonucleotide probes are synthesized in situ with non–contact printer. Dog chips were manufactured in 11K format (two individually hybridizable 11K arrays per glass covering 10,223 dog ophthalmic genes. Rabbit chips were manufactured in the 8–pack format (8 individually hybridizable 1.5K arrays per piece of glass) as two sets. The first set covers 1,578 cornea–specific genes while the second set covers 1,578 genes expressed in the rest of the eye. Results: Initial test of the arrays were performed using rabbit eyes with acute and latent herpes infection. Over 99% of genes represented by the probes were present above background levels under one of the experimental conditions. Statistical analysis of the data identified a number of genes whose expression is significantly altered by the disease process. Conclusions: Initial results demonstrate these new microarray chips detect mRNA levels for rabbit and dog ocular tissues and provide a valuable tool for ocular research.

Keywords: gene microarray