Abstract
Purpose: :
The Borate transporter NaBC1, encoded by SLC4A11 gene, has recently been implicated in recessive congenital endothelial dystrophy (CHED2) and Fuchs endothelial dystrophy. The precise function of NaBC1 in the corneal endothelium is unknown. The purpose of the present study is to clone and characterize SLC4A11 gene expression in bovine corneal endothelial cells (CE), which are extensively used to model endothelial function.
Methods: :
Total RNA was extracted from both cultured bovine CEs using Trizol reagents (Invitrogen). Poly A positive mRNAs were isolated using oligodT containing magnetic beads (Bioclone Inc.). Subsequently, poly A containing anti-sense of the NaBC1 mRNAs were used to concentrate the specific mRNA populations. Anti-sense fragments were firstly coupled with the magnetic beads and then mRNA populations were added in the mixture. First and second strands of the cDNA were synthesized using SuperScriptTM Double-Stranded cDNA Synthesis Kit (Invitogen). A primer-linker was ligated with the cDNAs and amplified using polymerase chain reactions. The products were then subcloned and subjected to sequencing. For interspecies comparison, human NaBC1 mRNA/cDNA sequences (Genbank, NM[[Unsupported Character - Codename ­]]_032034) were used for the blast search. Comparisons among the genomic sequences were done using CLUSTALW, multiple sequences alignment software (http://align.genome.jp/).
Results: :
Full length cDNAs were obtained from bovine corneal endothelial cells (reported in the Genbank). Comparisons among different species suggest that NaBC1 is highly conserved among different vertebrate species. The 5’ end was found to be less conserved with the potential of alternative translational initiation. The 3’ end was highly conserved among different species. One of the exons (human NaBC1 mRNA, Genbank, NM[[Unsupported Character - Codename ­]]_032034, exon 2115-2288) was found to have significant homology in the lower species (Heterodera Glycines, Soybean cyst nematode) suggesting that this particular area may act as the core essential part of the NaBC1 gene.
Conclusions: :
NaBC1 is highly conserved among different vertebrates with the potential of multiple splice variants. Anti-sense mediated concentrating of specific mRNAs may replace the troublesome post-cloning screening of cDNA libraries. This study indicates that bovine corneal endothelial cells can be used as a model for studying the role of borate transport in endothelial health and disease.
Keywords: cornea: endothelium • ion transporters • gene/expression