Differences in expression of retinal pigment epithelium mRNA between normal canines.
A reference database of differences in mRNA expression in normal healthy canine retinal pigment epithelium (RPE) has been established. This database identifies non-informative differences in mRNA expression that can be used in screening canine RPE for mutations associated with clinical effects on vision. Complementary DNA (cDNA) pools were prepared from mRNA harvested from RPE, amplified by PCR, and used in a subtractive hybridization protocol (representational differential analysis) to identify differences in RPE mRNA expression between canines. The effect of relatedness of the test canines on the frequency of occurrence of differences was evaluated by using 2 unrelated canines for comparison with 2 female sibling canines of blue heeler/bull terrier lineage. Differentially expressed cDNA species were cloned, sequenced, and identified by comparison to public database entries. The most frequently observed differentially expressed sequence from the unrelated canine comparison was cDNA with 21 base pairs (bp) identical to the human epithelial membrane protein 1 gene (present in 8 of 20 clones). Different clones from the same-sex sibling RPE contained repetitions of several short sequence motifs including the human epithelial membrane protein 1 (4 of 25 clones). Other prevalent differences between sibling RPE included sequences similar to a chicken genetic marker sequence motif (5 of 25), and 6 clones with homology to porcine major histocompatibility loci. In addition to identifying several repetitively occurring, noninformative, differentially expressed RPE mRNA species, the findings confirm that fewer differences occurred between siblings, highlighting the importance of using closely related subjects in representational difference analysis studies.
Hanik
Heather L J
HL
Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan.
Loewen
Matthew E
ME
Appleyard
Greg D
GD
Grahn
Bruce H
BH
Forsyth
George W
GW
D000818:Animals; D017931:DNA Primers; D018076:DNA, Complementary / Q000032:analysis; D004285:Dogs / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D010375:Pedigree; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012333:RNA, Messenger / Q000378:metabolism*; D012016:Reference Values
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger