Identification of novel bovine RPE and retinal genes by subtractive hybridization.
OBJECTIVE
Understanding of the specialized function of the retinal pigment epithelium (RPE) can be aided by the identification and characterization of genes that are preferentially expressed in the RPE. With this aim, we undertook a systematic effort to identify and begin characterization of such genes.


METHODS
A subtracted bovine RPE cDNA library was generated through subtractive hybridization using a single-stranded circular bovine RPE cDNA library as target and biotinylated mRNA from bovine heart and liver as alternate drivers. Approximately one thousand of the resulting subtracted cDNA clones were partially sequenced and analyzed, and a non-redundant set of one hundred of these cDNAs were examined for tissue expression pattern using a mini-Northern blot procedure and for identity by sequence analysis.


RESULTS
The subtraction method successfully allowed the enrichment of cDNAs that are preferentially expressed in the RPE. Out of the analyzed clones, expression of forty-five clones was verifiable by Northern blotting. Of these, a significant proportion of cDNAs were preferentially expressed in the RPE. We observed that the expression of some subtracted cDNAs was restricted to the retina and no expression was detected in the RPE. These retinal clones were obtained in addition to RPE clones presumably because the initial RPE RNA population was contaminated with a small proportion of retinal RNA. Two thirds of the identified RPE and retinal cDNAs are likely to represent novel genes because they do not have homology to known genes in the databases.


CONCLUSIONS
Genes that are specifically or predominantly expressed in the RPE/retina are likely to be important for retinal function. We have identified novel cDNAs from bovine RPE and retina by subtractive hybridization. These cDNAs can be used as starting material for the identification of corresponding human genes expressed in the RPE and retina. The human genes thus identified are likely to contain good candidate genes for retinal disease.
Department of Ophthalmology, Flinders University, Bedford Park, South Australia, Australia. shiwani.sharma@flinders.edu.au
Shiwani
S
Sharma
Jinghua T
JT
Chang
Neil G
NG
Della
Peter A
PA
Campochiaro
Donald J
DJ
Zack
D000818:Animals; D015152:Blotting, Northern; D002417:Cattle; D018076:DNA, Complementary / Q000032:analysis; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods; D015723:Gene Library; D009693:Nucleic Acid Hybridization / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012160:Retina / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein
D018076:DNA, Complementary; D005136:Eye Proteins; D012333:RNA, Messenger