Expression profiling and gene discovery in the mouse lens.
OBJECTIVE
Defects in the development and physiology of the lens can result in cataracts (opacification of the lens), which are currently treatable only by surgical removal. The lens is also an excellent system for understanding fundamental biological processes such as cellular differentiation and ageing. Here, microarrays have been used to gain insights into global patterns of gene expression in the mouse lens. Lens gene expression compared to non-lens tissues has been investigated in order to identify genes preferentially expressed in the lens and lenses of different ages have been compared to identify differentially regulated genes.


METHODS
Genes expressed in the lens were identified using mouse GeneFilters microarrays (GF400; ResGen). Each array comprises 5,184 mouse cDNAs representing sequence-verified known genes and uncharacterized ESTs spotted onto a nylon membrane. Target RNA (33P labeled) from lens and non-lens samples was hybridized to the arrays. The proportion of genes involved in various biological processes was investigated using Onto-Express to search for GeneOntology terms associated with them. Differential gene expression was investigated using K-means clustering analysis. Expression of known and uncharacterized genes selected from the arrays was investigated further using semi-quantitative RT-PCR.


RESULTS
1,668 genes were expressed in one or more of newborn, 7 day old, and adult mouse lenses at levels significantly above background. Raw data and bioinformatics data relating to these genes have been published herein. There were 543 (33%) known genes, 124 (7%) had some similarity to known genes, 400 (24%) were functionally uncharacterized, and the remaining 601 (36%) genes were novel (matching only existing ESTs). Onto-Express identified genes involved in various biological processes including several categories containing greater numbers of genes than would be expected by chance, such as transcription regulation and G-protein coupled receptor signaling genes. Semi-quantitative RT-PCR confirmed preferential expression of several genes in the lens compared to non-lens tissues and genes exhibiting significantly higher expression in the 7 day lens compared to either adult or newborn lenses. Expression in the lens of 10 genes involved in apoptosis was also confirmed and, intriguingly, expression of hemoglobin isoforms (Hba-a1, Hba-X, Hbb-b1, Hbb-b2, and Hbb-Y) was confirmed using isotype specific primers. Finally, we confirmed the expression in the lens of all additional novel, uncharacterized and known genes tested.


CONCLUSIONS
The present work has provided insights into global patterns of gene expression in the lens and the expression of a significant number of genes has been confirmed using semi-quantitative RT-PCR. Genes preferentially expressed in the lens compared to non-lens tissues have been identified as well as genes differentially expressed between lenses at different ages. Gene expression profiling and gene discovery in the lens are essential prerequisites for future functional studies aimed at gaining insights into the potential roles of these genes in lens development, maturation, physiology, and pathogenesis (using targeted mutagenesis in mice, for instance).
Wride
Michael A
MA
Cardiff University, Cell and Molecular Research Group, School of Optometry and Vision Sciences, Cardiff, Wales, UK. WrideMA@cardiff.ac.uk
Mansergh
Fiona C
FC
Adams
Steffan
S
Everitt
Rebecca
R
Minnema
Stephanie E
SE
Rancourt
Derrick E
DE
Evans
Martin J
MJ
D000818:Animals; D003459:Crystallins / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics*; D016208:Databases, Factual; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000502:physiology*; D015723:Gene Library; D005796:Genes; D007908:Lens, Crystalline / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D003459:Crystallins; D018076:DNA, Complementary; D008565:Membrane Proteins