A comprehensive analysis of the expression of crystallins in mouse retina.
OBJECTIVE
Crystallins are expressed at high levels in lens fiber cells. Recent studies have revealed that several members of the alpha, beta, and gamma-crystallin family are also distributed in many non-lens tissues, though at lower levels. We observed that the use of retinal RNA as target for both custom I-Gene microarrays and Affymetrix GeneChips revealed significant expression of many crystallin genes. This prompted us to undertake a comprehensive investigation to delineate the baseline expression of crystallin genes in the adult mouse retina.


METHODS
Quantitative RT-PCR was carried out using gene specific primers (derived from the mouse genomic sequence) for each crystallin gene. Immunofluorescence studies using frozen sections of the mouse retinas were performed with crystallin-specific antibodies. Retinal lysates were analyzed by immunoblotting using antibodies specific to alphaA and alphaB crystallins and those produced against total beta-crystallin and gamma-crystallin fractions of bovine lenses.


RESULTS
Microarray analysis followed by quantitative RT-PCR revealed that mouse retinal cells express transcripts for 20 different members of the crystallin gene family; these are alphaA, alphaA-INS, alphaA-nov1, alphaB, betaA1, betaA2, betaA3, betaA4, betaB1, betaB2, betaB3, gammaA, gammaC, gammaD, gammaE, gammaF, gammaS, mu, zeta, and lambda-crystallin. The gene products of alphaA, alphaB, beta-, and gamma-crystallins are detected in the outer and inner nuclear layers of the retina by immunofluorescence analysis. In addition, alpha and beta-crystallins are detected in the photoreceptor inner segments. Retinal expression of these proteins was further confirmed by immunoblot analysis. Interestingly, our studies also showed a significant animal-to-animal variation in the expression level of some of the crystallins.


CONCLUSIONS
Our results establish the expression of many crystallins in the adult mouse retina. Detection of crystallins in the retinal nuclear layers, though surprising, is consistent with their proposed role in cell survival and genomic stability. We suggest that crystallins play vital functions in protecting retinal neurons from damage by environmental and/or metabolic stress.
Xi
Jinghua
J
Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Farjo
Rafal
R
Yoshida
Shigeo
S
Kern
Timothy S
TS
Swaroop
Anand
A
Andley
Usha P
UP
D000818:Animals; D003459:Crystallins / Q000235:genetics* / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D019084:Fluorescent Antibody Technique, Indirect; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D015151:Immunoblotting; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D020411:Oligonucleotide Array Sequence Analysis; D012160:Retina / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D003459:Crystallins; D017931:DNA Primers