Brandon George, Jing-Hua Xi, Fang Bai, Usha Andley, Ophthalmology Department, Washington University School of Medicine, St. Louis, MO

Crystallins are a class of proteins best known for their function in the fibrous cells that make up the lens of the eye, where they assist in refraction so the image is focused on the retina. However, it has been recently discovered that many cells outside the lens also produce the various types of crystallin proteins. We were interested in what the function of crystallin proteins in the retina was. We were already aware that the α-family of crystallin (αA, αA-INS, αA-nov1, αB) functions as chaperone proteins outside the cell, but the functions of the crystallin families β (βA1, βA2, βA3, βA4, βB1, βB2, βB3), γ (γA, γC, γD, γE, γF, γS), μ, ζ, and λ remain unknown.

We performed two experiments on crystallins in the retina of mouse (mammalian model organism) eyes. The first examined the relationship between retinal crystallin expression and the age of the mouse. This was to test the hypothesis that in the C57 strain, crystallin expression drops as the mouse ages. We had previously found high levels of crystallin expression in young C57 mice, so next we had to test some older mice.

The other experiment was to determine what effect the α crystallin family has on the expression of other families of crystallin, specifically β and γ. This would be determined by taking a 129SV, the “wild type” strain, and comparing its β and γ crystallin levels to a mutant 129SV in which both αA and αB crystallin have been “knocked out.”

The processes for the two experiments were very similar. We would start with a frozen mouse retina and begin by performing RNA isolation. Next we treated the solution with DNase to eliminate any small fragments of DNA that remained. This makes it so that the only genetic material in the solution was in the form of mRNA. Since mRNA is the key intermediate in protein synthesis, it makes the perfect means to detect gene expression. After DNase treatment, we performed cDNA synthesis off the mRNA using reverse transcriptase. After we had some cDNA, we ran a PCR to amplify the cDNA. The primers were specially chosen for a variety of cyrstallin proteins and standards such as glyceraldehyde 3-phosphate dehydrogenase (GAPDH, a “housekeeping” gene) and hypoxanthine guanine phosphoribosyl transferase (HPRT). The age experiment used regular PCR, while the knock out used QRT-PCR. After we had a large amount of PCR product, we ran a gel electrophoresis to get a graphical representation of what genes are being expressed, since all the DNA in the gel came from mRNA.

In the age experiment, we found that the elderly mouse (1 year old) had no noticeable crystallin expression, a stark contrast to the high levels in the younger C57 mice. However, the elderly mouse was expressing both GAPDH and HPRT, two standard genes, so we can be fairly confident that our sample had some genes being expressed. This supporting of the hypothesis stated earlier is significant, since the decline of crystallin expression in the retina with age may also apply to humans.

The double knockout experiment yielded surprising results. We found that γC expression was far greater in the absence of αA and αB crystallin than in their presence. However, further experimentation is required to determine the function of γC crystallin; the best method for this would probably be the creation of a γC knockout based off the 129SV, so you could compare the phenotype to the wild type and other mutants of 129SV.