This lab has previously generated alpha-crystallin gene knockout mice to study the in vivo function of these remarkable proteins. The alpha-crystallins comprise a large fraction of the soluble protein in the vertebrate lens where they were, for many years, believed to function solely as structural proteins. Lenticular alpha-crystallin is comprised of two similar subunits alphaA and alphaB, each encoded by a single gene. They are related to the small heat shock proteins, and in vitro they exhibit molecular chaperone activity, autokinase activity, and interact with, and affect the state of, several cytoskeletal components. alpha-Crystallin, especially alphaB-crystallin, has been shown to be a normal constituent of many non-lenticular tissues, and has been detected in cytoplasmic inclusion bodies found in several human pathological conditions. alphaA-Crystallin, and indeed many of the formerly """"""""lens-specific"""""""" crystallins, have also been shown to be expressed on non lenticular tissues. Toward understanding the major roles of alpha-crystallin in vivo, we previously generated alphaA- and alphaB-crystallin gene knockout mice and alphaA-/alphaB-crystallin gene double knockout mice (DKO). We continue to maintain these lines as a resource to other investigators around the world who are studying these proteins in various tissues and disease states. Expression of other classes of crystallins have been discovered in nonlenticular eye tissues and a variety of nonocular tissues. In a continuing collaboration with Dr. Debasish Sinha at Johns Hopkins University the function of betaA3/A1-crystallin in retina is being investigated. Transgenic mice expressing the nuc1 mutant form of betaA3/A1 specifically in astrocytes, under control of the GFAP promoter, are recapitulating many of the observations made in the original nuc1 mutant rats, clearly demonstrating that a dysmorphic lens is not responsible for the retinal effects of this mutation. As a part of this collaboration, and in collaboration with Dr. Lijin Dong of the NEI Genetic Engineering Facility, conditional knockout mice in which the betaA3/A1 gene is specifically disrupted in astrocytes have been generated. We have attained germline transmission of the targeted mutation in lines of mice derived from two of the mutated ES cell lines. This approach avoids interference or complicating secondary effects from a disintegrating lens, when studying subtle phenotypes in the retina and other ocular tissues. The approach was to insert loxP recombinase sites flanking exons 4 and 5 of the mouse betaA3/A1-crystallin gene, and excising this portion of the gene by astrocyte-specific expression of cre recombinase, using the GFAP promoter. The next step will be to breed these mice to beta actin/Flpe mice to excise the frt-flanked selectable marker from the targeted chromosome to keep the gene functioning in non-cre-expressing tissues. Then, it will be bred to the astrocyte-specific cre line to generate mice in which betaA3/A1-crystallin is deleted specifically in astrocytes. Effects of this mutation can then be studied in retina and brain.
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