Transgenics and Genome Manipulation Section

5635 Fishers Lane
Room 1127, MSC 9402
Rockville, Maryland 20892-9402
Phone: (301) 496-8841
Fax: (301) 402-3603

• Activities/Accomplishments
• Staff
• Recent Publications

Activities/Accomplishments

The research done by this group has two major focuses 1) to understand the roles of the small heat shock proteins alphaA- and alphaB-crystallin in development and function of the eye and other tissues, and 2) to develop novel technologies for introducing targeted mutations into the genomes of mice.

1) We previously made knockout mice alphaA- and alphaB-crystallin and examined the phenotype of each, and of the double knockout (DKO) mice. Most recently we have been investigating, in the double knockout line, the role of alpha-crystallin in the attenuated apoptosis-like process of secondary lens fiber cell differentiation. We have shown that the lenses of DKO mice exhibit disintegration of fiber cells surrounding the lens nucleus, and have shown that these morphological abnormalities result from elevated DEVDase (caspase 3) and VEIDase (caspase 6) activities in lenses lacking alpha-crystallin, suggesting involvement of the apoptosis pathway in this pathology. We have also demonstrated significantly elevated APAF1 in DKO lenses on the mRNA and protein levels, compared to wild type, suggesting that the apoptosis pathway is upregulated. We have demonstrated direct interaction of alphaA-crystallin with caspases 6 and 9, which may contribute to its ability to inhibit the apoptosis pathway, and suggesting that, at least in the lens, alphaA may have equal, or even stronger, antiapoptotic activity compared to alphaB.

We have collaborated with many groups around the world in studying alpha-crystallin function in a variety of tissues. These collaborations have demonstrated that: absence of alphaB-crystallin increases severity of EAU (a mouse model for multiple sclerosis); alphaB-crystallin is not essential for normal heart function, but protects the heart under stressful conditions; absence of alphaB-crystallin exacerbates clinical symptoms in a mouse model of Alexander’s disease; alphaB-cryatallin is critical in tumor angiogenesis; both alphaA- and alphaB-crystallin are important in protecting the retina from various stressors.

2) To streamline procedures for inducing targeted mutations in the mouse genome, we have been using mutagenic oligonucleotides, in combination with biologically active proteins to effect chromosomal alterations. The procedure involves injecting the oligonucleotides and accessory factors into the pronuclei of fertilized mouse ova. We have demonstrated that inclusion of Rad51 & 54, the key proteins in the homologous recombination pathway, as well as antibodies against Ku70 & 86, proteins in the alternative pathway of non-homologous DNA end joining, increases modification frequency. To continue this work we have developed a new transgenic mouse model which gives fluorescence readout of an induced mutation. We are also investigating novel ways of achieving targeted gene mutations using more conventional double stranded DNA targeting constructs.

Staff

Recent Publications

• Morozov V, Wawrousek EF (2008) Single Strand DNA-Mediated Targeted Mutagenesis of Genomic DNA in Early Mouse Embryos is Stimulated by hRad51/54 and Antibodies Against Ku70/86. Gene Ther. 15(6):468-472.
• Morozov V, Wawrousek EF (2006) Caspase-dependent Secondary Lens Fiber Cell Disintegration in αA-/αB-Crystallin Double Knockout Mice. Development 133 (5): 813-821.
• Ousman SS, Tomooka BH, Robinson WH, Sobel RA, Van Noort JM, Wawrousek EF, Steinman L. (2007) Protective and αB-Crystallin in Therapeutic Role forAutoimmune Demyelination. Nature 448(7152): 474-479.
• Hagemann TL, Boelens WC, Wawrousek EF, Messing A (2009) Suppression of GFAP toxicity by αB-crystallin in mouse models of Alexander disease. Hum. Molec. Genet. 18(7):1190-1199.
• Whiston E, Sugi N, Kamradt M, Sack C, Heimer S, Engelbert M, Wawrousek EF, Gilmore MS, Ksander BR, Gregory M (2008) αB-Crystallin Protects Retinal Tissue During S. aureus-Induced Endophthalmitis. Infect. Immun. 76(4):1781-1790.
• Cox CA, Shi G, Yin H, Vistica BP, Wawrousek EF, Chan CC, Gery I (2008) Both Th1 and Th17 are immunopathogenic but differ in other key biological activities J. Immunol. 180(11):7414-7422.
• Rao NA, Saraswathy S, Wu GS, Katselis GS, Wawrousek EF, Bhat S (2008) Elevated retina-specific expression of the small heat shock protein, αA-crystallin, is associated with photoreceptor protection in experimental uveitis. Invest. Ophthalmol. Vis. Sci. 49(3): 1161-1171.