␣B-crystallin has been demonstrated, in tissue culture experiments, to be a caspase 3 inhibitor; however, no animal model studies have yet been described. Here, we show that morphological abnormalities in lens secondary fiber cells of ␣A-/␣B-crystallin gene double knockout (DKO) mice are consistent with, and probably result from, elevated DEVDase and VEIDase activities, corresponding to caspase 3 and caspase 6, respectively. Immunofluorescence microscopy revealed an increased amount of caspase 6, and the active form of caspase 3, in specific regions of the DKO lens, coincident with the site of cell disintegration. TUNEL labeling illustrated a higher level of DNA fragmentation in the secondary fiber lens cells of DKO mice, compared with wild-type mice. Using a pull-down assay, we show interaction between caspase 6 and ␣A-but not ␣B-crystallin. These studies suggest that ␣-crystallin plays a role in suppressing caspase activity, resulting in retention of lens fiber cell integrity following degradation of mitochondria and other organelles, which occurs during the apoptosis-like pathway of lens cell terminal differentiation.

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Low and variable efficiency is a major problem in targeted gene alteration, which is used as a primary tool in gene therapy and animal model studies. We tested several types of constructs alone, or in combination with other factors, to introduce a point mutation into the aB-crystallin gene in onecelled mouse embryos. We found that co-injection of ssDNA along with antibodies against Ku70/86, or supplementing the system with hRad51/hRad54, increases efficiency of targeted mutagenesis. These findings suggest that proteins in the homologous recombination DNA repair pathway contribute, and that proteins involved in the alternative nonhomologous end-joining pathway inhibit, ssDNA-mediated targeted mutagenesis. This is the first successful demonstration of targeted mutation in early mouse embryos. This novel methodology of supplying protein factors to stimulate gene modification in the nucleus has not been previously reported. Gene Therapy (2008) Keywords: targeted mutagenesis; animal models; gene correction/modification; oligonucleotide-based therapies Site-specific gene alteration by precise gene repair mechanisms is the ultimate goal in somatic gene therapy and in generating animal models of human genetic diseases via knockout/knock-in technology. Targeted genome alteration in the one-celled embryo would be ideal, however, attempts to accomplish this have not yet been successful. 1 Currently, the production of knockout and knock-in animals utilizes gene recombination techniques in mouse embryonic stem cells, followed by transferring selected stems cells into blastocysts to create a chimeric animal. Several laboratories have studied gene correction in mouse embryonic stems cells. 2,3 Even if the mutation efficiency can be increased sufficiently, the selection and screening of embryonic stem cell clones and the microinjection of selected stem cells into blastocysts are still required. Gene alteration directly in one-celled embryos will eliminate many costly and timeconsuming steps in this process. However, early mouse embryos have never been demonstrated to potentiate targeted gene alteration. This is a unique, rapidly developing and changing system distant from any other described for genomic DNA alteration. There is limited data for mouse ova and early embryos suggesting a rapid change in double-strand DNA (dsDNA) break repair pathways, 4 which could greatly affect gene correction efficiency.In other model systems, a variety of synthetic oligonucleotide constructs has been successfully used: triple helix-forming oligonucleotides (TFO), 5,6 chimeric RNA/DNA constructs, 7-10 peptide nucleic acids, 11 short DNA fragments, 12 and single-strand DNA (ssDNA). [13][14][15] However, the necessity of having a specific sequence of targeted DNA, and restrictions for triplex formation under physiological conditions, 16 impose serious limitations on the universality of TFOs as mutation mediators. Moreover, the previously reported efficiency of gene alteration mediated by chimeric RNA/DNA constructs cannot be reproduced by many e...