TAKEDA LABORATORY | Osaka University Graduate School of Medicine
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The mouse is an excellent animal model extensively used in laboratories for the understanding of mechanisms underlying human diseases. Our laboratory has developed novel technologies for rapid analysis of gene function in mice: A novel in vivo mutagenesis method using Sleeping Beauty transposon system, and conditional regulation of Bloom's syndrome gene for biallelic mutagenesis. We are improving these methods in order to expand their application for various aspects of biological research. We are also using embryonic stem cell technologies for creating mutant mice in order to address gene functions in vivo.
A novel in vivo mutagenesis method using the Sleeping Beauty transposon system
Generation of mutant mice is important in determining the function of genes in the post-genome era. We have developed a novel forward genetics system using the Sleeping Beauty (SB) transposon for tagged mutagenesis in mice. SB is a synthetic transposon reconstructed from evolutionally inactivated transposon sequences found in the salmoid fish. The SB transposon system consists of two elements: transposon, which acts as a mutagen, and transposase, which recognizes and mobilizes the transposon - known as a transposition event. Mice with transposon inserted into functional genes can be screened rapidly and non-invasively using GFP fluorescence. Current data indicates the usefulness of this system for region-specific saturated mutagenesis, which is currently impossible with other large-scale mutagenic systems. We are currently analyzing >50 mutant lines with transposon integration in both known and novel genes by generating homozygotes and analyzing the resulting phenotype. To date, we have several mutant lines which are either embryonic lethal at various stages of development or non-lethal. Some of these non-lethal lines display advert phenotypes such as strange behavior pattern or infertility, while some are clearly non-essential genes. Taken together, SB transposon system is a rapid and efficient method for large-scale tagged mutagenesis, as well as for saturated mutagenesis within a defined region.
Conditional regulation of Bloom’s syndrome gene for s
At present, the chief limitation of phenotype-based genetic screening in mammalian systems is the diploid nature of the genome. As it is known that cells deficient in the Bloom's syndrome gene (Blm) show an increased rate of loss of heterozygosity, we have developed a novel system based on this phenotype to generate a tetracycline-regulated Blm allele in order to introduce bi-allelic mutations across the genome in mouse embryonic cells. Transient loss of Blm expression induces homologous recombination not only between sister chromatids but also between homologous chromosomes. In combination using ENU mutagenesis and transient loss of Blm expression, we were able to generate an ES cell library with genome-wide bi-allelic mutations. Our results have shown that this novel system is very efficient for identifying gene functions in ES cells.
Creating mutant mice using embryonic stem cell technologies
We have generated various mutant mice using embryonic stem (ES) cells. We have also utilized the Cre/loxP system for tissue-specific conditional knockout models. For example, skin and T lymphocyte. These studies revealed novel findings for the further understanding of human diseases.
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