|Research and Education
|Forward and Reverse genetics is a method for the functional analysis of various proteins. Forward genetics aims to identify mutants with particular malfunctions, that are subsequently used to identify the gene responsible for the observed phenotype. The approach of reverse genetics starts at the opposite end with a gene, whose function needs to be analyzed. Gene disruption by gene targeting technologies in cells or animals, and phenotype analysis of the resulting mutants is a powerful way to learn about the molecular role of genes. We have developed Reverse genetic research systems using the chicken B lymphocyte line DT40, and recently the Medaka fish. We are using these systems to understand the function of a variety of genes in DNA repair, recombination, replication, cell cycle, and its checkpoint regulation. A defect in these processes often leads to tumor genesis. The understanding of the molecular details of these defects is thus useful for development of tailor-made therapy. Moreover, reverse genetic approaches will be used more and more in clinical applications, with the development of efficient gene disruption techniques by siRNA and viral gene therapies.|
Among cells of higher eukaryotes, DT40 is an ideal model system for reverse genetic studies, because the frequency of gene targeting is increased by two orders of magnitude when compared to mammalian cells. Another advantage is the very stable phenotype of these cells that make it easy to compare different mutants to wild type cells. Using DT40 cells, Ph.D. students can learn all basic molecular and cellular biology techniques in projects involving gene disruption and phenotype analysis, as shown in Figure 1 as an example. Moreover, our students are publishing their results in high-qualified journals. Hence, most first authors in our labs publications are Ph.D. students.
Medaka (Figure 2) as well as Zebra-fish as genetic model systems have several advantages over rodents in medical studies. Using this system one can quickly perform a large number of genetic experiments, such as genetic crosses, creation of transgenic fishes, injection of anti-strand RNA into fertilized eggs to specifically suppress any given genes, and storage of mutant lines by making frozen sperm aliquots. Medaka and zebra-fish are complementary to each other because studies of each species have advantages and disadvantages when compared to each other. Our lab was the first to create gene disrupted Medaka fishes in 2005. This break-through will certainly make our medical school the center of excellence in the medical research of Medaka in the world. Making gene disrupted Medaka lines from scratch in any research field of your interest, you will be able to become a pioneer just like the first settlers in America 200 years ago. Once you found a way in this unique system, you will feel no reason to stick to the rodent experimental system, where there are too many old die-hard experts similar to the aristocrats in 18th century Europe.
Reverse genetics in these unique experimental systems is highly educative particularly for beginner students. Avoiding fierce competition as in studies on mice and human materials, you will be able to learn effectively from your colleagues because everybody in our laboratory shares the same experimental methods and materials while studying different genes. You can study both systems and publish data in three years to defend your thesis. Seminars are done in English twice a week. We will also support a few months externship in top laboratories in Europe or America during the Ph.D. course.
||Ph.D. students can learn all techniques necessary for performing the following experiment within three years. To visualize dynamics of chromosomes during mitosis, Green fluorescent protein (GFP) gene was knocked-in towards the endogenous locus of a kinetochore protein gene, CENP-H in the chicken B lymphocyte line DT40. This GFP tagged CENP-H substituted for loss of wild-type CENP-H protein, indicating that the chimeric protein works normally. Using this newly developed phenotypic assay, we evaluated the role of Scc1 protein by conditional inactivating Scc1, whose homolog is known to be essential for mitotic cell division in yeast. From this result, we conclude that Scc1 promotes association of the spindle body with mitotic chromosomes thereby contributing to accurate transmission of chromosomes to daughter cells.|
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||Yoshikiyo K. et al. (2010) KIAA1018/FAN1 nuclease protects cells against genomic instability induced by interstrand cross-linking agents. Proc Natl Acad Sci USA. 107: 21553-7.|
||Narita T. et al. (2010) Human replicative DNA polymerase δ(delta) can bypass T-T (6-4) ultraviolet photoproducts on template strands. Genes Cells 15: 1228-39.|
||Iijima J. et al. (2010) RAP80 acts independently of BRCA1 in repair of topoisomerase II poison-induced DNA damage. Cancer Res. 70: 8467-74.|
||Kohzaki M. (2010) DNA polymerases ν(nu) and θ (theta)are required for efficient Immunoglobulin V gene diversification in chicken. J Cell Biol. 189: 1117-27.|
||Nakamura K. et al (2010) Collaborative action of Brca1 and CtIP in elimination of covalent modifications from double-strand breaks to facilitate subsequent break repair. PLoS Genet. 6: e1000828.|