Tanimoto’s Laboratory

 

Themes:

1. Genomic imprinting (Igf2/H19 genes)

2. Hypertension-responsive gene regulation (renin)

3. Transvection in mammals (beta-globin genes)


Research:

Many eukaryotic genes (such as globin, hox, imprinted genes, etc.) are organized into gene clusters and the coordinated expression of these genes during development is critically important for their functions.  The temporal and spatial control of gene expression in such loci is known to involve long-range chromatin interaction by cis-regulatory elements (such as enhancer, imprinting control region (ICR) and insulator sites), as well as the establishment of preferable epigenetic states (DNA and histone modifications).  However, the way in which these factors coordinate complex gene expression pattern is not well understood.  The focus of our laboratory is to analyze the mechanistic basis for such regulations and we have been studying the beta-globin, renin (both for elucidating the enhancer-promoter interaction mechanism) and Igf2/H19 gene loci (for mono-allelic gene expression mechanism).  In order to analyze long-range gene expression mechanisms, we introduced non-conventional vector systems capable of cloning a huge DNA insert (i.e. YAC and BAC) and a recently-developed CRISPR/Cas genome editing system for generating transgenic and knock-out/in model organisms, respectively.  Because genomic imprinting is a trans-generation phenomenon existing only in mammals, and because renin gene expression in the kidney is regulated by blood pressure and body fluid volume, it is also important to employ a host system that allows both temporal and spatial assessment of gene expression and a mouse genetics system is appropriate for such a purpose.  We use biochemical, molecular biology, and cell culture techniques in combination with mouse molecular genetics to identify and functionally characterize cis-regulatory DNA elements and trans-acting components involved in the regulation. (Partial list is shown below)


Host organisms:

-  microorganisms (E. coli, Yeast)

-  Mammalian cultured cells (Cancer cells, ES cells, MEF cells, etc.

-  Mouse


Techniques (biochemistry, molecular biology and cell culture):

-  DNA cloning and transformation in E.coli

-  Mammalian cell culture and transfection (plasmid and siRNA)

-  Yeast artificial chromosome (YAC) modification by homologous

    recombination in yeast

-  Manipulation of large DNA molecules using pulse field gel

    electrophoresis

-  Southern blotting

-  DNA methylation analyses (methylation-sensitive restriction enzymes,  

    bisulfite sequencing)

-  Northern blotting

-  Real-time quantitative RT-PCR

-  DNaseI hypersensitive site mapping

-  Luciferase reporter assay

-  Western blotting

-  Recombinant protein production in E.coli

-  GST-pull down assay

-  Immunoprecipitation

-  Chromatin immunoprecipitation (ChIP)

-  Electrophoresis mobility shift assay (EMSA or gel-shift assay)

-  Affinity purification of DNA binding proteins

-  Analysis of protein posttranslational modifications

-  Immunocytochemistry analysis of mouse tissues

-  Yeast one-/two-hybrid screening


Techniques (molecular genetics, developmental engineering):

-  Generation of transgenic mice by pronuclear DNA injection

-  Gene targeting in mouse embryonic stem (ES) cell

-  Generation of chimeric mice (by blastocyst injection or by

    aggregating of ES cells with 8-cell stage embryos)

-  Generation of gene knock-out/ knock-in mice

-  Genome editing by CRISPR/Cas9 system (cultured cells and mice)

In vitro fertilization of mouse embryos

-  Freezing mouse embryos for long term stock in LN2


Recruitment:

Graduate School of Life and Environmental Sciences,

University of Tsukuba

     Agro-Bioresources Science and Technology

(http://www.agbist-tsukuba.jp/?lang=english)

     Life Sciences and Bioengineering

(http://www.agbi.tsukuba.ac.jp/~seibutsukinou/english/index.html)


Contact information:

Keiji Tanimoto, Ph.D.

Faculty of Life and Environmental Sciences

Life Science Center, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba, Ibaraki, 305-8577, Japan

keiji[at]tara.tsukuba.ac.jp