CHIBA

Development of New Technologies
Based on Genome Information

Established inFY2001

R&D Targets

Background

Chiba prefecture is conducting research projects
into advanced technologies for the post-genome
era that are based on the genetic resources andassociated technologies that have been accumulated at the Kazusa DNA Research Institute. With
the aid of such projects, we aim to create aregional Center of Excellence (COE) specializingin biotechnology by developing new technologies
and products in the medical field.

Chiba prefecture is promoting the development of
new regional industries by assembling biotechnology
related research functions at the Kazusa Academia
Park. As part of this development, we will establish
the core laboratory at the Kazusa DNA Research Institute, which is located in the park. Our aim is to promote the development of new technologies, innovative products and to contribute to the developmentof the new biotech industries in collaboration with
regional businesses, public research institutions andthe universities.

Project Director
Tsugio Kimijima
Coordinator for newbusiness development
Chiba Industry
Advancement Center

R&D Themes and Organizations

1. Obtaining Long Mouse cDNAs, Analyzing their Structures,and Developing Technologies for Cloning and the Analysis oflong cDNAs with High Efficiency

Research Director
Michio Oishi
DirectorKazusa DNA ResearchInstituteThis project aims to acquire 2,000 types of long mouse cDNAs that correspond to the long human cDNAs,
which have been accumulated at the Kazusa DNA Research Institute. The laboratory strains of the mouse
have been used most frequently in the research area of mammalian biology or medical science as a model
animal. Genetically modified mice by using mouse long cDNA will contribute in research for the development of pharmaceuticals. We will construct several sets of gene mouse bank consisting of long cDNAs(> 4,000 bp) that are not easily obtained.
(Kazusa DNA Research Institute, Institute of Research and Innovation, Chiba University)

2. Development of Technologies for the Production of Antibodies Against
Proteins Encoded by the Long Mouse cDNAs, their Productionand Evaluation
We will establish reliable technologies for the efficient production of a large number of antibodies and produce more than several hundreds of the antibodies that can be applied for practical use.

Primary R&D Achievements

1

Development of Efficient Long Mouse cDNA
Cloning Technology, and Determination ofthe Entire Sequences (R&D Theme 1)

The Kazusa DNA Research Institute has been accumulating human libraries of longcDNA ( > 4,000 bp). The reason for this strategy is that we believe the functions ofbiological phenomena in diseases such as cancers, brain diseases and life-style related diseases might be clarified in terms of the expression of genes that code for large
proteins. So far in this project, we have achieved the following. 1) The Development of an efficient method of cloning the genes of mice, which are homologous
to human genes. 2) The Screening of about 130,000 clones in the mouse cDNA
libraries constructed with long cDNAs derived from the brain and other organs. 3)
Beginning to obtain the targeted mouse 2,000 long cDNAs, the selection was completed and the 1,750 candidates were picked up for the next sequencing step. 4)
Completion of the entire sequencing of approximately 900 clones. 5) For 500 of all
the 900 clones, the related information of entirely sequenced is available on theWorld Wide Web at http://www.kazusa.or.jp/rouge .

Throughput Recombinant Antigen Preparation
2 HighTechnologies(R&D Theme 2)

In order to improve the efficient production of a large number ofantibodies, we have first developed the requisite technology for thehigh throughput preparation of antigen proteins. Specifically, we
have developed a new in vitro homologous recombination methodthat is capable of processing simultaneously the reactions of 96
samples. (Conventional methods restricted the processing of only
one reaction sample at a time.) We have also developed technologies for expressing and purifying recombinant proteins with highefficiency. The recombinant proteins obtained by this method areconfirmed to be advantageous for the preparation of antibodies.
The results of the electrophoretic analyses of recombinant proteins
expressed in E.coli(Kazusa DNA Research Institute, Protein Express Co., Ltd., Institute of Research and Innovation, National Centerof Neurology and Psychiatry, Japan)
Technology Facilitator
Noboru Tomioka
Senior Chief Researcher
Kazusa DNA ResearchInstitute

3. Development of Technologies for the Production of DNA andAntibody Array and their Production and Evaluation
In this theme, we will aim to achieve the following. 1) The establishment of technologies for the immobilization of DNAs or antibodies. 2) Development of microarrays with 2,000 different cDNAs or antibodies. 3)
Improvements to a microarray spotter and assemble of an array detector with a high sensitivity. By using
these microarrays and equipments, we will analyze long mouse cDNAs(mKIAA clones), the most of theirfunctions being as yet known.
(Kazusa DNA Research Institute, Kaken Geneqs, Inc., Fuji Photo Film Co., Ltd., Chiba University, Tokyo University of Science, University of Tokyo, Tokyo Institute of Psychiatry)

4. Construction and Management of a Database for all of
the Joint Research Projects
We will construct a relational database which includes the general databases covering our mouse long cDNA
clones, antibodies and microarrays. The mouse relational database will be systematically linked with thedatabase of the human long cDNAs (KIAA clones) in the Kazusa DNA Research Institute.
(Kazusa DNA Research Institute, NS Solutions Co., Ltd., Mathematical Systems, Inc.)
For details, please refer to our Website. (http://www.ccjc-net.or.jp/~create/)

Central Project Organization
Chiba Industry Advancement Center
Administrative Departments in Charge
Industrial Promotion Division, Commerce, Industry and Labor Department, Chiba Prefecture

32Core Laboratory
Kazusa DNA Research InstituteComparison of the structures of mouse (M) andhuman (H) long cDNAs (The blue boxes indicate theprotein coding regions)of Specific Protein Expression Patterns Using
3 Analysis
an Immunohistochemical Staining Method(R&D Theme 2)

In this theme, we have analyzed specific localization of the antigens in the mouse brain,using an improved immunohistochemical
method. As a result of the analyses of about 100
antibodies, it has been confirmed that specificor characteristic immunohistochemical stains
for 90% of the antibodies can be obtained fromvarious regions of the brain.

4 Development of an Improved DNA Arrayer
In order to conduct high-density microarrays ofthe DNAs obtained in R&D Theme 1 or antibodies prepared in R&D Theme 2, it is necessary
to develop an improved and reliable array spotting system. The improved arrayer system isIn this case, specific localization of
the antigen in the hippocampus
region of mouse brain wasobserved.

(R&D Theme 3)

capable of spotting at twice the speed and witha higher accuracy than has been possible with
our previous system.

Construction of a Consistent Process Management System
for the Shotgun Sequencing of Mouse Long cDNAs and5 thePreparation of Antibodies

An improved DNA arrayer(R&D Theme 4)

To help increase the throughput of analyses of
cDNA sequences and the preparation of antigens, we have built a database with a process

management application that is potentially useful for identifying the status of all the required
experimental processes.

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