論文 / 著書情報
Article / Book Information
題目(和文)

枯草菌ゲノムベクターシステムを用いたクラスＩ嗅覚受容体遺伝子の発現制御領域の解析

Title(English)

Studies on the cis-element for mouse class I odorant receptor genes
using the Bacillus subtilis genome vector system

著者(和文)

岩田哲郎

Author(English)

Tetsuo Iwata

出典(和文)

学位:博士(工学),学位授与機関:東京工業大学,
報告番号:甲第9686号,
授与年月日:2014年12月31日,
学位の種別:課程博士,
審査員:廣田 順二,中村 聡,和地正明,福居俊昭,蒲池Citation(English)

Degree:Doctor (Engineering),
Conferring organization: Tokyo Institute of Technology,
Report number:甲第9686号,
Conferred date:2014/12/31,
Degree Type:Course doctor,
Examiner:,,,,学位種別(和文)

博士論文

Category(English)

Doctoral Thesis

種別(和文)

論文要旨

Type(English)

Summary

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利章

（博士課程）Doctoral Program

論	 文	 要	 旨	 ＴＨＥＳＩＳ ＳＵＭＭＡＲＹ	 
専攻：	 
Department of

学生氏名：	 Student’s Name

生物プロセス	 岩田 哲郎	 

専攻	 	 

申請学位（専攻分野）：	  博士	 
Academic Degree Requested	 	 

指導教員（主）：	 
Academic Advisor(main)	 

指導教員（副）：	 
Academic Advisor(sub)Doctor of（	  工学	  ）	 廣田	  順二	 	 要旨（英文 800 語程度）	 
Thesis Summary （approx.800 English Words ）Technological developments in chromosome engineering are essential for the manipulation and functional
analysis of genomic DNA fragments. Artificial chromosomes, such as bacterial and yeast artificial chromosomes(BACs and YACs), have been used for these purposes in combination with transgenesis. However, there exist
several technological limitations in cloning size, genetic modification and insert stability. The Bacillus subtilis
genome (BGM) vector is a novel cloning system for large DNA fragments, in which the entire 4.2 Mb genomeof B. subtilis functions as a vector. The BGM vector system has several attractive properties, such as a large
cloning capacity of over 3 Mb, stable propagation of cloned DNA and various modification strategies using
RecA-mediated homologous recombination. However, genetic modifications using the BGM vector system havenot been fully established, and this system has not been applied to transgenesis. To explore the potential of theBGM vector, I focused on the mouse class I odorant receptor (class I OR / fish-like OR) family, which consistsof 158 genes and forms a single gene cluster in the genome. Although a cis-acting locus control region isexpected to activate singular OR gene expression, such element has not been experimentally identified yet. In
this study, I provided a complete genetic modification method of cloned DNA fragments, including insertion,deletion, inversion and fusion to elongate a DNA fragment. I further demonstrated that the modified, enlarged
genomic DNA fragments could be used to generate transgenic mice.
First, I developed a complete genetic manipulation method for the BGM vector system by demonstrationof a target insertion and an inversion modification. Using two contiguous BAC clones containing several class I
OR genes, I constructed two transgenes in the BGM vector by inserting a reporter gene cassette IRES-tauEGFP
into one class I OR gene, MOR42-3. Because they were oriented in opposite directions, I aligned theirorientation by inversion modification, and reconstructed a ~252 kb transgene of a part of class I OR gene cluster
for transgenic reporter assay. The DNA sequencing analysis of the constructed transgenes revealed that nomutations occurred during gene manipulation and all targeted modifications were precisely conducted.
Second, I applied the BGM vector system to mouse transgenesis (BGM transgenesis). I established a newprotocol to purify BGM transgenes from the BGM vector. I succeeded to establish several transgenic lines by
the pronuclear-microinjection of the purified transgenes into mouse eggs. Transgenic mice carrying the enlarged
transgene recapitulated the expression and axonal projection patterns of the target class I OR gene in the main
olfactory system, indicating that a cis-acting element for the class I OR gene is present in the extended region
from the original transgene.
Third, to identify the cis-acting element for the class I OR gene, I performed the deletion mutagenesis of
the BGM transgene and the bioinformatic analyses. I successfully shortened the putative cis-acting element byseveral transgenic analyses. Functional analyses revealed that the identified sequence was necessary for thetransgene expression and was capable of recapitulating the expression pattern of class I OR genes in the main
olfactory epithelium.
Through this study, I demonstrated the utility of the BGM vector for engineering large DNA fragments tomodify and reconstruct genomic structure and to generate transgenic mice. Using the BGM vector system, I
provided the first experimental evidence of a cis-acting element for class I OR gene expression. Since thissystem can be applied to any BAC and other library resources of any species, the BGM vector system provides anovel tool for recapitulation of genomic structure and functional studies of genomic DNA.
備考	 :	 論文要旨は、和文 2000 字と英文 300 語を 1 部ずつ提出するか、もしくは英文 800 語を 1 部提出してください。	 
Note : Thesis Summary should be submitted in either a copy of2000 Japanese Characters and 300 Words (Engｌish) or 1copy of 800

Words (English).
注意：論文要旨は、東工大リサーチリポジトリ（T2R2）にてインターネット公表されますので、公表可能な範囲の内容で作成してください。
Attention: Thesis Summary will be published on Tokyo Tech Research Repository

Website (T2R2).