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 利章

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

Type(English)

Doctoral Thesis

Powered by T2R2 (Science Tokyo Research Repository)

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

A DOCTORAL THESIS
Submitted to the
Department of Bioengineering
Tokyo Institute of Technology
2014

Tetsuo IWATA
Department of Bioengineering
Tokyo Institute of Technology
Nagatsuta-cho 4259, Midori-ku
Yokohama 226-8501, Japan

SUMMARY
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. Through this study, I demonstrated the utility of the Bacillus subtilis genome(BGM) vector for engineering large DNA fragments to modify and reconstructgenomic structure and to generate transgenic mice.
The BGM vector is a novel cloning system for large DNA fragments, in which
the entire 4.2 Mb genome of B. subtilis functions as a vector. The BGM vector system
has several attractive properties, such as a large cloning capacity of over 3 Mb, stablepropagation of cloned DNA and various modification strategies using RecA-mediatedhomologous recombination. However, genetic modifications using the BGM vector
system have not been fully established, and this system has not been applied totransgenesis. Through this study, I provided a complete genetic modification methodof cloned DNA fragments, including insertion, deletion, inversion and fusion toelongate a DNA fragment. I further demonstrated that the modified, enlarged
genomic DNA fragments could be used to generate transgenic mice.
To explore the potential of the BGM vector, I focused on the mouse class I
odorant receptor (class I OR / fish-like OR) family, which consists of 158 genes andforms a single gene cluster in the genome. Although a cis-acting locus control region
is expected to activate singular OR gene expression, such element has not been

experimentally identified yet. Using the BGM vector system, I modified andreconstructed genomic structure of a part of class I OR gene cluster for transgenic
reporter assay. Transgenic mice carrying the enlarged transgene recapitulated theexpression and axonal projection patterns of the target class I OR gene in the main
olfactory system. The deletion analysis showed that the approximately 13 kb region
upstream the class I OR gene contains a cis-acting element necessary for thetransgene expression. Further functional analyses revealed that the 3.8 kb region in
the 13 kb upstream sequence was capable of recapitulating the expression pattern ofclass I OR gene in the main olfactory epithelium. In addition, bioinformatics analyses
showed that the 3.8 kb region contained a highly homologous sequence between
human, and which was conserved in at least 13 mammalian species. Thus, using the
BGM vector system, I provided the first experimental evidence of a cis-acting element
for class I OR gene expression. Since this system can be applied to any BAC andother library resources of any species, the BGM vector system provides a novel tool
for recapitulation of genomic structure and functional studies of genomic DNA.

ACKNOWLEDGEMENT
I would like to take this opportunity to thank everyone who has helped me over thelast six years. First of all I would like to thank my supervisor, Dr. Junji Hirota, for his
continuous support, discussion and encouragement since I began my research works at theTokyo Institute of Technology in 2009. Under his supervision, I thoroughly enjoyed myworks and felt my research at his laboratory meaningful. I express my gratitude to Dr.
Shinya Kaneko (Tokyo Institute of Technology) for valuable advice to BGM vector
experiments and for instruction of handling the B. subtilis step by step. I would like tothank Drs. Yuh Shiwa (Tokyo University of Agriculture (current affiliation: Iwate Medical
University)) and Hirofumi Yoshikawa (Tokyo University of Agriculture) for great support
in the DNA sequencing analysis. I would like to thank Drs. Yoshihito Niimura andKazushige Touhara (The University of Tokyo) for great support in the large-scalebioinformatics analysis. I would like to thank Dr. Takayuki Enomoto (Tokyo Institute ofTechnology) for great support in immunohistochemistry and for useful suggestions. I
would like to thank Drs. Naoki Kobayashi (Tokyo Institute of Technology) and Kenta
Sumiyama (National Institute of Genetics (current affiliation: RIKEN)) for instruction of
the pronuclear-microinjection. I thank Dr. Yoshihiro Yoshihara (RIKEN) for the gift ofplasmids and for internship for three month. I also thank Dr. Mitsuhiro Itaya (Keio
University) for the gift of B. subtilis strains and plasmids and for fruitful discussion.
Finally, I engrave the kindness of the members in the Hirota laboratory on my mind.
September 2014

Tetsuo IWATA

CONTENTS

ABBREVIATIONS
I

II

1

GENERAL INTRODUCTION
1.

General overview of manipulation of genomic DNA

42.

The Bacillus subtilis genome vector system

63.

Mouse transgenesis

9

4.

Mouse main olfactory system and odorant receptor genes

11

5.

Purpose of this study

15

DEVELOPMENTOFA

COMPLETEGENETICMANIPULATIONMETHOD FOR THE BGM VECTOR SYSTEM
1.

Introduction

2.

Experimental procedures

3.

4.

26

1) Strains and preparation of competent B. subtilis cells

282) One-step transfer of BAC inserts to the BGM vector283) I-PpoI/CHEF analysis

294) Southern blot analysis of the BGM clones

295) Construction of plasmids

306) DNA sequencing

31

Results
1) Cloning of genomic DNA fragments into the BGM vector

342) Targeted insertion of a reporter gene353) Inversion of the insert in the BGM vector

364) Fusion of two inserts to reconstruct genomic structures

385) DNA sequencing of modified and reconstructed BGM clones

39

Discussion

40

III

THE BGM VECTOR-BASED MOUSE TRANSGENESIS
1.

Introduction

2.

Experimental procedures

3.

511) Preparation of the transgenes for pronuclear injection

522) Production and analyses of transgenic mice523) Southern blot analysis of transgenic mice534) Immunohistochemistry

53

Results
1) Generation of the BGM transgenic mice562) Experimental evidence of a cis-acting element for a class I OR gene 57
4.
IV

Discussion

60

IDENTIFICATION AND FUNCTIONAL ANALYSIS OF CIS-ELEMENT
FOR A CLASS I ODORANT RECEPTOR GENE
1.

Introduction

2.

Experimental procedures

3.

681) Nucleotide identity plots

692) Construction of a deletion series of Tg-220

703) Characterization of the identified 13 kb region704) Construction of a series of 13 kb reporter transgenes

715) Generation and analysis of transgenic mice726) Bioinformatics analysis of the Core region in other species

73

Results
1) Comparative analysis of the Tg-220 mouse genomic sequence762) Construction of a series of deletion mutants of the Tg-220

773) The 13 kb region is necessary for transgenic MOR42-3 expression

774) Functional analysis of the 13 kb region by conventional transgenesis 79
4.
V5) The Core region is conserved in 13 placental mammals

81

Discussion

83

CONCLUSIONREFERENCES

92100

ABBREVIATIONS

BAC

bacterial artificial chromosome

BCIP

5-bromo-4-chloro-3-indolyl phosphate

BGM

Bacillus subtilis genome

BReT

Bacillus Recombinational Transfer

Bsblasticidin S (resistance gene: bsr)

CHEF

contour-clamped homogeneous electric fields (gel electrophoresis)

ChIP

chromatin immunoprecipitation

CIlambda (λ) phage CI repressorCmchloramphenicol (resistance gene: cat)Cmchloramphenicol resistance gene for E. coliCsClcesium chloride

CTCF

CCCTC-binding factor

DAPI

4',6-diamidino-2-phenylindoleDNA

deoxyribonucleic acid

EDTA

ethylenediamine -N,N,N’,N’-tetraacetic acid, disodium salt

EGFP

enhanced green fluorescent proteinEmerythromycin

Emx2

empty spiracles homeobox 2

ES cellembryonic stem cell

EtBr

ethidium bromide

GpBR

genomic pBR322 sequence

HAC

human artificial chromosome

IHC

immunohistochemistry

IRES

internal ribosome entry sitekpkilo base pair

Lhx2

LIM-homeobox protein 2

Mbmega base pairMOEmain olfactory epithelium

MOR

mouse odorant receptor

NBT

nitro-blue tetrazolium chloride

Nmneomycin (resistance gene: neo)OB

olfactory bulb
1

OGG

orthologous gene groupOMP

olfactory marker proteinORodorant [olfactory] receptor

OSN

olfactory sensory neuron

PAC

P1 derived artificial chromosome

PBS

phosphate buffered saline

PCR

polymerase chain reaction

PFA

paraformaldehydePhlphleomycin (resistance gene: phl)Prlambda (λ) phage Pr promoterRNA

ribonucleic acidSDS

sodium dodecyl sulfate

Spc

spectinomycin (resistance gene: spc)

Tettetracycline (resistance gene: tet)Tgtransgene / transgenic mouse

TSS

transcription start site

YAC

yeast artificial chromosome2

CHAPTERI

GENERAL INTRODUCTION

3

I-1

General overview of manipulation of genomic DNA
DNA cloning is the first step for investigation of genes and their functions.

Traditional genetic engineering based on restriction enzymes and DNA ligase to cut andfuse DNA fragments using multi-copy plasmid vectors, has made a great success using
Escherichia coli (E. coli). This technology has been confined to clone and manipulate
small DNA fragments, because naked large DNA fragments rarely have unique restriction
enzyme sites and are exposed to physical shearing in liquid. Thus, alternative technologies
for manipulation and engineering of large genomic DNA molecules have been required forthe physical mapping, manipulation and functional analysis of genomic DNA fragments.
To manipulate large genomic DNA fragments, several artificial chromosomes have beendeveloped such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes(BACs), P1 derived artificial chromosomes (PACs) (Sternberg, 1990) and human artificial
chromosomes (HACs) (Harrington et al., 1997). Of these, the YACs and BACs have been
often used to generate genomic libraries and to manipulate genomic DNA fragments.
These two systems have complementary advantages over each other in terms of cloning
capacity and insert stability.
The YAC system is a high-capacity cloning system based on Saccharomyces
cerevisiae (Burke et al., 1987). The YAC is composed of some functional units of yeast
chromosomes: a centromere, an autonomously replicating sequence and two sequences thatseed telomere formation (Figure 1-1A). Genomic DNA inserts of up to 2 Mb can be usedwith the YAC system. In yeast, the DNA double-stranded-break and -repair reaction arevery efficient, allowing easy modifications of the inserts by homologous recombination.
However, the YAC system has some problems in terms of stability of the cloned inserts.

4

YACs are often chimeric, i.e., single YAC clone often contains genomic fragments derived
from multiple loci because of co-cloning events (Green et al., 1991). YACs often sufferfrom unwanted rearrangements due to a potent and constitutive recombinase activity(Kouprina et al., 1994). In addition, the isolation of intact YACs is difficult because of
their linear structure and contamination with endogenous yeast chromosomes. Thus, theYAC system has an advantage in cloning capacity, and has disadvantages in insert stability
and purification (Table 1-1).
The BAC system is based on E. coli and its single-copy plasmid F factor (Shizuya etal., 1992). Replication of the F plasmid in E. coli is strictly controlled by some regulatory
genes including oriS, repE, parA and parB (Kline, 1985). Because the BAC vector
contains these genes, BAC clones are maintained in low copy number (one or two copy percell) in a recA deficient stain, reducing the potential for unwanted recombination events(Shizuya et al., 1992) (Figure 1-1B). BAC clones are easy to manipulate and retrieve dueto their plasmid form. The BAC system can generally accommodate up to 300 kb genomic
inserts. Modifications of BAC inserts require additional recombination components, e.g.,
RecA of E. coli (Yang et al., 1997; Gong et al., 2002) or phage-derived recombination
proteins, RecE and RecT carried by the cryptic Rac prophage (called ET cloning) (Zhang
et al., 1998) and Redα and Redβ carried by the bacteriophage-λ (RedET or defective λprophage system) (Muyrers et al., 1999; Copeland et al., 2001; Lee et al., 2001). These
phage-encoded recombination factors are capable of modifying BAC inserts using
PCR-generated linear double strand DNA targeting cassettes (Zhang et al., 1998; Copeland
et al., 2001). The recombination based genetic manipulations of BACs are very attractive
techniques. However, introduction of such additional components complicates the5modification procedures. Thus, the BAC system has advantages in handling and insert
stability, and has disadvantages in cloning size and insert modification. (Table 1-1)

I-2

The Bacillus subtilis genome vector system
The Bacillus subtilis genome (BGM) vector system has been developed as a novel

cloning system using a unique concept in which the entire 4.2 Mb genome of B. subtilis
functions as a vector (Itaya, 1993; Kaneko et al., 2005; Itaya et al., 2008; Itaya and Tsuge,2011). The cloning strategy for the BGM vector system is based on natural competence of
B. subtilis (Dubnau, 1999; Chen et al., 2005). B. subtilis expresses competence-related
genes at the late stage of cell growth, and their products, transformation machinery
molecules (Com proteins), are assembled in the cell membrane. The transformation
machinery non-specifically binds and imports extracellular DNA fragments into thecytoplasm in single-stranded form. The recombinogenic DNA is incorporated into the B.
subtilis genome by homologous recombination (Figure 1-2). In the BGM vector, the B.
subtilis genome is engineered to have homologous cloning site sequences so that a target
DNA fragment flanked by homologous sequences can be cloned into the BGM (Itaya andTsuge, 2011). B. subtilis can be cultivated under the same conditions as E. coli in LB brothat 37°C. Competent B. subtilis cells can be prepared by merely cultivating the cells in a
special medium for several hours, and these cells are transformed by simply mixing in
DNA fragments without additional heat-shock or electroporation (Itaya and Tsuge, 2011).
Thus, the competency of B. subtilis/BGM vector enables easy transformation procedures
and efficient recombination reactions.

6Cloning principles in the BGM vector
To utilize the B. subtilis genome as cloning vehicles, it should be engineered to havethe cloning site for exogenous DNA, because such exogenous DNA generally would haveno homologous sequences with the host B. subtilis genome. The first BGM vector series
were constructed by inserting an E. coli plasmid vector pBR322, which cannot replicate inB. subtilis as plasmid, to mediate homologous recombination reaction (Itaya, 1993; Itaya,
1995). The classical BGM vector series can clone pBR322 inserts composed of exogenous
DNA fragment and antibiotic resistance genes via homologous recombination at pBR322
sequences (genomic pBR: GpBR). For general use, a counter-selection system wasdeveloped to clone DNA segments without antibiotic resistance markers for B. subtilis(Itaya, 1999; Itaya et al., 2000). This system uses two cassettes. One is a bacteriophage
lambda Pr promoter directed neomycin resistance gene, Pr-neo cassette. The other is a
cI-spc cassette composed of bacteriophage lambda CI repressor gene and spectinomycin
resistance gene. The original BGM strains carrying the cI-spc cassette are resistant tospectinomycin (Spc) and sensitive to neomycin (Nm), because the constitutively expressed
CI repressor protein specifically binds to Pr promoter and repress the promoter activity.
Once the cI-spc cassette is replaced with the incorporated inserts, the recombinant BGM
clones become resistant to Nm and sensitive to Spc due to loss of the repressor. In
combination with the internal selection system, the specific BGM vector for cloning the
BAC insert was established by inserting a BAC vector pBAC108L (Kaneko et al., 2005;
Kaneko et al., 2009) (Figure 1-3). These BGM vectors can utilize BAC library clones in a
one-step transfer. In addition, two I-PpoI recognition sequences, ATGACTCTCTTAA/GGTAGCCAAA, are introduced at the both side of the cloning site, allowing simple

7

evaluation of the inserts.

Genome DNA cloning and gene manipulation
The BGM vector system is notably valuable for cloning giant DNAs (Table 1-1).
Several genome DNA have been cloned into the BGM vectors, including 3.5 Mb
Synechocystis whole genome (Itaya et al., 2005), mouse genomic DNAs (Itaya et al., 2000;
Kaneko et al., 2003), 150 kb rice chloroplast DNA (Itaya et al., 2008), 16 kb mousemitochondrial DNA (Itaya et al., 2008), lambda phage DNA (Itaya, 1995), mouse BAC
clones (Kaneko et al., 2009) and Arabidopsis BAC clones (Kaneko et al., 2005). The
cloned inserts in the BGM vector show high structural and genetic stability, because the
DNA fragments are directly inserted into the single circular host genome (Kaneko et al.,2005). To achieve genome DNA cloning and extension, there are some large DNA
reconstruction/bottom-up approaches named as the ‘inchworm method’ (Itaya et al., 2005)
and ‘domino method’ (Itaya et al., 2008). In the ‘inchworm method’, natural guest DNA
directly fills a gap between the two separate fragments to elongate DNA (Itaya et al., 2005).
As the gap ranges about 40-50 kb, the elongation rate is faster. But this method requires
long continuous DNA (>100 kb) as template. In contrast, ‘the domino method’ connects anumber of DNA pieces (5-10 kb, called domino clones) one-by-one by homologous
recombination between overlapping sequences of pBR322 sequence and guest DNA (Itaya
et al., 2008). Therefore, the elongation rate in the domino method is slower than that in the
inchworm method. However, this method can utilize DNA fragments prepared by PCR or
by chemical synthesis, thus allowing application in all organisms and even in generation of
totally artificial genomes.

8The inserts in the BGM vector can be engineered through various modification
strategies based on RecA-mediated homologous recombination. Using the BGM vector
system, genetic modifications of insertion of B. subtilis marker gene, deleting and fusing
cloned inserts have been achieved (Kaneko et al., 2003; Kaneko et al., 2009). Assembled
and modified DNA molecules in the BGM vector are to be isolated for general applications
such as biomaterial production and further genetic experiments. Some retrieval methods
for BGM inserts were proposed including direct isolation in test tube by I-PpoI digestion(Kaneko et al., 2009), direct isolation in vivo by genome segregation (Itaya and Tanaka,1997) and copying the insert by Bacillus Recombinational Transfer (BReT) (Tsuge and
Itaya, 2001) (Figure 1-4). Overall, the BGM system is a novel and an alternative platform
for chromosome engineering (Table 1-1).

I-3

Mouse transgenesis
The generation of transgenic animals is a routine method in many laboratories.

Transgenesis is often used to study gene functions in vivo and to provide animal models ofgenetic diseases (Giraldo and Montoliu, 2001). The size of transgenes is generally ~10 kb
because of the capacity of multi-copy plasmid vector. However, mammalian functional
gene units composed of exons, introns and regulatory elements often reach as long as
several hundred kb. To harbor these genomic regions, artificial chromosome transgenesis,
including BAC and YAC transgenesis, have contributed greatly to genome researches,
because transgenesis with large DNA molecules allows transgene expression torecapitulate the endogenous gene expression patterns (Schedl et al., 1993; Mendez et al.,
1997; Yang et al., 1997; Giraldo and Montoliu, 2001; Kuroiwa et al., 2002).

9

Methods for generating transgenic mouse
The most conventional method of introduction of transgenes is the pronuclear
microinjection of purified DNA (Schedl et al., 1992; Peterson et al., 2007) (Figure 1-5A).
In this method, linearized DNA molecules are directly introduced into fertilized mouseeggs with a fine glass capillary, and then are randomly integrated into mouse chromosomes.
There is the upper limit in size of DNA, because long DNA molecules are easily damagedby physical shearing during purification and injection. For introduction of larger DNA
transgenes such as YACs, other methods were reported. One is lipofection of purified
DNA into embryonic stem (ES) cells (Strauss et al., 1993; Lee and Jaenisch, 1996). In thismethod, purified YACs are mixed with cationic lipids, and the YAC-lipid complex is
introduced into ES cells by endocytosis. The transfectant ES clones are used to generatechimeric mice. By crossing the chimeric mice with wild type, transgenic lines are
established. Another is the yeast spheroplast fusion with ES cells (Jakobovits et al., 1993;
Li and Blankenstein, 2013). In this strategy, intact Mb-sized YACs can be introduced into
the germline without purification of YAC DNA. Because ES cells retain their pluripotency
after yeast spheroplast fusion, the fused ES cells can be used to produce chimeric mice.
Although spheroplast fusion method reliably yield large numbers of transfectants with
intact YAC transgenes, the incorporation of endogenous yeast genomic DNA potentiallycomplicates interpretation of results. In contrast, pronuclear microinjection and lipofection
use purified DNA but find high frequency of transgene deletion because of physicalshearing.

10

Tol2-transposon mediated transgenesis
Generally, the overall transgenic efficiency is low, only 2-5% of pronuclear- injected
fertilized eggs. To improve transgenesis in mice, new methods have been developed
including virus-based vector and transposon systems. Recently, it was reported that theTol2 transposon system could be applied to conventional and BAC transgenesis in mice
(Suster et al., 2009; Sumiyama et al., 2010). The Tol2-mediated cytoplasmic injection
method uses a special plasmid vector including minimal Tol2 transposable elements(Urasaki et al., 2006). A foreign DNA cloned in the Tol2 vector is injected into thecytoplasm of fertilized eggs with the transposase mRNA, reducing physical damages to
eggs (Figure 1-5B). The foreign DNA is efficiently transposed from plasmid to the genome
by synthesized Tol2 transposase. The efficient DNA integration and small physical damage
to injected egg greatly improve the overall transgenic efficiency: more than 20%(Sumiyama et al., 2010). The Tol2 transposon system could offer following possible
advantages; a transgene is integrated as a single copy but not as a concatemer;transposition does not cause gross rearrangements around integration sites; the integrationsites are identifiable. Note that transgenic mice generated by the Tol2 method generally
have several independent integration sites because of the potent transposition activity,
whereas those generated by the pronuclear injection method generally are single
integration (Sumiyama et al., 2010).

I-4

Mouse main olfactory system and odorant receptor genes
The mammalian main olfactory system can detect millions of different types ofchemicals in the environment. Animals perceive these odorous molecules to find food11sources and offspring and to avoid predators and rotten foods. Olfactory sensory neurons(OSNs), the primary sensory neurons in the main olfactory epithelium (MOE) can detectodorants to initiate the sense of smell (Figure 1-6A). The detection is mediated by odorant
receptors (ORs), which are G-protein coupled receptors with a putative seventransmembrane domain and are located on the cilia of OSNs. To distinguish a vast numberof odorants, the vertebrate genome possesses numerous OR genes, which form the largest
multi-gene family in vertebrates.
There are two principles underling the functional organization of the olfactory
system. One is the one-neuron–one-receptor rule; each OSN expresses only one functional
odorant receptor (OR) gene from a huge repertoire of OR genes in a monoallelic manner(Chess et al., 1994; Malnic et al., 1999). This singular OR gene expression is considered tobe established by a negative feedback regulation, in which functional expression of one
OR gene inhibits additional OR genes expression (Serizawa et al., 2003; Lewcock andReed, 2004). The other is OR-instructed axonal projection. Targeted mutagenesis
demonstrated OSNs expressing the same OR were shown to converge their axons to a
specific set of glomeruli in the olfactory bulb (OB) (Vassar et al. 1994; Ressler et al. 1994;
Mombaerts et al. 1996) (Figure 1-6B). In addition, the dorsal-ventral arrangement ofglomeruli in the OB is correlated with the locations of OSNs in the MOE (Miyamichi et al.,2005). Therefore, whereas OSNs are scattered in the MOE, olfactory signals from OSNs
are reorganized and projected as a topographical map in the OB.

12

Two classes of OR genes
In mouse, ~1400 OR genes have been identified (Zhang et al., 2007). Phylogenetic
analysis classified OR family into two broad but distinct groups, class I and class II (Zhang
and Firestein, 2002). The class I (also called fish-like) OR family is a phylogeneticallyancient mammalian OR family (Glusman et al., 2001) and includes 158 genes, forming anapproximately 3 Mb gene cluster on chromosome 7 (Zhang et al., 2007) (Figure 1-6C, 1-7).
Class I OR genes are expressed almost exclusively by OSNs in the dorsal MOE, and these
OSNs project their axons to a specific subset of glomeruli in the dorsal domain of the OB(Tsuboi et al., 2006; Bozza et al., 2009). There are at least two exceptions called as
non-classical class I OR, which are expressed in a more ventral region (Tsuboi et al., 2006).
In contrast, class II OR genes are terrestrial specific and are distributed throughout themouse genome (Niimura and Nei, 2005; Zhang et al., 2007) (Figure 1-6C). Class II OR
genes are expressed throughout the dorsal and ventral MOE, and the expression areas are
specific to each class II OR genes (Miyamichi et al., 2005).

Cis- and trans-regulatory elements for OR genes
The molecular mechanisms underlying OR genes expression remain unclear. To
uncover this issue, several cis-regulatory elements have been identified for class II OR
genes expression. Transgenic analyses using minimal OR gene regions showed that a few
hundred bp of upstream regions of the transcription start site (TSS) are sufficient for someclass II OR transgene expressions including M71, MOR23 and MOR262-12 (Vassalli et al.,
2002; Rothman et al., 2005; Zhang et al., 2007) (Figure 1-6D). Indeed, the ~300 bp
promoter segments of some class II OR genes are capable of driving expression of a13

reporter gene (Vassalli et al., 2011). In contrast, some class II OR genes need more distal
regulatory regions to activate OR genes; H region at ~60 kb upstream of the MOR28
cluster on chromosome 14 (Serizawa et al., 2003) and P element at position between P3
and P4 OR genes on chromosome 7 (Bozza et al., 2009) (Figure1-6D). Mutagenesis of theregulator regions revealed that cis-element in proximal position activates only one
proximal OR gene (Rothman et al., 2005), whereas the distal cis-elements, such as H
region and P element can activate one of several class II OR genes in a cluster (Serizawa etal., 2003; Fuss et al., 2007; Nishizumi et al., 2007; Khan et al., 2011). Thus, there are two
type regulatory regions in proximal and distal positions depending on OR genes.
The class II OR regulatory regions were shown to contain conserved motifs ofhomeodomain and O/E binding sites (Vassalli et al., 2002; Rothman et al., 2005;
Nishizumi et al., 2007; Vassalli et al., 2011). The comprehensive promoter analyses
suggested involvements of the two motifs in a large proportion of OR gene promoters(Clowney et al., 2011; Plessy et al., 2012). Mutagenesis of theses motifs in M71 promoter(proximal) and H region (distal) demonstrated that the homeodomain and O/E-like motifs
played important roles in the class II OR gene expression (Rothman et al., 2005;
Nishizumi et al., 2007).
Regarding to the trans-elements for OR gene expression, LIM-homeobox protein 2(Lhx2) and empty spiracles homeobox 2 (Emx2) were identified as binding proteins inhomeodomain site of M71 promoter region (Hirota and Mombaerts, 2004). Analysis of
Lhx2-deficient mutant mice demonstrated that Lhx2 was required for all tested class II OR
gene expression and OSN development (Hirota and Mombaerts, 2004). Further analysis
showed that class II OR and non-classical class I OR expression were abolished in the14mutant mice, but class I OR expression was largely spared, suggesting mechanistic
differences in class I versus class II OR gene choice (Hirota et al., 2007). Loss of Emx2
decreased many OR genes expression irrespective of their classes and increased some class
II OR genes expression (McIntyre et al., 2008). Studies on OR gene expression have been
so far conducted on mainly class II OR genes, and there are several findings of regulatory
elements for class II OR genes. By contrast, neither cis- nor trans-elements for class I OR
genes have been identified yet.

Ⅰ-5

Purpose of this study
The BGM vector system has several attractive properties including large cloning

capacity, insert stability and amenability of various modification strategies. However, thissystem is a developing technology, i.e., genetic modifications have not been fully
established and this system has not been applied to transgenesis. Therefore, I explored thepotential utility of the BGM system in chromosome engineering and mouse transgenesis.
For this purpose, I focused on the class I OR gene family (Figure 1-7A, B). Although a
cis-acting locus control region is expected to regulate transcription, such a region has notbeen found (Hirota et al., 2007; Rodriguez, 2007) (Figure 1-7C).
In this study, I performed two new genetic modifications in the BGM system tocomplete the BGM-based gene manipulation methods including insertion, deletion,
inversion and fusion (Chapter II). Using these techniques, I constructed some transgenes
including a 252 kb reconstructed transgene, which was generated by fusing two BAC
clones whose inserts were initially oriented in opposite direction. Mutations during DNA
manipulation was examined by next-generation DNA sequencing.

15

To evaluate the potential utility of the BGM system in transgenesis, I applied thissystem to generate transgenic mice using two transgenes constructed in Chapter II(Chapter III). I established a protocol to retrieve the BGM insert and to generate transgenic
mice by microinjection into fertilized mouse eggs. By analyzing the transgenic mice, I
showed a first experimental evidence of a cis-acting element of a class I OR gene.
Finally, by combining the functional analyses of transgenes and the bioinformatics
analyses, I identified the cis-element for the class I OR gene, MOR42-3 (Chapter IV).
Comparative analyses revealed that the cis-element consists of some functional elements tocontrol the class I OR gene expression.

16

A

B
Genomic0DNA0insert!

TEL!

ARS!

Genomic0DNA0insert!

Cmr!

TEL!
parB!

TRP1! CEN4!

pBAC108L!

URA3!

pYAC2!

parA!

oriS!
repE!

Figure 1-1. Structures of the YAC and BAC vectors.
(A) Schematic structure of a YAC clone (pYAC2, Burke et al., 1987). The YAC vector contains anautonomously replicating sequence (ARS), a centromere (CEN4) and two telomere seedsequences (TEL) for replication and maintenance of YAC clone; and two selectable marker genes(TRP1 and URA3). (B) Schematic structure of a BAC clone (pBAC108L, Shizuya et al., 1992). The
BAC vector contains several replication-related genes including oriS, repE, parA and parB, and achloramphenicol resistance gene (Cm). The triangles indicate cloning sites.

17Figure 1-2. A model for the molecular mechanism of transformation in B. subtilis. (fromChen et al., 2005)
The transformation machinery molecules are preferentially located at the cell poles. The
pseudoprepilins (ψ-pilins) are processed by the peptidase (ComC) and translocate to the outer faceof the membrane. With the aid of the other proteins (ComA and ComB), the pseudopilins assemble
into the psudopilus, which attaches exogenous DNA via a hypothetical DNA binding protein.
Incorporation of the DNA requires retraction of the psudopilus, driven by the proton motive force(PMF), and DNA binding to the receptor (ComEA). The one strand of DNA is transported throughthe membrane channel (ComEC) while the other is degraded by a nuclease existing the cell. The
helicase/DNA translocase (ComFA) assists the process, along with single-strand DNA binding (ssb)proteins interact with the incoming DNA. RecA forms a filament around the single-strand DNA, andmediates a search for homology with chromosomal DNA.
	
 

18

A

oriC

oriC

BEST310)

BEST6528)

Pr#neo(~4.2)Mb))(~4.3)Mb))

I

GpBR)

[R])

cI#bsrspacer)DNA)

I
[R])

GpBR)

[L])

I

cI#spc

I

[L])

B
Pr#neoPr#neoTransforma3on)(Nm)selec3on)

BEST310)ICI)repressor

I

BEST310/BAC)

I

BAC)
clone)

insert)DNA)cI#spcI
BEST310

Spc:R,)Nm:S

Spc:S,)Nm:R

Figure 1-3. Strategies of BAC cloning into the BGM vectors.
(A) Structures of two BGM vectors for BAC cloning. BEST310 has two extra DNA segments(Kaneko et al., 2005). One is a 4.3 kb pBR322 sequence, the genomic pBR (GpBR) sequence, in
the NotI site of the proB locus at the position of 1,378 kb (amp-half, blue stripe; tet-half, brawn
stripe) (Itaya, 1993; Kunst et al., 1997). A modified pBAC108 plasmid was also inserted in thisposition with cI-spc cassette, which is composed of λ-phage cI repressor gene and spectinomycin
resistance gene (cyan circles). The other segment is a Pr-neo cassette, which is composed of
λ-phage Pr promoter and neomycin resistance gene, in the NotI site of yvfC-yveP locus at theposition of 3,516 kb (Itaya, 1999). BEST6528 is another BGM vector for large BAC handling,
contains a 100 kb spacer DNA (mtDNA of Arabidopsis thaliana) and two cI gene cassettes (spc andblasticidin S resistance gene, bsr) (Kaneko et al., 2009). The BAC vector sequences are indicatedby closed and open arrows (3.5 kb each), and the right half contains chloramphenicol resistancegene for E.coli (open arrow). The I-PpoI sites are introduced to flank the BAC vector sequences(designated as “I”). (B) Schematic illustration of BAC cloning. Before transformation, the BGM
vector is resistant to Spc and sensitive to Nm (Spc:R, Nm:S) due to repression of the Pr-neo
cassette by the CI repressor. The BAC inserts are integrated into the BGM vector via double
crossings-over recombination with the BAC vector sequences. The recombinants become resistant
to Nm and sensitive to Spc (Spc:S. Nm:R), because of replacement of the cI-spc cassette with the
BAC insert.

19

A
oriC

oriC

Linear,fragment,
I"PpoI
I

insert,DNA,

I

I

I

B
oriC

oriC

Sub,genome,(circular,DNA),,tet

te,&,et,fragments,

,

oriN

,

e

C
oriC

oriC

BReT,clone,（recovered,BAC,clone）,
pBReT,vector,

ori

,

,

tet

Figure 1-4. Methods for the insert retrieval from the BGM vector.
The BGM vector system has several retrieve methods. (A) A liner insert DNA can be retrieved by
I-PpoI digestion (Kaneko et al., 2009, Chapter III). The I-PpoI sites are represented by “I”. (B) An
insert DNA can be separated from the BGM vector in a circular form by the targeted insertion of thete and et cassettes into the ends of BAC vector sequences (Itaya and Tanaka, 1997). (C) BGM
inserts can be reformed into a plasmid by transformation with a linearized pBReT vector (Tsuge and
Itaya, 2001).

20

A
Holding)pipe2e)

Fer$lized)egg)

Injec$on)needle)

Zona)pellucida)Pronuclei)BTol2)transposase)mRNA)
AAAA)

Tol2%

(4682)bp))200)bp)(Tol2L))

Holding)pipe2e)150)bp)(Tol2R))

Pronuclei)

pTol2:transgene)
circular)plasmid)

Transposi$on)&)Integra$on)

Injec$on)needle)

Cytoplasmic)injec$on)

Transposase)mRNA)

excision)(vector)

Genome)Figure 1-5. Methods for microinjections in the mouse transgenesis.
(A) A view of pronuclear microinjection. A linearized transgene is introduced into a pronucleus of a
fertilized mouse egg using a fine glass needle. (B) Tol2-transgenesis system. The top illustrationshows the structure of the full-length Tol2 transposable element and the minimal Tol2 sequences(orange, L and R) for Tol2 vector. RNA transcribed from Tol2 that encodes a transposase protein is
depicted by lines (exons) and dotted lines (introns); coding regions, blue boxes; untranslated
regions, cyan boxes; 12 bp terminal inverted repeats (TIRs), black triangles. The lower portion
shows the Tol2-mediated cytoplasmic injection method. A transgene cloned in a Tol2 transposon
vector together with the transposase mRNA is injected into cytoplasm of fertilized eggs. The
transgene is transposed from the plasmid to the host genome.

21

A

B

C
Number'of'OR'genes









            

Mouse'chromosomes

DProximal'regulatory'regions'

1'kb'

TSS'

405'bp'

MO

H'

M71'
(Chr.9)'161'bp'TSS'Distal'regulatory'regions'

MOR23'

8'
R2

O

O

71
lfr

1'
O

2'
lfr

A'
29

R
MO

(Chr.14)'~60'kb'6'
lfr

10'kb'
0'
3'
R1
R8
MO MO

O

71
lfr

3'(

)'
P3

P'

O

71
lfr

4'(

)'
P4

2)'
'(P 15'
7
1
fr
fr7
Ol Ol

(Chr.7)'(Chr.1)'~11'kb'Figure 1-6. Mouse olfactory system and OR genes.
(A) Two major olfactory systems in mouse (from Mombaerts, 2004). Green color indicates the main
olfactory system for detection of odorants; main olfactory epithelium (MOE) and main olfactory bulb
(MOB). Red color indicates the vomeronasal system for detection of pheromones; vomeronasalorgan (VNO) and accessory olfactory bulb (AOB). (B) Schematic illustration of the axonal
connectivity pattern between MOE and OB (from Mori et al., 2006). OSNs expressing a given OR
are distributed widely in the MOE and converge their axons onto a few topographically fixed
glomeruli that are located within a corresponding region of the OB. “Zone1-4” are traditional namesof compartments of the MOE. (C) Distribution of OR genes in mouse genome (Zhang et al., 2007).
(D) Identified regulatory regions for class II OR genes. In proximal types (left), two mini-ORtransgene regions of M71 and MOR23 are depicted (Vassalli et al., 2002; Rothman et al., 2005).
Red bars, regulatory regions; Cyan, non-coding regions; Blue, coding regions. In distal types (right),
H region in MOR28 cluster and P element in P centromeric cluster are depicted (Serizawa et al.,
2003; Bozza et al., 2009).

22

A

Mouse'chromosome'7

E3'

B
Centromere!61O11'(2)!327E18'(4)'!392H7'(1)'!306C18'(3)'!125E6'(6)



P'
!8
14 0'
!1
14 P'
4
0! 5P'
!
14!2'
14
'
!1
14 '
!2
18 '
!1
11!2'
11!1'
18!3'
18

'
!1
41 '
!1
10
'
!1
25

'
!2
25 1'
!
28 '
!1
29 '
!1
16
'
!3
31 3'
!1
31 4'
!
31

$
#1
42#3$
42#2$
42

100'kb!480A17'(8)!211E6'(9)

!282D19'(5)!310P1'(7)!347I6'(11)

'
!1
39!1'
23 5'
!
23!2'
23



P'
!6
32 0'
!1
32!2'
24
3'
!1
32 1P'
!
24 P'
1
0! 3'
!
32 '
1
6! P'
3
7! '
7
8!
'
!4
14 '
!2
30

'
!6
14 3'
!
30 '
8P
8! P'
4
8!9P'
8!6P'
8! '
1
7! '
2
7!5'
8!1'
8! !9'
14!4'
10 '
!2
10
'
!1
21 '
3
8! '
!1
30 3'
!
14 4P'! '30!7P
14
!419H14'(10)!344M21'(12)!539K4'(13)





'
!1
31 3'
!
22 2'
!
22 1'
!
17 2'
!
26 '
!1
22
'
!4
22 P'
!4
266P' P'
4! !15
31 P'
2
4!
4'
1!
'
!1
26 P'
3
4!
1'
4! !5'
31!9'
31 10'
!
31
'
!1
19!2'
19!1'
20 '
!3
15
'
!1
15
2'
9!
1'
9!
'
!3
26 3P'
!
33!2'
17!5'
24

'
!2
32 P'
!3
23 3'
!
24



!266J11'(14)!104I11'(16)!125B17'(15)



4'
!1
31
'
!7 '
31!12
31 P'
5
0!

'
!2
33

'
!5
13 '
!1
13!2'
13 6'
!
13!3'
13
'
!4
13!1'
121' '
5! !3P
'
12!2P
12 '
2
5!
'
!4
12 5'
!
121'
2!

1'
3! '
1
1! '
2
1! '
3
1!



!180O9'(18)!103C10'(17)!157I3'(19)!475C24'(20)

'
0P
!1
34
2'
!1 '
40!13P
34!4' '
40!18P
40!4'
34

'
!5 '
34!13
40
'
!1
27
'
!1
38!2'
38!1'
33 '
!8
31 11'
!
31







!68E7'(22)!111G17'(21)!397L11'(24)!492N18'(23)



'
!6
31!2'
31 '
!3
40

'
!5
40

1'
!1
40
'
!1
35
'
!1
37
P'
!8
40 '
!1
40 9P'
!1 '
40!14
40
'
0P
!1
40!2'
40
'
!5
32 11'
!
32

'
!1
32 P'
!9
32 '
P
!7
32 '
!4
32
2'
!1
32
'
!8
32
'
!6
34 '
!3
34!2'
34

'
!1
34 7'
!
34

Telomere

'
!1
36

C

transcripRon'
acRvaRon''complex

gene$choice$&$ac+va+on

regulatory'region

promoterclass'I'OR'gene'clusterFigure 1-7. Mouse class I OR gene cluster and BAC clones.
(A) The ideogram of mouse chromosome 7. The class I OR gene cluster is located in the E3 region.
(B) Structure of ~3 Mb mouse class I OR gene cluster. BAC clones, whose full-length insert
sequences are available, are represented. In this study, the MOR42 family at most centromere
position (blue) was chosen as a target for the transgenic analysis. Numbers in parentheses are BAC
numbers in this study (ex. first BAC clone RP24-392H7 means ‘BAC1’). (C) A hypothetical model forthe mechanism of class I OR gene regulation. One regulatory region controls expression ofmulti-class I OR genes such as the locus control region.

23

Table 1-1. Properties of cloning platforms for the large genomic DNA.
SystemB.#sub'lis#genome+
vector+(BGM)

Cloning+
capacity+

Advantages

over+3+Mb

?+targeted+cloning+?+easy+manipulaAon+?+intrinsic+homologous+
recombinaAon?based+modiﬁcaAon+?+stability+of+insert+?+preservaAon+?+developing+tool

plasmid+(E.coli)+++++
BAC+vector+~+300+kb+(generally+++~200+kb)

?+genome+DNA+library+?+easy+manipulaAon+?+stability+of+insert+?+isolaAon+?+requirement+of+extra+factors+for+
modiﬁcaAon+?+potenAality+of+loss+?+relaAvely+small+cloning+size+

parAal+genome+(S.#cerevisiae)++
telomere+telomere++
ARS,+centromere+~+2+Mb?+genome+DNA+library+?+instability+of+inset+?+intrinsic+homologous+?+isolaAon+
recombinaAon?based+modiﬁcaAon

Vector+(host+cell)
genome+(B.#sub'lis)++
foreign+DNA+fragment++++

B.#sub'lis#genome+

Bacterial+arAﬁcial+chromosome+(BAC)

Disadvantages

(including+ori)+

Yeast+arAﬁcial+chromosome+(YAC)

Properties, advantages and disadvantages in the BGM, BAC and YAC systems are summarized.
The BAC and YAC systems have complementary advantages over each other in term of cloning
capacity and insert stability. The BGM system is an attractive alternative of the manipulation of large
DNA, but this system is a developing technology.

24

CHAPTERIIDEVELOPMENT OF A COMPLETE GENETIC
MANIPULATION METHOD FOR THE BGM
VECTOR SYSTEM

25

ⅠI - 1Introduction
The Bacillus subtilis genome (BGM) vector system has been developed as a novel

cloning system using a unique concept in which the entire genome of B. subtilis functions
as a vector. The specific features of the BGM vector system include a cloning capacity
over 3 Mb, the stable propagation of cloned DNA fragments in a single copy per cell andthe amenability of various modification strategies based on RecA-mediated homologous
recombination. These advantages make it an attractive alternative for the manipulation oflarge DNA. It has been used to clone genomic DNA from several species, including
cyanobacteria (Itaya et al. 2005), Arabidopsis (Kaneko et al. 2005) and mouse (Itaya et al.
2000; Kaneko et al. 2003). Modifications consisting of deleting and fusing cloned inserts
have also been achieved (Kaneko et al. 2003; Kaneko et al. 2009). However, genetic
modification methods have not been fully established in the BGM vector system; targeted
insertion and inversion modifications remain to be demonstrated, and the fusion of two
contiguous DNA fragments is limited to clones that are orientated in the same direction(Kaneko et al. 2009). Thus, the BGM vector system is a developing technology withattractive potential, which includes its megabase-cloning capacity and homologous
recombination-based genetic modification.
In this study, I have added two new genetic modifications to the BGM system tool
kit to establish BGM-based gene manipulation methods (Figure 2-1). One is targeted
insertion of a reporter gene without introducing an unwanted selectable marker at the target
site when constructing the transgene. Another is inversion of the cloned insert to align its
orientation so that two contiguous BAC clones in opposite directions can be fused toreconstruct the genomic structure. In fact, inversion of a cloned insert has not been26

reported in any other system. Through all experiments in Chapter II, I finally constructedtransgenes for analyzing cis-elements for a class I OR gene.

27

ⅠI - 2

Experimental proceduresII-2-1)

Strains and preparation of competent B. subtilis cells

The B. subtilis strains and the BAC-specific BGM vectors BEST310 and BEST6528(Kaneko et al., 2005; Kaneko et al., 2009) were kindly provided by Dr. Itaya. These strains
are derived from the restriction modification-deficient strain RM125 to possess a cI
repressor gene cassette flanked by pBAC108-based BAC vector sequences (Shizuya et al.,
1992; Kaneko et al., 2005) and are identical with the exception that BEST6528 contains a
100 kb spacer sequence and an additional cI repressor gene cassette (Kaneko et al., 2009).
For defining insert orientation, the half of the BAC vector containing the chloramphenicol
resistance gene for E. coli is defined as right side (open arrows in Figures). The preparationof competent cells and transformation of B. subtilis were performed as described elsewhere(Itaya and Tsuge, 2011). Strains containing multiple antibiotic-resistance genes were tested
using the replica plating method. B. subtilis was routinely grown in 1-5 ml Luria-Bertani(LB) broth at 37°C by rotating (>50 rpm) or shaking (200 rpm). Antibiotic Medium 3(Difco) was used for the selection of the BGM transformants with the following antibiotics,
as appropriate: neomycin (Nm, 5 μg/ml, Sigma), spectinomycin (Spc, 50 μg/ml, Sigma),
erythromycin (Em, 5 μg/ml, Sigma), phleomycin (Phl, 0.5 μg/ml, Sigma), tetracycline (Tet,
10 μg/ml, Nacalai) and blasticidin S (Bs, 250 μg/ml, Funakoshi).

II-2-2)One-step transfer of BAC inserts to the BGM vector

Two contiguous BAC clones, RP24-392H7 and RP23-61O11 (designated BAC1 and
BAC2, respectively), were purchased from the Children’s Hospital Oakland ResearchInstitute. The BAC DNA was prepared by the alkaline lysis method and subsequent28

equilibrium centrifugation in a CsCl-ethidium bromide gradient (Kaneko et al., 2003).
BGM strains were transformed with the purified BAC DNAs (Kaneko et al., 2005). BAC1and BAC2 were cloned into BEST310 and BEST6528, respectively.

II-2-3)

I-PpoI/CHEF analysis

The cloned inserts in the BGM vectors were analyzed by I-PpoI digestion followedby CHEF electrophoresis. Agarose plugs containing BGM clones were prepared as
described (Itaya and Tsuge, 2011). A sliced block of the agarose plug was soaked in 200 μlof I-PpoI digestion buffer for 15 min to replace the TE buffer in the agarose plug. Afterdiscarding the soaking buffer, the block was immersed in 100 μl of digestion buffer
containing 20 U of I-PpoI (Promega) and incubated for 1-1.5 hours at 37°C. The plug
containing the digested DNA was embedded in a well of a 1% (w/v) agarose gel andseparated by CHEF (Bio Craft) in 0.5× TBE buffer (50 mM Tris–borate (pH 8.0), 1.0 mM
EDTA). Electrophoresis was performed at 4 V/cm at 14°C under the following conditions:
12 sec for 21 h (BAC1, Tg-110), 30 sec for 22 h (BAC2, Tg-220) and 25 sec for 22 h
(Tg-250SB).

II-2-4)Southern blot analysis of the BGM clones

Genomic DNA from the BGM clones was prepared using the liquid isolation method(Itaya and Tsuge, 2011). The genomic DNA was digested with EcoRI, HindIII or BamHI(TaKaRa). The digested DNA was separated in pulse-field gels at 3 V/cm, 18 sec for 14 h
at 14°C, and the DNA was transferred onto a Hybond-N (GE Healthcare) membrane filter.
The preparation of digoxigenin (DIG)-labeled DNA probes, Southern hybridization and29

detection with NBT/BCIP were performed using a DNA labeling and detection kit (Roche).
A BAC probe was prepared by the random prime labeling method using the target BAC
clone as a template. A full-length R arm probe and MOR42-3 coding probe (190-1091
from NM_020289) were prepared by PCR labeling method.

II-2-5)Construction of plasmids

To construct the reporter cassette insertion plasmid, a polylinker containing AscI,
SpeI, BglII, NdeI and SphI sites was inserted between the XbaI and SacI sites of
pBluescript II SK(+) (Stratagene) to construct the pT1 vector. An EcoRI-XbaI fragment of
IRES-tauEGFP from the iTGFP-ACNF plasmid (Bozza et al., 2002) was cloned into pT1
to generate pT1-iTGFP. The 1.0 kb left (L) and right 1.0 kb (R) arms for the targeted
insertion of IRES-tauEGFP into the 3 bp downstream of the MOR42-3 stop codon wereprepared by PCR and contained sequences that were homologous to the upstream anddownstream MOR42-3 insertion sites, respectively. The L arm was first cloned into the
SalI-EcoRI site of pT1-iTGFP, and then the R arm was cloned into the SpeI-BglII site to
generate the plasmid piTG423. The piTG423-CISP plasmid was constructed by inserting a
selection marker-containing the cI-spc cassette into the AscI-SpeI site of the piTG423
plasmid.
To construct the inversion plasmid, the new BAC plasmid p108IPpoI-HPNSB was
generated by inserting a linker containing PmlI, NotI and SphI sites between the HindIII
and BamHI sites of the BAC plasmid p108NHBN-MIM (Kaneko et al., 2005). The te-erm
cassette, which was obtained from a NotI-digested fragment from pBEAZ191 (Kuroki etal., 2008), was inserted into the PmlI site of this plasmid after blunt-end treatment to30

generate the plasmid p108Term. Similarly, the phl-et cassette, a NotI fragment frompBEAZ195 (Kuroki et al., 2008), was inserted to generate the plasmid p108Phlet. A 0.9 kb
fragment homologous to the left end of Tg-110 was cloned into the NotI site of p108Term.
A 0.8 kb fragment homologous to the right side of Tg-110 (1.3 kb from the right end) was
then cloned into the HindIII site of p108Phlet.
Fusion plasmids were also constructed based on p108IPpoI-HPNSB. The p108CISP
plasmid was constructed by inserting the cI-spc cassette, a HindIII-BamHI fragment
excised from pCISP310B (Kaneko et al., 2005), into the PmlI site after blunt-ending.
Similarly, p108CIBS was constructed by cloning the cI-bsr cassette, a PstI fragment
derived from pBEST10007. A 1.3 kb PCR fragment homologous to the left end of Tg-220
was cloned into the NotI site of p108CISP, and a 1.3 kb fragment homologous to the rightend of the inverted Tg-110 was cloned into the MluI site of p108CIBS.
All of the plasmids were linearized with the appropriate restriction enzymes(TaKaRa or Toyobo) and used for transformation. The all homologous arms
(approximately 1 kb) and cI-spc cassette were amplified by PCR (PrimeSTAR HS DNA
polymerase, TaKaRa, or KOD plus DNA polymerase, Toyobo) using the BAC clones and
pCISP310B as templates, respectively. Primer sequences and PCR conditions are
summarized in Table 2-1. The orientations of the inserts were determined by restriction
enzyme digestion or PCR. The accuracy of the sequences generated by PCR was
confirmed by DNA sequencing.

II-2-6)

DNA sequencing

Sequences of the original BACs and modified BGM clones (Tg-110CIBS,31

Tg-220CISP and Tg-250SB) were verified using next-generation sequencing. Briefly,
genomic DNA (3 µg) from the above clones was fragmented to an average length of 300
bp using the Covaris S2 system (Covaris). After purification, end-repairing, A-tailing,
paired-end adapter ligation and 12-cycle PCR were performed using the NEBNext DNA
Library Prep Master Mix Set and NEBNext Multiplex Oligos for Illumina (New England
Biolabs). All libraries were quantified using an Agilent Bioanalyzer 2100 (Agilent
Technologies) and pooled to provide equal genome coverage from each library. Pooled
libraries were sequenced in a single lane of the Illumina Genome Analyzer IIx (Illumina),
which produced 102 paired-end reads, in accordance with the manufacturer’s instructions.
Reads obtained from the Illumina Genome Analyzer IIx were analyzed using CLC
Genomics Workbench 5.5 (CLC Bio). Reads were trimmed and mapped to each reference
sequence with default parameters. The reference sequences used in this study included thefollowing: BAC1, original BAC clone RP24-392H7 [GenBank: AC132096]; BAC2,
original BAC clone RP23-61O11 [GenBank: AC102535]; B1TgCIBS and B2TgCISP,
predicted modified mouse genomic Tg-110CIBS and Tg-220CISP insert sequences; andTg250SB, a predicted fused mouse genomic Tg-250SB insert sequence. The mapping
results are detailed in Table 2-2. After mapping the reads, variant calling was performed
using the probabilistic variant detection function with default parameters, and variants withfrequencies of at least 90% were considered. All read data have been deposited in the
DDBJ Sequence Read Archive (DRA) under accession number [DRA000859].

32

Table 2-1. Primer sequences and PCR conditions used in Chapter II.
Annealing temp
Extension time

Purpose

PCR condition

Forward TTTGTCGACATGTCAGGGTGGAGCAATG
Reverse TTGAATTCAATCATCCAGGAAGCTCTC

59.2 °C
1 min

homology arm (L-arm)
to 5' side of the insertion site

PrimeSTAR HS

J532_RFJ533_RR

Forward TACTAGTCCAGAATTTGTGGGTCTCA
Reverse TTTAGATCTAGAGTTGAGTGCAAGCTG

55.5 °C
1 min

homology arm (R-arm)to 3' side of the insertion site

PrimeSTAR HS

J554_CISPF
J555_CISPR

Forward GGCGCGCCAGCCGTCATCCTCGGCAC
Reverse TACTAGTAACCAATTCCGACAGGCTTTG

55 °C
2 min 30 sec

subcloning of cI-spc cassette
check (cI-spc cassette: F3/R3)

PrimeSTAR HS
Genotyping

J476_MOR42-3
J477_MOR42-3

Forward GCAATGGCACCTACAATGAGT
Reverse CTGCCTGATTTCCTTGGTTC

60 °C
1 min

check (MOR42-3 coding: F1/R1)
MOR42-3 coding probe

Genotyping

GFP-F
GFP-R3Forward GGCATCAAGGTGAACTTCAAGATCC
Reverse CTTTACTTGTACAGCTCGTCCATGC

62 °C
30 seccheck (GFP: F2/R2)

Genotyping

J554_R seq.
J533_RR

Forward ACATGGTCCTGCTGGAGTTC
Reverse TTTAGATCTAGAGTTGAGTGCAAGCTG

55 °C
3 min 30 sec

check (GFP_42-3 R-arm: F4/R4)

Genotyping

Inversion of Tg-110
J591_BAC1_LF
J592_BAC1_LR

Forward AATGCGGCCGCGATCTCAGCTCCTCAAACTC
Reverse AATGCGGCCGCCTGGATACATTCCTCTTCC

54.5 °C
1 min

homology arm (L-arm)to left-end of Tg-110

KOD plus

J593_BAC1_RF
J594_BAC1_RR

Forward CCCAAGCTTGGATTTCCAGCTTGCTTCC
Reverse CCCAAGCTTAGGCAGTGATGACCTTTGC

59.2 °C
1 min

homology arm (R-arm)
to right-end of Tg-110

KOD plus

191F-teA2
195F-etA

Forward GATAGCCCATTATATTCATTGGTCC
Reverse CAAGTATCCCACCAATGTAGCCG

61 °C
1 min 40 sec

check of tet genetet probe

Genotyping

191R-ermB
195R-PhlB

Forward GTAAAATGGCCTTTTCCTGAGCCG
Reverse GGCAATGCCGGGATAGACTG

61 °C
1 min 40 sec

check of erm-e-phl

Genotyping

Fusion of Tg-220 and Inverted Tg-110
J629_BAC2_LNotF
Forward ATGCGGCCGCGAATTCCTGATCTTTCCAAGAC
J630_BAC2_LNotR
Reverse ATGCGGCCGCAGACCACCATGGCCTAATG

58.4 °C
1 min 10 sec

homology arm (L-arm)to left-end of Tg-220

KOD plus

J631_BAC1Inv_RMluF Forward CCGACGCGTAGCACACCTGAAAGCTCTAG
J632_BAC1Inv_RMluR Reverse CCGACGCGTGATCTCAGCTCCTCAAACTC

55.5 °C
1 min 10 sec

homology arm (R-arm)
to right-end of Tg-220

KOD plus

Primer name

StrandMOR42-3 targeting
J530_FF
J531_FR

Sequence

Poly linkers
Linker 1
Linker 2

Sense CTAGAGGCGCGCCACTAGTAGATCTCATATGGCATGCGAGCTAntisense TCCGCGCGGTGATCATCTAGAGTATACCGTACGC

linker for pT1 vector
containing AscI-SpeI-BglII-NdeI-SphI

HPNSB-1
HPNSB-2

Sense AGCTTCACGTGCGGCCGCATGCG
Antisense GATCCGCATGCGGCCGCACGTGA

linker for p108IPpoI-HPNSB pasmid
containing PmlI-NotI-SphI

PrimeSTAR HS (TaKaRa)
94 °C 5 min

KOD plus ver.2 (Toyobo)
94 °C 2 min

Genotyping, Check of BGM clones
94 °C 5 min[30 cycles]
98 °C 10 sec(Annealing temp.)* 5 sec
72 °C (Extension time)*[30 cycles]
98 °C 10 sec(Annealing temp.)* 30 sec
68 °C (Extension time)*[30 cycles]
94 °C 30 sec(Annealing temp.)* 30 sec
72 °C (Extension time)*

72 °C 5 min

68 °C 5 min

72 °C 5 min

Primers and oligonucleotides used in Chapter II for constructing homology arms, cassettes and new
BAC plasmids, screening of BGM clones are summarized. PCRs with PrimeSTAR and KOD plus
kits were basically performed according to the manufacturer's recommendations; all PCR conditions
are summarized in bottom of Table. Genotyping PCR used a recombinant Taq DNA polymerase.
The annealing temperature and extension time were described in Table.

33

ⅠI - 3Results

II-3-1)

Cloning of genomic DNA fragments into the BGM vector

Mouse genomic BAC libraries have been constructed that cover nearly the entire
mouse genome, and each clone contains a genomic DNA fragment with an average size of
approximately 150 kb (Osoegawa et al., 2000). The BGM vector system can utilize thesevaluable DNA resources in a one-step transfer (Kaneko et al., 2005). I prepared two BAC
clones, designated BAC1 and BAC2, which overlapped each other via 82 kb region andcarried 114 kb and 220 kb mouse genomic DNA fragments containing two and ten class I
OR genes, respectively (Figure 2-2). I transferred these BAC inserts into BGM vectors,
which harbor BAC vector sequences, to clone the BAC inserts by homologous
recombination (Figure 2-1) (Kaneko et al., 2005; Kaneko et al., 2009). Briefly, the originalBGM vector is resistant to spectinomycin (Spc) and sensitive to neomycin (Nm) due torepression of the Pr-neo cassette by the CI repressor. The BAC inserts taken up by the B.
subtilis are integrated directly into the genome (BGM vector) via double crossings-overrecombination with the BAC vector sequences of the BGM vector. Once a BAC insert iscloned into the BGM vector, the recombinants become resistant to Nm and sensitive to Spc,
because the BAC insert replaces the cI-spc cassette. This selection mechanism can be used
in cI cassette-mediating modifications, such as the targeted insertion technique. Because
two 23-bp sequences “ATGACTCTCTTAA/GGTAGCCAAA” recognized by therare-cutting endonuclease, I-PpoI are introduced at the both sides of the BAC cloning site,
I-PpoI digestion enables to excise out the cloned insert from the BGM vector. The inserts
were evaluated by digesting the genomic DNA of the recombinant vectors with I-PpoI
followed by contour-clamped homologous electric field (CHEF) gel electrophoresis

34

(Figure 2-3). Three of seven candidate clones (resistant to Nm and sensitive to Spc) in
BAC1 and one of 29 candidate clones in BAC2 contained BAC inserts. The efficiency ofcloning the BAC insert into the BGM vector differs between BAC clones used (Table 2-3).
I cloned 10 different BAC clones with 3 to 100% efficiency (average 40%). The cloned
inserts were further confirmed by Southern blot analyses using the original BAC clones asprobes; the results indicated that the digest patterns of the BGM recombinants wereidentical to those of the original BAC clones, except for the fragments derived from theends of the insert (Figure 2-3). Thus, the BGM vector system imported the BAC insert bysimple transformation, which enabled the use of already established valuable BAC
resources.

II-3-2)

Targeted insertion of a reporter gene

To construct transgenes for the analysis of cis-acting elements of class I OR genes, I
inserted an IRES-tauEGFP reporter gene cassette (Bozza et al., 2002), which consisted of
an internal ribosome entry site and the tauEGFP fusion protein coding sequence, into theclass I OR gene MOR42-3 locus by two-step recombination using the cI-spc cassette(Figure 2-4A). In the first step, the BGM clones containing the BAC inserts weretransformed with a reporter cassette with the selection marker cI-spc flanked by 1.0 kb
homology regions to the target OR locus (L and R arms) to generate Tg-110CISP and
Tg-220CISP. Four of 15 Spc-resistant Tg-110CISP clones and six of 15 Spc-resistantTg-220CISP clones were sensitive to Nm. In the second step, the counter-selection was
performed to remove selection marker using a reporter gene cassette without a cI-spc
cassette to generate two transgenes, Tg-110 and Tg-220. Nm-resistant and Spc-sensitive35

clones were screened by PCR to amplify the region between EGFP and R arm (Figure 2-5).
The cI-spc cassettes were correctly removed in four of 15 tested clones for Tg-110 and twoof 48 clones for Tg-220. Negative clones were no PCR product, suggesting that insert
DNA sequences had been also deleted along with the cI-spc cassettes. Relatively low
success of the recombination events in the counter-selection was previously reported in the
BGM system (Itaya et al., 2000) as well as the BAC system (Zhang et al., 1998).
Accordingly, two transgenes, Tg-110 and Tg-220, which were derived from BAC1 and
BAC2, respectively, were constructed by inserting IRES-tauEGFP 3 bp downstream of the
MOR42-3 stop codon. Southern blot analyses of representative clones with the MOR42-3
coding or R arm probes indicated the correct insertion of the reporter cassette (Figure 2-4
B, C). Thus, I established a targeted insertion method using the BGM vector system withhomologous recombination using several antibiotic resistance genes and the cI genewithout leaving any trace of selection markers or site-specific recombination sites, such as
loxP and FRT, in the target sequence (Figure 2-1B) (Zhang et al., 1998; Lee et al., 2001).

II-3-3)Inversion of the insert in the BGM vector

The Tg-110 and Tg-220 shared approximately 82 kb of overlapping sequence(Figure 2-2), which would enable the fusion of these two fragments to create a longer
transgenic construct in the BGM vector system if the two clones were cloned in the same
direction. This technique extends one insert by homologous recombination between theoverlapping region of another insert and the common vector region (Kaneko et al., 2009).
However, the Tg-110 and Tg-220 inserts were cloned in opposite directions in the BGM
vector. In this case, the two clones could not be fused to extend the insert by the double

36crossing-over recombination. Insert inversion techniques have not been reported in the
BGM or other conventional systems. To fuse these two fragments, I first inverted theTg-110 insert to orient it in the same direction as Tg-220 using two incomplete fragmentsof the tetracycline resistance gene (tet), te (5´ side) and et (3´ side), which shared an
approximately 1.1 kb overlap region designated e (Kuroki et al., 2008). The erythromycin
resistance (erm) and phleomycin resistance (phl) genes were added as selection markers to
the te and et fragments, respectively. The two inversion cassettes, te-erm and phl-et, weresequentially inserted at the ends of the Tg-110 insert in the reverse direction (Figure 2-6A).
Briefly, phl-et cassette was inserted to the right end of the Tg-110 insert by transforming
Tg-110 with the linearized BAC plasmid containing the phl-et cassette and a 0.8 kb
homology region. The phl-et cassette was inserted in all five Phl-resistant clones assessedby PCR. Subsequently, I inserted the te-erm to the left end of the insert using the linearized
BAC plasmid containing the te-erm cassette with a 0.9 kb homology region, and obtained
Tg-110 T/P (1 clone / 1 tested). Intrachromosomal homologous recombination between teand et via the overlapping region e (Tg-110 T/P) resulted in an inversion of the insert andthe formation of a complete tet gene, enabling the selection of the inverted clone(Tg-110-Inverted) using Tet (Figure 2-6A). I obtained over 100 Tet-resistant colonies from
20 µl of Tg-110 T/P overnight culture. The formation of tet and erm-e-phl cassettes was
confirmed by PCR in all four randomly picked clones from these colonies. As expected,
Southern blot analysis using a tet probe revealed changes in size of the tet cassettefragments, indicating the successful inversion of the Tg-110 insert (Figure 2-6B). Because
the direction of the inserts is not coordinated in the BAC library, the inversion techniquefor insert fragments is essential to fuse and reconstruct genomic structures using BAC

37

clones.

II-3-4)Fusion of two inserts to reconstruct genomic structures

I then fused Tg-220 and the inverted Tg-110 to enlarge the mouse genomic DNA
fragment (Figure 2-7A). The cI-spc and cI-bsr marker gene cassettes were inserted into theleft end of the Tg-220 insert and the right end of the inverted Tg-110 insert, respectively. I
transformed Tg-220 with the linearized BAC plasmid containing the cI-spc and a 1.3 kb
homology region, and obtained Tg-220CISP clones (9 clones from 10 Spc-resistant clones).
Tg-110-Inverted was similarly transformed with the cI-bsr cassette fragment containing a
1.3 kb homology region, and obtained Tg-110CIBS clones (2 clones from 2 Bs-resistantclones). The two transgenes were fused by genetic transformation of Tg-220CISP with thepurified genomic DNA of Tg-110CIBS. Homologous recombination of the 82 kb
overlapping region and the common sequence of the BGM vectors resulted in theextension of the insert to generate Tg-250SB. Twenty-six of 29 Spc- and Bs-resistantcolony recombinants were sensitive to Nm. I selected two representative clones for further
examinations. The two Tg-250SB clones turned to be sensitive to Tet, whose resistancegene exists in the left end of the Tg-110-Inverted insert and was removed by the fusion,suggesting correct recombination occurs to extend the insert. I-PpoI/CHEF analyses of theTg-250SB genomic DNA demonstrated a larger band corresponding to the 252 kb fused
insert fragment (Figure 2-7B). Southern blot analysis showed that Tg-250SB was
composed of two transgenes, Tg-220 and Tg-110 (Figure 2-7C), indicating that the two
inserts were successfully fused to extend the insert size.

38II-3-5)

DNA sequencing of modified and reconstructed BGM clones

To verify the reliability of the BGM vector system in manipulating genomic DNA
fragments and its accuracy in genetic engineering, I performed DNA sequencing of theoriginal BAC clones (BAC1 and BAC2), the modified BGM clones (Tg-110CIBS and
Tg-220CISP) and the fused BGM clone (Tg-250SB). The read mapping results are
summarized in Table 2-2. No mutation was observed in the cloned and modified mouse
genomic inserts during manipulations of the BGM vector, and all targeted modifications
were accurately introduced. A few nucleotides differed from the reference sequence data inboth BAC1 and BAC2. Two single-nucleotide differences were found between BAC1 andthe reference sequence [GenBank: AC132096.4]: the replacement of G at positions 10349
and 113530 by A and T, respectively. Four differences were found between BAC2 and thereference data [GenBank: AC102535.17]: G at 210780, G at 210788, A at 210815 and G at
210840 were replaced by A, A, G and A, respectively. Therefore, starting from targeted
insertion, all possible genetic modifications, i.e., insertion, deletion, inversion and fusion to
the targeted site, could now be introduced using the BGM system without leaving
unnecessary sequences in the insert DNA.

39

ⅠI - 4

Discussion
In this chapter, I developed a complete genetic manipulation method for the BGM

vector system by demonstration of a target insertion and an inversion modification. The
DNA sequencing analysis of the constructed transgenes revealed all targeted modification
were precisely conducted. Because the reliable inversion modification was notdemonstrated in any other system including BAC or YAC, the BGM vector system has
many advantages in not only the property of a vector system but also the availability ofmodification approaches.
A concern is the efficiencies of each modification. Overall, the efficiency of theinsertion of a fragment with a selection maker was high (~100%), whereas that of theremoval of a selection marker was low in BAC cloning or targeted insertion modification.
The major cause of this phenomenon is considered to be mutations to cI gene, which
increase the number of Nm-resistant false-positive colonies. These clones are resistant toboth Nm and Spc. I also observed another false-positive colonies, which clones areresistant to both Nm and Spc. This is likely to be caused by an accidentally deletion of
genomic region including a selection marker cassette cI-spc. This unwanted recombination
may result from an unexpected recombination between neighbor regions of the selection
marker in B. subtilis genome or mouse genomic sequences (Bird et al., 2011). Because thecurrent BGM vector uses the proB locus for cloning, the change of the cloning site toalternative locus, such as leuB may increase cloning efficiencies of counter-selection steps.
But it is note that the efficiency of counter-selection using the BGM vector in this study iscomparable in the BAC system (Zhang et al., 1998; Bird et al., 2011).

40

A Transfer BAC clone to BGM vector
BAC

I

I

I

I
cI-spc

Nm: R
Spc: S

Pr-neocI-spcPr-neoBGM clone

BGM vector

B Modifications of genomic insert

Established
Insertion
I

I

I
G

BGM

Yes

This study

Yes

Yes

Phl: R
Em: R
Tet: R

No

This study

Nm: S
Spc: R
Bs: R

Yes

Yes

Yes

This study

I
G

G

Nm: S
Spc: RPr-neoG cI-spc

BAC / YAC

Nm: R
Spc: S

Pr-neocI-spcDeletion
I

I

I

I

cI-spc
Nm: S
Spc: RPr-neoPr-neoI

Inversion

I

I

I

erm

I

I

I

I

I

I

te
phl
et

Phl: R
Em: R

Pr-neoPr-neoFusion of two BAC clones
I

I

I

I

cI-spcI

Nm: S
Spc: RPr-neoPr-neoI

I

I

Pr-neocI-bsrPr-neoPr-neo

I

I

Nm: S
Bs: R

C Applications; Retrieval and Microinjection
I

I
G

I-PpoI

MicroinjectionG

Pr-neoTransgeneTransgenic mouse

Figure 2-1. Cloning and various modification methods of the BGM vector system.
(A) The cloning strategy used to introduce BAC inserts into the BGM vector. BGM vectors possess
two antibiotic gene cassettes: Pr-neo, a lambda Pr promoter fused to the neomycin resistance gene(neo), and cI-spc, which contains cI encoding the CI repressor protein, which binds to the Pr
promoter, fused to the spectinomycin resistance gene (spc). The cI-spc is flanked by two BAC
vector sequences that function as the cloning site for BAC inserts. The original BGM strains areresistant to Spc and sensitive to Nm. Once a BAC insert is cloned into the BGM vector, therecombinant becomes resistant to Nm and sensitive to Spc because the cI-spc cassette is replacedby the BAC insert. (B) Modifications of large DNA fragments. The BGM vector system allows
various manipulations, including insertion (e.g., EGFP is designated as “G” in the box), deletion andthe inversion and fusion of two overlapping clones. (C) The BGM inserts can be retrieved by several
methods, and the recombinant DNA fragments can be used for the generation of transgenic animals
by microinjection (described in Chapter III). The right column represents the summary ofachievements for each modification using the BAC, YAC and BGM vector systems. The closed
triangles represent a target gene and its direction. The BAC vector sequences are indicated by theclosed and open arrows. The half part of BAC vector containing chloramphenicol resistance genefor E. coli is depicted by open arrow (defined as right side). The I-PpoI sites, designated as “I” areintroduced to flank the BAC vector sequences.

41

Chr.7

50 kb

BAC1 (RP24-392H7)~82 kb overlap

BAC2 (RP23-61O11)

Figure 2-2. Genomic structures of mouse chromosome 7 and the BAC clones used in thisstudy.
The BAC clones cover a portion of the class I OR locus on chromosome 7, share 82 kb overlapping
region and are oriented in opposite directions in the BAC vector. A large triangle depicts MOR42-3,
black triangles indicate other OR genes, and the gray triangle represents the pseudo-OR gene
(Zhang et al., 2007).

42

A I-PpoI/CHEF
M

[kb]

#1

B I-PpoI/CHEF

Southern blot
EcoRI
[kb]
23

M BAC1 #1

Southern blot

HindIII

EcoRI

BAC1 #1

M #28

HindIII[kb] M BAC2 #28 BAC2 #28
23

9.4

9.4

6.6

6.6
4.4

4.4
[kb]

194

242
145
2.3
97

2.0

194

2.3

145

2.0

97
48

48

Figure 2-3. Cloning of BAC inserts into the BGM vector.
The cloning of BAC1 and BAC2 into the BGM vector was confirmed by I-PpoI/CHEF (left panels in A
and B) and Southern blotting (right panels in A and B). The representative BGM vectors withtransferred BAC1 and BAC2 inserts were digested with I-PpoI and resolved by CHEF. The open
arrowheads indicate BAC insert bands, and the closed arrowheads indicate the BGM vector.
Purified original BAC clones and genomic DNA from representative BGM clones were digested with
EcoRI or HindIII and hybridized with the original BAC clones as probes. The BGM recombinants
showed a band pattern identical to the original BAC clones (lane: BAC), except for the bands
corresponding to the BAC end sequences from EcoRI-digested BAC1; the closed arrowhead
indicates a BAC-specific signal, and the open arrowheads indicate BGM-specific signals from
EcoRI-digested BAC1. In other digestion patterns, the bands of the BAC end sequences wereoverlapping and indistinguishable from insert signals predicted from restriction maps of BAC clones
and the BGM vector. Numbers above lanes are the BGM clone numbers. In lane M, lambda/HindIII
fragments or a lambda DNA concatemer was used as a size marker.

43

A BGM/BAC
B

B

Spc: S
Nm: R

OR

MOR42-3 locus

L arm

cI-spc

IRES-tauEGFP

R arm

1st step

BGM/Tg-CISP

B

B

OR

B

IRES-tauEGFP

cI-spcSpc: R
Nm: S

IRES-tauEGFP

2nd step

BGM/Tg

B

B

OR

B

Probe (in C)

Probe (in B)

B
[kb]
23

M

Spc: S
Nm: R

IRES-tauEGFP

C

P
P
IS
IS
0C 20
0C 10
2
2
1
1
C
C
-2
-1
-1
-2
BA
Tg
BA
Tg
Tg
Tg

[kb]

Predicted
sizes [kb]

23

9.4
6.6
4.4

9.4
6.1 (Tg-CISP, Tg)
5.6 (BAC)

M

BA

C1

P
P
IS
IS
0C 20
0C 10
2
2
1
C
-2
-1
-1
-2
BA
Tg
Tg
Tg
Tg
Predicted
sizes [kb]6.6
5.6 (BAC)
4.4

4.5 (Tg-CISP)

2.3

2.3

2.0

2.0

2.0 (Tg)

Figure 2-4. Targeted insertion of a reporter gene.
(A) In the first step, IRES-tauEGFP was inserted into the targeted site 3 bp downstream of the
MOR42-3 stop codon by transformation with a purified DNA fragment consisting of L and R arms
(1.0 kb each) homologous to MOR42-3 and the cI-spc selection cassette. In the second step, theselection cassette was removed by transformation with a fragment lacking the selection marker. (B,C) The targeted insertion of the reporter gene was confirmed by Southern blot analysis using a
MOR42-3 coding region or R homology region as a probe. The genomic DNA of each BGM clone
was digested with BamHI and resolved by CHEF, and the changes in the sizes of the BamHI
fragments were as expected, indicating the successful targeted insertion of IRES-tauEGFP. The
sensitivity to antibiotics is shown. A bold arrowhead in panel B represents bands corresponding to
the cross-hybridized MOR42-1 coding region, which sequence is contained in BAC2 but not in
BAC1. R, resistant; S, sensitive. The BamHI sites are represented by “B”. Lane M, lambda/HindIII
fragments were used as a size marker.

44

A
BGM/BACOR

Spc: S Nm: R

BGM/Tg-CISP

F4 F3

OR

IRES-tauEGFP

F1

IRES-tauEGFP

Spc: S Nm: R
R2

R1

R4

OR ORF (F1/R1): 900 bp
C1 -1
BA Tg

GFP (F2/R2): 240 bp

SP
CI 0
0
2
2
2
-2
-1 AC g-2
B
Tg
T
Tg
P
IS

C
10

M

R4

F2 F4
OR

[kb]
1.0

Spc: R Nm: S
R3

BGM/TgB

cI-spc

10

C1

BA

P
P
IS
IS
0C 20
0C 10
2
2
1
-2
-1 AC g-2
-1
B
Tg M
T
Tg
Tg

0.5

1.0
0.5

CISP marker (F3/R3): 2.4 kb

M

BA

C1

GFP_R arm (F4/R4): 3.5 kb or 1.1 kb
P
P
IS
IS
0C 20
0C 10
2
2
1
1
C
-2
-1 AC g-2
-1
B
Tg M
T
Tg
BA Tg

P
P
IS
IS
0C 20
0C 10
2
2
1
-2
-1 AC g-2
-1
B
Tg
T
Tg
Tg

3.0

3.0

2.0

2.0

1.0

1.0

Figure 2-5. Verification of each targeted insertion step by PCR.
(A) Primer binding positions to verify recombinant BGM clones are as indicated. Primer sets wereused as follows; F1/R1 for MOR42-3 coding sequence, F2/R2 for the 3´ side of EGFP, F3/R3 for thecI-spc cassette and F4/R4 for the region between inside EGFP to the 3´ end of the R. (B) PCR
analyses of each insertion step confirm the insertion and removal of the reporter and marker genes
in the Tg-110 and Tg-220 constructs. Representative results of BGM/BAC clones (BAC1 and BAC2),
BGM/TgCISP clones (Tg-110CISP and Tg-220CISP) and BGM/Tg clones (Tg-110 and Tg-220) are
shown, and verifying the correct targeted insertion of the reporter cassette. A F4/R4 primer set wasused to screen of Nm-resistant and Spc-sensitive clones in removal step of a cI-spc cassette.

45

A Tg-110

Phl: S
Em: S
Tet: S

OR
G

erm

te

et

B
[kb]

phl

6.6
phl

G

B

B

B

B

2.8 kbPhl: R
Em: R
Tet: S

1-1

Predicted
sizes [kb]4.3 (tet)

5.0 kb

3.4 (e)phl

G

2.8 (te)erm

Tg-110-Inverted

2.3

tet

G
B

4.3 kbTgrve

5.0 (et)
4.4

Inversion

B

10
-1
g
T

d
te

In
0-

9.4

Tg-110 T/P
erm

M

P
T/

erm

e

phl

B

B

Phl: R
Em: R
Tet: R

2.0

3.4 kbFigure 2-6. Inversion of the Tg-110 insert.
(A) Schematic diagram of inversion strategy. Two incomplete fragments of the tetracycline
resistance gene (tet), te (5’ side) and et (3’ side), which contained an overlapping region e, wereused for inversion. The inversion cassettes, phl-et, consisting of et and the phleomycin resistancegene (phl), and te-erm, consisting of te and the erythromycin resistance gene (erm), weresequentially inserted into the ends of the Tg-110 insert. A 0.9 kb fragment in the left end and a 0.8
kb fragment in the right end were used as homology arms. Homologous recombination between teand et in Tg-110 T/P results in the inversion of the insert to form an intact tet gene. The about
positions of BamHI sites are represented by “B”. (B) Inversion was confirmed by Southern blotting
using the tet gene as a probe. The genomic DNA of the represented clones was digested with
BamHI. Changes in the size of tet cassette fragments represented inversion by intrachromosomal
homologous recombination. The open (2.8 kb te-erm cassette and 2.3 kb phl-et plus 2.7 kb Tg-110
fragment) and closed (3.4 kb erm-e-phl cassette and 1.6 kb tet plus 2.7 kb Tg-110 fragment)arrowheads indicate signals before and after inversion, respectively.

46

A

Tg-110-Inverted
I

I

I
G

Tg-220

I

erm

B

C

phl
cB

I

I

Phl: R
Em: R
Nm: R
Bs: S

G

Spc: S
Nm: R
cS

I

I
G

Tg-220CISP
cS

cB

Overlap(~82 kb)

Genetic fusion

Spc: R
I
Nm: S

242
194
145

I
G

291

2.3
2.0

97

I
cS

23
9.4
6.6
4.4

Phl: S
Em: S
Nm: S
Bs: R

G

I

Tg-250SB

S SP
IB
I
B
0C 20C 50S
1
1
2
2
Tg Tg Tg[kb]

Tg-110CIBS
I

M

S P
IB IS B
0C 20C 50S
1-1 -2 -2
Tg Tg Tg[kb] McB

Spc: R
Bs: R
Nm: S

48

Figure 2-7. The genetic fusion of the inverted Tg-110 and Tg-220 fragments to reconstruct
the genomic structure of the MOR42-3 locus.
(A) Schematic diagram of fusion of Tg-110-Inverted and Tg-220 fragments. The selection marker
cassettes cI-bsr, consisting of the cI and blasticidin S resistance genes (depicted as cB), and cI-spc(depicted as cS) were inserted to the right end of the inverted Tg-110 and to the left end of Tg-220,
respectively. 1.3 kb fragments in the left end of Tg-220 and in the right end of Tg-110-Inverted wereused as homology arms. Genomic DNA was isolated from the bsr-labeled Tg-110 clone(Tg-110CIBS) and added to the spc-labeled Tg-220 competent cells (Tg-220CISP). The Tg-220
insert was extended to 252 kb by homologous recombination at the overlap region. (B) The fusedTg-250SB insert was evaluated by I-PpoI digestion followed by CHEF. Tg-250SB shows a larger
band that corresponds to the fused insert fragment. The closed and open arrowheads represent
bands from the 4.2 Mb BGM vector and inserts, respectively. (C) The structure of the Tg-250SB
insert was confirmed by Southern blot analysis. Genomic DNA was digested with EcoRI andhybridized with Tg-110 and Tg-220 probes. The genetic fusion of the two inserts is shown as thesum of the respective Tg-250SB bands. The I-PpoI sites are represented by “I”. Lane M,
lambda/HindIII fragments or a concatemer of lambda DNA was used as a size marker.

47

Table 2-2. Summary of the read mapping in DNA sequencing.
BAC1# reads

BAC2%# reads

Tg110CIBS%

220,003# reads

Tg220CISP%

115,372# reads

Tg250SB%

222,532# reads%

Total reference length

114,183

254,658

Mapped reads

8,341,236

91.1

8,604,424

95.9

1,028,479

2.7

1,737,127

5.2

504,510

5.8

Unmapped reads

814,8248.9

370,790

4.1

37,166,207

97.3

31,564,383

94.8

8,227,736

94.2

Reads in pairs

8,327,110

91.0

8,594,722

95.8

960,578

2.5

1,714,062

5.2

498,152

5.7

Broken paired reads

14,126

0.2

9,702

0.1

67,901

0.2

23,065

0.1

6,358

0.1

Total number of reads

9,156,060

8,975,214

38,194,686

33,301,510

8,732,246

Total read length (Mb)

915.6

897.5

3,819.5

3,330.2

873.2

Fraction of reference covered

1.00

1.00

1.00

1.00

1.00

Minimum coverage

241

125

28

22

6

Maximum coverage

15147

5631

2211

1514

574

Average coverage

7100.73812.3

851.5

752.2

191.0

Reads obtained from the Illumina Genome Analyzer IIx were analyzed and mapped to eachreference sequence using CLC Genomics Workbench 5.5 (CLC Bio). The number of mapped reads
in each sample was enough to evaluate mutations. Analyzed DNA samples are follows: original
BAC clones of BAC1 and BAC2 and BGM clones of Tg110CIBS, Tg220CISP and Tg250SB.

48

Table 2-3. Cloning efficiency of BAC inserts into the BGM vector.
BAC #clone ID

BAC1
BAC2
BAC3
BAC5
BAC7
BAC8
BAC9
BAC10
BAC11
BAC14

RP24-392H7
RP23-61O11
RP24-306C18
RP24-282D19
RP23-310P1
RP23-480A17
RP24-211E6
RP24-419H14
RP23-347I6
RP24-266J11

insert (kb) BGM vector
114
220
149
165
165
177
159
141
202
163

BEST310
BEST6528
BEST6528
BEST6528
BEST6528
BEST6528
BEST6528
BEST310
BEST6528
BEST6528

total tested clones
insert positive cloning efficiency (%)(Nm-resistant, Spc-sensitive)
7
3
43%
29
1
3%
3
3
100%
15
6
40%
32
3
9%
10
3
30%
4
4
100%
10
2
20%
25
7
28%
24
6
25%
Average40%

Efficiency of cloning of BAC clones into the BGM vector are summarized; clone names and ID,
length of BAC inserts, BGM vector used for cloning and the numbers of tested and insert-positive
clones.

49

CHAPTERIII

THE BGM VECTOR-BASED MOUSE
TRANSGENESIS

50

ⅠII - 1

Introduction

The BAC and YAC systems have been recognized as powerful tools for functional
analysis of genomic DNA fragments in combination with transgenesis, and they havecontributed greatly to life science researches, including functional analyses of genomic
DNA, production of recombinant proteins and studies on genetic diseases (Schedl et al.,
1993; Mendez et al., 1997; Yang et al., 1997; Giraldo and Montoliu, 2001).
The BGM vector is now available for cloning and manipulation of various DNA
fragments including cyanobacteria genome, mouse mitochondrial genome and rice
chloroplast genome DNA (Itaya et al., 2005; Itaya et al., 2008). Although some retrieve
methods for BGM inserts were previously proposed (Itaya et al., 2008; Kaneko et al.,2009), the BGM system has not been applied to transgenesis in any species. Because ofmany advantages such as the Mb cloning capacity and amenability of modifications, theestablishment of the BGM vector-based transgenesis (BGM transgenesis) must provide a
new approach in various research fields. In this study, I established the method to generatetransgenic mice using the BGM vector system.

51

ⅠII - 2

Experimental proceduresIII-2-1) Preparation of the transgenes for pronuclear injection
Genomic DNA carrying the transgene in an agarose plug was prepared, digested with
I-PpoI and resolved by CHEF in a 1% (w/v) agarose gel using sterile 0.5× TBE, asperformed for the I-PpoI analysis. To concentrate the transgene fragment, the band was
excised from the agarose gel, turned vertically and embedded in 4% (w/v) agarose;electrophoresis was then performed at 3.3 V/cm for more than 10 hours in 0.5× TBE. The
excised concentrated transgene band was placed in a prepared dialysis tube hydrated with
0.5× TBE, and the transgene was electroeluted from the gel under the same conditions. The
eluate was dialyzed with a high salt injection buffer (10 mM Tris-HCl (pH 8.0), 0.1 mM
EDTA, 100 mM NaCl) at 4-6°C overnight. The concentration and integrity were estimatedbased on the control band intensity in the CHEF analysis. The purified transgenes wereused immediately. Alternatively, 75 μM spermidine and 30 μM spermine were added forlong-term storage. The stock transgenes were diluted to 0.3-1.5 ng/μl with injection buffer
before use.	
 III-2-2) Production and analyses of transgenic mice
The purified transgenes were microinjected into the pronucleus of B6C3F1(C3H/HeSlc male × C57BL/6NCrSlc female) mouse zygotes in a standard protocol(Sumiyama et al., 2010). Olympus IX-71 microscope equipped with the micromanipulatortransgenic system (Narishige) and Eppendorf FemtoJet system were used for
microinjection. Injected eggs were transferred to the oviducts of pseudopregnant female
ICRs. The founders were screened by PCR with the following three primer sets that52

specifically amplified the internal and left and right ends of the transgenes: EGFP: GFP-F
and GFP-R; the left end of the BAC sequence: SacB-F and SacB-R, or BAC108L-F and
BAC108L-R; and for the right end of the BAC sequence: CmR-F and CmR-R (Table 3-1).
All founder mice positive for these three primer sets were selected as candidates containing
intact transgenes. The founder mice were crossed with C57BL/6 mice. The transgenic mice
were dissected and fixed in 4% paraformaldehyde (PFA)/PBS for 10-15 min on ice. Line
#8 of two EGFP-expressing Tg-250SB transgenic lines was used for the expression
analyses in Figure 3-5.

III-2-3) Southern blot analysis of transgenic mice
10 µg of genomic DNA extracted from a tail was digested with EcoRI (TaKaRa) andseparated on a 1% agarose gel. The DNA was transferred onto a membrane filter, andhybridized with a DIG-labeled MOR42-3 coding probe (nucleotides 190 – 1091 from
NM_020289). The signals were detected by chemiluminescence (CSPD, Roche) using a
CCD camera (ChemiDocTM XRS, Bio-Rad). The copy numbers were estimated bycomparing the intensities of transgenic and endogenous signals.

III-2-4) Immunohistochemistry (IHC)
Mice were fixed by perfusion with 4% PFA/PBS and infiltration in the same fixative
solution at 4°C for 30 min. After cryoprotection with 15% and 30% sucrose/PBS, tissues
were embedded in Frozen Section Compound (Surgipath FSC22, Leica microsystems).
Serial cryosections (20 µm) were collected on MAS-coated microscope glass slides(Matsunami) and dried for 1 h at room temperature. IHC was performed by a standard

53

protocol (Enomoto et al., 2011). After post-fixation, permeabilization and antigen-retrieval
pretreatment, sections were blocked with 10% normal horse serum and incubated withprimary antibody at 4°C overnight. The following primary antibodies and dilutions wereused: rat anti-GFP (1:2000, catalog #04404-84, Nacalai) and goat anti-OMP (1:5000,
catalog #544-10001, Wako). After incubation of the primary antibodies, sections were
washed in PBS containing 0.01% Tween 20 and stained by the following Alexa
Fluor-conjugated secondary antibodies (1:500, Invitrogen): Alexa Fluor 488-conjugateddonkey anti-rat IgG and Alexa Fluor 546-conjugated donkey anti-goat IgG. Nuclear
staining was performed with DAPI (1:1000, Vector Laboratories), and CC/Mount(Diagnostic BioSystems) was used for mounting.
Fluorescent images of endogenous GFP and IHC signals were taken with Olympus
SZX10 fluorescent stereomicroscope with a DP71 digital CCD camera and Leica SPE
confocal microscope. Confocal images were collected as z-stacks and projected into asingle image for display. Images were analyzed and adjusted using Photoshop CS4(Adobe). All of the mouse studies were approved by the Institutional Animal ExperimentCommittee of the Tokyo Institute of Technology and performed in accordance withinstitutional and governmental guidelines.

54

Table 3-1. Primer sequences and PCR conditions used in Chapter III
Sequence

Annealing temp
Purpose
PCR condition
Extension time
62 °C
genotype
Genotyping
30 secEGFP (intermediate of transgenes)

Primer name

Strand

GFP-F
GFP-R3Forward GGCATCAAGGTGAACTTCAAGATCC
Reverse CTTTACTTGTACAGCTCGTCCATGC

J619_SacB_F
J620_SacB_R

Forward GCTGAATACAACGGCTATCACG
Reverse TCTCTCAGCGTATGGTTGTCG

62 °C
30 secgenotype
Left end of Tg-110

Genotyping

J621_CmR_F
J622_CmR_R

Forward GAGGCATTTCAGTCAGTTGCTC
Reverse CGGCATGATGAACCTGAATCG

62 °C
30 secgenotype
Right end of Tg-250SB and Tg-110

Genotyping

J709_BAC108L_F
J710_BAC108L_R

Forward CGTATTCAGTGTCGCTGATTTG
Reverse TTAGCGATGAGCTCGGACTTC

60 °C
30 secgenotype
Left end of Tg-250SB

Genotyping

Genotyping
94 °C 5 min[30 cycles]
94 °C 30 sec(Annealing temp.)* 30 sec
72 °C (Extension time)*
72 °C 5 min

Primers and PCR conditions used for genotyping Tg-110 and Tg-250SB transgenic mice aresummarized. PCR with a recombinant Taq DNA polymerase was performed according to thecondition described under the Table.

55

ⅠII - 3

Results

III-3-1) Generation of the BGM transgenic mice
In contrast to BAC and YAC systems, the BGM vector system utilizes the entire
endogenous genome as a vector. Therefore, the transgene can be obtained by digesting the
genome with I-PpoI, whose recognition sites are introduced to flank the BAC vector
sequence (in Chapter II, Figure 2-1). But its concentration is expected to be low because of
the single copy of the transgene per genome/cell. Thus, I prepared linearized transgenes by
excision from the BGM vector using I-PpoI digestion and CHEF and concentrated thetransgenes by conventional electrophoresis followed by electroelution and dialysis with ahigh-salt injection buffer (Figure 3-1). Using the method established in this study,
linearized microinjection-grade transgenes were readily prepared from the BGM clones.
The purified transgenes were microinjected into fertilized mouse eggs to obtain foundertransgenic mice. Newborn mice were genotyped by PCR to amplify transgene-specificsequences. I designed three primer sets to amplify BAC vector sequences (left and rightend of the transgene) and EGFP (middle of the transgene) to assess the integration of thetransgene into chromosome (Figure 3-2). Assuming that all three PCR positive mouse
would carry an intact transgene, I obtained six Tg-110 founders from 45 newborn mice.
Partial integrations of the transgene were also observed in 2 mice, which were PCR
positive for the left end of the transgene only. For Tg-250SB, four of 26 pups carried anintact transgene, and one lacked the left end sequence (Table 3-2). These transgenic ratios
are comparable to a conventional BAC transgenesis (Yang et al., 1997). Germline
transmission of the transgenes was obtained in 5 transgenic founders for Tg-110 (from 6
founders) and two for Tg-250SB (from 4 founders).

56III-3-2) Experimental evidence of a cis-acting element for a class I OR gene
An individual olfactory sensory neuron (OSN) expresses only one OR gene in a
monoallelic and mutually exclusive manner (Chess et al., 1994). Class I ORs are expressedalmost exclusively by OSNs in the dorsal main olfactory epithelium (MOE), and these
OSNs project their axons to a specific subset of glomeruli in the dorsal domain of theolfactory bulb (OB) (Tsuboi et al., 2006; Bozza et al., 2009). The transgenes of the
MOR42-3 locus are designed to activate the bicistronic expression of MOR42-3 andtauEGFP, a fusion of the microtubule-associated protein tau with EGFP, to visualize the
MOR42-3 transgene expression and OSN axonal projections. Thus, if the transgenes
contain a MOR42-3 cis-acting element, MOR42-3 expression can be monitored by EGFP
fluorescence. For Tg-110, I analyzed 5 transgenic lines and 1 founder. None of the Tg-110
transgenic mice displayed labeled OSNs. For Tg-250SB, two transgenic lines establishedfrom two founders were EGFP positive (Figure 3-3), indicating expression of MOR42-3
transgene. Remaining two founders were EGFP negative. Whole-mount images of the
MOE and the OB of Tg-250SB showed a punctate EGFP expression pattern within thedorsal region of the MOE and axonal projections into the dorsomedial and dorsolateralglomerulus of each OB (Figure 3-3B and 3-5A). To assess the copy number and integrityof the transgene, I analyzed Tg-250SB by Southern blot using MOR42-3 coding region as aprobe (Figure 3-4), which cross-hybridizes to highly homologous gene MOR42-1 (97%
identical). Southern blot analysis showed that the specific band corresponding to thetransgene was observed in expected size and the bands corresponding to endogenous
MOR42-3 were the same between control and Tg-250SB, indicating that the intact
transgenes were integrated into chromosome, and this is not due to a gene targeting event.

57

Ratio of signal intensities (transgene / endogenous gene) were 1.05 for line #5 (n=1) and
0.46 ± 0.02 for line #8 (n = 4), indicating the copy number of transgene of two and one forline#5 and line#8, respectively. Because the Tg-250SB transgene carries MOR42-1,
intensities of the band corresponding to MOR42-1 increased 1.61 ± 0.18 fold in Tg-250SB
line #8 with reference to the control, confirming one copy of the transgene was integrated.
OSNs expressing highly homologous ORs tend to project their axons to near but
distinct subsets of glomeruli in the OB (Feinstein and Mombaerts, 2004). Because
MOR42-3 and MOR42-1, members of the MOR42 subfamily, share 97% amino acidsimilarity in their coding sequences, the axonal projection site of MOR42-3-expressing
OSNs was expected to be close to that of MOR42-1. Using axonal projection of OSNs
expressing endogenous MOR42-1 as control, we examined the MOR42-1-iTG
gene-targeted mouse (Bozza et al., 2009) in which an IRES-tauEGFP reporter was inserted
downstreamof

MOR42-1.

The

glomerular

positionsoftransgenic-MOR42-3(Tg-MOR42-3) in Tg-250SB transgenic mice were similar to those of endogenous
MOR42-1 (Figure 3-3B, 3-5A). I further analyzed the detailed structure of the
Tg-MOR42-3 glomeruli. Glomeruli are innervated exclusively by axons from OSNs
expressing

the

same

OR

(Treloaretal.,2002).

I

performed

double-label

immunohistochemistry on coronal cryosections with antibodies against olfactory marker
protein (OMP) to stain all mature OSN and antibodies against EGFP to label MOR42-3
transgene- or MOR42-1 endogenous gene-expressing OSNs. Tg-MOR42-3 glomeruli
contained a mixture of EGFP-negative and EGFP-positive axons, whereas MOR42-1
glomeruli of homozygous MOR42-1-iTG mice were completely EGFP positive (Figure
3-5B), suggesting that axons of OSNs expressing Tg-MOR42-3 formed glomeruli together

58with those of OSNs expressing endogenous MOR42-3 (Vassalli et al., 2002). The
projection site and converged glomerular structure of OSNs expressing the EGFP reportersuggest proper expression of the MOR42-3 transgene. Taken together, these results
indicate that a cis-acting element for MOR42-3 is present in the extended region fromTg-110 and provide the first experimental evidence of a cis-acting element for a class I OR
gene.

59

ⅠII - 4

Discussion

In this chapter, I established the BGM transgenesis using the transgenes, Tg-110 andTg-250SB constructed in Chapter II. This is the first time that modified BGM inserts weretransferred into a mammalian genome. The purified DNA from BGM vector can be
introduced into not only mouse but also other species. Using the BGM transgenic mice, I
provided the first evidence of a cis-element for a class I OR gene.
The transgenic efficiency (the ratio of the number of transgene-positive pups in total
number of pups) was comparable previous BAC transgenesis methods (Yang et al., 1997).
The BGM vectors used in this study are derived from the restriction modification-deficientstrain RM125 (Uozumi et al., 1977; Kaneko et al., 2005). Thus, it is conceivable that thedifference of host cells between B. subtilis and E. coli does not affect the transgenic
efficiency.
In the YAC system, a similar pronuclear microinjection method succeeded in a
generation of~460 kb YAC transgenic mouse (Serizawa et al., 2000). Thus, it is possible

to speculate that my protocols can be applied to several hundreds kb transgene. However,as a long DNA molecule is easy to shear, the upper limit of size for microinjection is
expected to be approximately 500-800 kb (Peterson, 2007; Li and Blankenstein, 2013). In
addition, the purification of an intact long DNA fragment is extremely difficult. Thus
generation of transgenic animals carrying ~1 Mb transgene must require different
approaches for DNA introduction such as lipofection of purified transgene into embryonicstem cells following chimera mouse production (Lamb et al., 1999) or sperm-mediatedtransfer methods (Garrels et al., 2012).

60

A

Agarose(plugs((BGM(carrying(transgene)(
I7PpoI(diges:on,(Separa:on(by(CHEF((
Cut(out(transgene(band(
Concentra:on(by(second+dimension+electrophoresis+(2D6E.P.)((
Cut(out(transgene(band(
Electroelu:on(in(dialysis(bag(by(E.P.+
Dialysis(with(injec:on7buﬀer(
BGM+transgene+(linear+fragment)+

B

C

Separa:on((CHEF)((Electric(
ﬁeld(

Concentra:on((2D6E.P.)(

B(
T(

1%(Agarose/0.5xTBE(

D

4%(Agarose/0.5xTBE(

E

Elu:on((E.P.)(B
0S 0
25 -11
M Tg Tg[kb]

Dialysis(tube((in(0.5xTBE)(

291
242
194
145
97
48

Figure 3-1. Preparation of transgenes from BGM clones.
(A) Procedures of the preparation method of transgene for pronuclear-microinjection for the BGMtransgenesis. Linearized transgene is isolated from agarose plugs in 4-steps. First, the transgene isseparated from the BGM vector (band B) by CHEF electrophoresis of I-PpoI digested agarose plugs(illustrated in B). Second, the transgene band (band T) is excised, and the agarose strip is placed
vertically and embedded in a high-percentage (4%) agarose gel. The transgene is concentrated bya second dimension (2D) electrophoresis (illustrated in C). Third, the concentrated transgene bandis excised and placed into a dialysis bag with electrophoresis buffer. By electrophoresis, thetransgene is eluted from a 4% agarose plug (illustrated in D). Dotted or solid bands represent
positions of DNA bands before or after electrophoresis. Finally, the transgene solution in the dialysis
bag is dialyzed with a high-salt injection buffer. (E) Quality and yield of the purified transgene formicroinjection are confirmed by CHEF. The concentration of retrieved transgenes ranged from 0.3
to 3 ng/µl.

61

A
I.PpoI

mouse&genomic&sequence&

L&

CmR

L.end&(SacB&or&BAC108L)&

EGFP(
||

IRES

tau EGFP

R&

I.PpoI

R.end&(CmR)&

inside&(EGFP)&

B

Pups&#&[kb]&

L.end:&SacB&P N 1 2 3& 4& 5& 6& 7& 8& 9& 10& 11&

1.0&.&
0.5&.
0.1&.&

1.0&.&

inside:&EGFP& 0.5&.
0.1&.&

1.0&.&

R.end:&CmR&

0.5&.
0.1&.&

Figure 3-2. Screening of founder mice.
(A) Schematic representation of transgenic construct and targets for genotyping PCR. To assess
the integration of the transgene, three primer sets are used; SacB or BAC108L vector sequence forleft end, EGFP for middle, CmR sequence for right end of the transgene. (B) A representative resultof genotyping of newborn mice for Tg-110 founder. Mouse#10 was all three PCR positive, would
carry an intact transgene. In contrast, Mouse#7 was PCR positive for the left end only, indicating apartial integration of the transgene.

62

Figure 3-3. Expression of the EGFP reporter gene in transgenic mice.
(A) Schematic diagram of Tg-110 and Tg-250SB. Tg-250SB transgenic mice express the EGFP
reporter gene, whereas Tg-110 transgenic mice do not. The number of EGFP-positive independent
founders and lines among the total analyzed is shown in parentheses. (B) Whole-mount images ofthe medial aspect of the OB in transgenic Tg-110 (line #10, left panel) and Tg-250SB mice (line #8,
medial panel) and the MOR42-1-iTG (Bozza et al. 2009) gene-targeted mouse (right panel). Tg-110
transgenic mice do not show EGFP fluorescence, whereas Tg-250SB transgenic mice do, allowing
the visualization of axonal projections of transgene-expressing OSNs to the OB. EGFP-labeledaxons in Tg-250SB converge on the dorsal side of the OB and form a glomerulus. Arrowheads
indicate glomeruli. D, dorsal; V, ventral; A, anterior; P, posterior.

63

[kb] M
23
9.4
6.6

SB
l
ro 250tn Co Tg

Tg

#5
ne
i
L
SB

[kb] M
23
9.4
6.6

Tg

#8
ne
i
L

l
0
tro -25nog
C T

MOR42-1(13.6 kb)

4.4
4.4
2.3
2.0

Endo
(2.0 kb)

2.3

Tg(1.2 kb)

2.0

0.6

Figure 3-4. Southern blot analysis of two Tg-250SB lines.
Southern blot analysis of transgenic mouse of Tg-250SB line #8 and littermate control mouse. The
MOR42-3 probe detected the endogenous (2.0 kb, open arrowhead) and the transgenic (1.2 kb,
closed arrowhead) MOR42-3 genes, and also detect both endogenous and transgenic MOR42-1
gene (13.6 kb, arrow) due to cross-hybridization. By comparing the intensities of the hybridized
signals, the copy numbers of the transgene in Tg-250SB line #5 and #8 was estimated to be two
and one, respectively.

64

Figure 3-5. Analysis of the EGFP-expression pattern in Tg-250SB.
(A) Confocal images of Tg-250SB line #8 transgenic and MOR42-1-iTG gene-targeted mice (Bozzaet al. 2009). Mosaic patterns of EGFP-expressing OSNs in the dorsal MOE (medial view, left panel)and glomeruli formed by axons in the OB (dorsal view, right panel) were observed, indicating thefunctional expression of transgenic MOR42-3. (B) Tg-MOR42-3 (top panel) or endogenousMOR42-1 glomeruli (bottom panel) were detected by immunostaining coronal cryosections. Red,
OMP immunoreactivity, which designates olfactory sensory axons. Green, EGFP immunoreactivity,
which designates Tg-MOR42-3 or endogenous-MOR42-1 axons. Blue, DAPI nuclear staining,which marks the glomeruli structure. The glomeruli in the Tg-250SB transgenic mouse showed anintermingling of red (EGFP-negative) and yellow (EGFP-positive) axons, whereas the glomeruli inthe homozygous MOR42-1-iTG mouse were fully doubly labeled. Scale bars: A, 500 µm; B, 30 µm.

65Table 3-2. Summary of screening of Tg-110 and Tg-250SB founder mice.
Marker&genes&!EGFPSacBCmR!

Germline&transmission&(Tg+&/&pups)&

Tg#110&Founder

Sex

#&7&



#

+

#

N.D.

#&10&



+

+

+

2&/&9

#&34



+

+

+

6&/&8

#&35



#

+

#

N.D.

#&37



+

+

+

0&/16

#&41



+

+

+

1&/&3

#&44



+

+

+

4&/&9

#&45



+

+

+

5&/&17

Tg#250SB&Founder SexMarker&genes&&EGFPBAC108LCmR!

Germline&transmission&(Tg+&/&pups)&

#1



+&

#&

+&

N.D.&

#&3



+&

+&

+&

0&/&31&

#&5



+&

+&

+&

3&/&20&

#&8



+&

+&

+&

9&/&18&

#&11



+&

+&

+&

0&/&26&

Results of the genotyping PCR and germline transmission of founder mice are summarized. Totally,
eight Tg-110 and five Tg-250SB transgenic founders were obtained from 45 and 26 newborn mice,
respectively; the efficiencies of founder in newborn mice were 18% for Tg-110 and 19% for
Tg-250SB. +, PCR-positive; -, PCR-negative; N.D., not determined.

66

  

CHAPTERIV

IDENTIFICATION AND FUNCTIONAL ANALYSIS
OF CIS-ELEMENT FOR A CLASS I ODORANT
RECEPTOR GENE

67

IV - 1Introduction

Cis-elements are regulatory sequences to control spatial and temporal gene
expression, and also provide important clues for exploration of the trans-elements. Several
regulatory regions including cis-elements have been identified for class II OR genes
expression (Vassalli et al., 2002; Serizawa et al., 2003; Rothman et al., 2005; Bozza et al.,2009). Some of these have been shown to activate single class II OR gene in proximal
position of each cis-element, others can select and activate one of class II OR genes among
the gene cluster in distal position (Vassalli et al., 2002; Serizawa et al., 2003; Rothman etal., 2005; Fuss et al., 2007; Nishizumi et al., 2007; Khan et al., 2011). Regarding to thetrans-elements for class II OR genes, some transcription factors have been identified,including Lhx2 (Hirota and Mombaerts, 2004) and Emx2 (McIntyre et al., 2008). The lossof function mutation in Lhx2 precludes expression of all tested class II OR genes. By
contrast most of class I OR genes are still expressed in Lhx2-deficient mutants, indicatinga different regulatory mechanism between two classes of OR genes (Hirota et al., 2007).
Loss of Emx2 decreases many class I and class II OR genes expression and increases someclass II OR genes expression, suggesting class-independent regulatory function of Emx2.
Whereas, the cis-elements and critical trans-elements for class I OR genes have not been
identified.
In the previous chapter, I demonstrated that the cis-element for the class I OR gene,
MOR42-3 exists in the Tg-220 mouse genomic region. Identification of cis-element forclass I OR genes will allow us to understand the regulatory mechanism of this gene family.
In this chapter, I identified the cis-element for MOR42-3 by a deletion mutagenesis of
Tg-220 in combination with bioinformatics analyses, and investigated its function.

68

ⅠV - 2

Experimental proceduresIV-2-1) Nucleotide identity plots
The nucleotide sequence of an approximately 220 kb mouse genomic region (BAC2:
RP23-61O11, Accession #: AC102535), in which the cis-element for the MOR42-3 geneexists, was compared with those of the class I OR cluster region in other species by using
the web-based VISTA program (http:// genome.lbl.gov/vista/mvista/submit.shtml). I used
the Shuffle-LAGAN for the alignment program and the following settings of VISTA
parameters; conservation identity: 70%; calculation window and minimal conserved width:
100 bp for mammals or 30 bp for other vertebrates. The 220 kb mouse genomic region
contains a part of class I OR gene cluster, and non-OR genes Rrm1 and Stim1. Because
class I OR gene cluster and non-OR genes compose a syntenic region among mammals, I
used approximately 2 Mb genomic sequences from Stim1 locus to the class I OR genecluster to analyze homology between mammalian species. For zebrafish, western clawedfrog and fugu, I used genomic regions or contigs containing orthologous genes of mouse
MOR42 subfamily (β group) up to 2 Mbp. I compared the following sequences; mouse(Mus musculus, mm9) chromosome 7: 109,558,891 - 109,778,893; human (Homo sapiens,
GRCh37) chromosome 11: 3,873,375 - 5,867,373; rhesus macaque (Macaca mulatta,
rheMac2) chromosome 14: 67,553,852 - 69,553,842; horse (Equus caballus, equCab2)
chromosome 7: 71,917,076 - 73,917,066; dog (Canis familiaris, canFam3) chromosome
21: 26,492,740 - 28,491,838; cow (Bos taurus, bosTau7) chromosome 15: 48,532,134 50,532,124; opossum (Monodelphis domestica, monDom5) chromosome 4: 348,999,211 350,999,201; zebrafish (Danio rerio, danRer7) chromosome 15: 4,465,100 - 6,464,551;
western clawed frog (Xenopus tropicalis, xenTro3) contig GL173612: 1 - 241,636; fugu

69

(Takifugu rubripes, fr3) contig HE593413: 1 - 9,721. Repetitive elements of the mousebase sequence were masked with RepeatMasker in VISTA server.

IV-2-2) Construction of a deletion series of Tg-220
To construct upstream deletion plasmids, I used a BAC plasmid p108BGMC(provided from Dr. S. Kaneko), which contains a cat gene for B. subtilis. 1.2 kb homology
arms homologous to -79, -29 or -16 kb position of Tg-220 were cloned into p108BGMC as
HindIII-BamHI fragments. To construct downstream deletion plasmid, the new BAC
plasmid p108Tet was generated by inserting a tet gene of a PstI-EcoRI fragment derived
from pBEST307 (Kuroki et al., 2008) into the PmlI site of p108IPpoI-HPNSB after
blunt-ending treatment. A 1.2 kb homology arm to +19 kb position of Tg-220 was cloned
into p108Tet as a HindIII fragment. All homology arms were amplified by PCR (KOD
plus DNA polymerase) using the BAC2 DNA as a template. Primer sequence and PCR
conditions are summarized in Table 4-1. All deletion plasmids were linearized with BamHI
or MluI, and used for transformation.
Transformation and the I-PpoI/CHEF analysis of the BGM clones were performed as
described in Chapter II. The transformants were screened with chloramphenicol (Cm, 5
μg/ml, Wako) or 10 μg/ml tetracycline. Electrophoresis was performed at 4 V/cm at 14°C
under the following conditions: 20 sec for 24 h (single deletion clones) and 12 sec for 21 h(double deletion clones).

IV-2-3) Characterization of the identified 13 kb region
To characterize the 13 kb region containing the cis-element for the MOR42-3 class I

70

OR

gene,

I

used

the

UCSC

GenomeBrowser

(UCSC

web

page,

http://genome.ucsc.edu/cgi-bin/hgGateway). I analyzed the 13 kb region locating thepositions of 109,653,017 - 109,666,061 on chromosome 7 (NCBI/mm9) with the deposited“Expression

andRegulation”

datasets

includingchromatin

immunoprecipitation

sequencing (ChIP-seq) results of CTCF (ENCODE mouse project, Mouse ENCODE
Consortium et al., 2012), and compared the information from mouse genome data with mynucleotide identity plot of human.

IV-2-4) Construction of a series of 13 kb reporter transgenes
To construct a vector plasmid, a linker containing EcoRI, PacI and AscI sites wasinserted in the SacI site of the PAKI3 plasmid, in which the KpnI site of the pBluescript II
SK(+) (Stratagene) was replaced with AscI and PmeI sites. Then, another linker containing
XbaI, SacI and SacII sites was inserted between the HindIII and BamHI sites. The
gapVenus-pA fragment (provided from Dr. Y. Yoshihara) was inserted into the
BamHI-PacI sites of the vector plasmid, and then the PCR-amplified 423p promoter
fragment was inserted into the XbaI-SacII sites to construct the 423pgVpA plasmid.
Following the reconstructed 13 kb region and its deletion fragments were inserted into thisplasmid to construct a series of reporter transgenes.
To clone the identified 13 kb region, the 2.4 kb 5´-end and the 0.9 kb 3´-end of the
13 kb region were subcloned into pBluescript II SK vector as SalI-BamHI and
BamHI-XbaI fragments, respectively. Both fragments have an endogenous PacI site. The
10 kb PacI fragment from BAC2 clone was inserted into the subcloned plasmid to generate
the pBS-13k plasmid, which contains a full-length 13 kb region as a SalI-XbaI fragment.

71

The 9.1 kb fragment was generated by deleting a SalI-PmlI fragment of the pBS-13k
plasmid. Similarly, the 7.9k and 3.9k fragments were generated by deleting SalI-NheI and
SalI-NcoI fragments of pBS-13k, respectively. The ΔCore fragment was generated by
self-ligating the vector part after NcoI digestion of the pBS-13k plasmid to remove the 3.8
kb NcoI fragment, designated as the Core (Nco) containing the Core region. The 13k, 9.1k,
7.9k, 3.9k and ΔCore fragments were transferred to the 423pgVpA plasmid as XhoI-XbaI
fragments to construct transgenes. The Core only transgene, the Core (Nco) wasconstructed by directly inserting the Nco fragment containing the Core region into the
XhoI and XbaI sites after blunt-ending. The 13k-423pgV, 9.1k-423pgV, Core(Nco)-423pgV and ΔCore-423pgV were transferred into a Tol2 donor plasmid, pTol2-EPA.
The vector was constructed by inserting a linker containing EcoRI, AscI and PacI sites
between SalI and BglII sites of the pT2 vector (provided from Dr. H. Nishihara). The 423
promoter, the 5´-end and 3´-end fragments of the 13 kb region were amplified by PCR(PrimeSTAR HS) using the BAC2 DNA as a template (Table 4-1).

IV-2-5) Generation and analysis of transgenic miceTransgenic mice carrying Tg-220 and its deletion mutants were generated as
described in Chapter III. The founder mice of Tg-220 were screened by PCR with the sameprimer sets for Tg-110. The same primer set for Tg-250SB was used of deletion series of
Tg-220 (Table 4-1).
For a series of 13 kb reporter transgenes, I took two microinjection methods of theconventional pronuclear microinjection and the Tol2-mediated cytoplasmic microinjection
method (Sumiyama et al., 2010). For 7.9k-423pgV and 3.9k-423pgV, the linear transgenes

72were excised from the vector plasmids by AscI digestion, and purified with the QIAquick
Gel Extraction Kit (QIAGEN), phenol-chloroform extraction and ethanol precipitation.
The purified transgenes were dialyzed with the TG buffer (5 mM Tris-HCl (pH 8.0), 0.1
mM EDTA) at 4-6°C overnight. The transgenes were diluted to 5 - 10 ng/μl with injection
buffer before use, and were microinjected into the pronuclei of fertilized eggs (pronuclear
microinjection). For high efficient transgenesis, I used the Tol2 cytoplasmic microinjection
method as described elsewhere (Sumiyama et al., 2010). I took this method to generate the
13k-423pgV, 9.1k-423pgV, Core (Nco)-423pgV and ΔCore-423pgV transgenic mice.
Briefly, the Tol2 transgene plasmids were purified by phenol-chloroform extraction andethanol precipitation, and then dissolved a TG buffer prepared with DEPC-treated water.
Tol2 transposase mRNA was synthesized in vitro using the NotI-linearized pCS-TP(Kawakami et al., 2004) with the mMESSAGE mMACHINE SP6 Kit (Ambion). DNA
solution containing 20 ng/μl circular Tol2-trangene plasmid and 25 ng/μl transposasemRNA was injected into the cytoplasm of fertilized eggs (cytoplasmic microinjection). In
both methods, the newborn mice were screened by PCR with a primer set for the Venus
gene (Table 4-1).
The founders and lines obtained by crossing with C57BL/6 mice were used foranalysis. The procedures of expression analysis of the Venus reporter are described in
Chapter III.

IV-2-6) Bioinformatics analysis of the Core region in other species
A genome alignment was performed using 59 vertebrate genomes provided fromUCSC regarding the mouse genome (Mus musculus, mm10, released in December 2011)73

as a reference. Two continuous regions in the mouse genome were picked up fromalignable regions between group β and α class I OR genes among mouse, human and dog,
and defined as core1 and core2. Then, I mapped the two core sequences to 13 placental
mammalian genomes. The class I OR gene cluster of cow was divided because ofincomplete assembly. Names of orthologous gene groups (OGGs) were used as describedin a previous report of the analysis of OR genes among 13 placental mammals (Niimura etal., 2014). Briefly, The OGG1-60 and OGG1-105 belong to group β class I OR genes(MOR42 family in mouse), whereas other OGGs including OGG1-47, OGG1-107 andOGG1-120 belong to group α class I OR genes.

74

Table 4-1. Primer sequences and PCR conditions used in Chapter IV.
Annealing temp
Extension time

Purpose

PCR condition

Forward CCCAAGCTTATCACCAGGGACAACTTGG
Reverse CGGGATCCAGAGGTGCAAGAAACTGAC

55.5 °C
1 min 20 sec

homology arm
to -79 kb of MOR42-3 (ΔTg-U80)

KOD plus

J633_B2Del2_F
J634_B2Del2_R

Forward CTCAAGCTTGTCTGCCCTCTCTGAC
Reverse CGGGATCCTTGACGGTGGCTCTGATTG

60.6 °C
1 min 20 sec

homology arm
to -16 kb of MOR42-3 (ΔTg-U20)

KOD plus

J635_ B2Del3_F
J636_ B2Del3_R

Forward CCCAAGCTTGGCTCGCTGAGACATTAGC
Reverse CCCAAGCTTCCATCTTCTGCTCCCTAGAC

61 °C
1 min 20 sec

homology arm
to +19 kb of MOR42-3 (ΔTg-140Δ3')

KOD plus

J675_B2Del4_F
J676_B2Del4_R

Forward GGCCATTCAAGCTTCTACTCTCTAC
Reverse CGGGATCCACTTCCATGACCTGAGTC

58 °C
1 min 20 sec

homology arm
to -29 kb of MOR42-3 (ΔTg-U30)

KOD plus

55 °C
2 min 30 sec

5'-region of 13 kb region
for reconstruction

PrimeSTAR HS

Primer name

StrandDeletion of Tg-220
J631_B2Del1_F
J632_B2Del1_R

Sequence

Short transgene for reporter assay
J734_13k_5F
Forward ACGCGTCGACTCAGTATCAGGCCATTC
J735_13k_5R
Reverse CGGGATCCTAGTTCAGCTGTCGCTTTCC
J736_13k_3FJ737_13k_3R

Forward CGGGATCCATAGATCCGCCTACTTCCAC
Reverse GCTCTAGAGAGGGCTCAACATTGCTTTG

57 °C
1 min

3'-region of 13 kb region
for reconstruction

PrimeSTAR HS

J738_423p-F
J739_423p-R

Forward GCTCTAGAGCACACCCCTTCAGCTCCTC
Reverse TCCCCGCGGCAGGACTCACACAGGCAGGG

62 °C
1 min

42-3 promoter region

PrimeSTAR HSPoly linkers
J740_Hind-XSS-Bam_F
Sense AGCTTTCTAGAGAGCTCCCGCGGG
J741_Hind-XSS-Bam_R Antisense GATCCCCGCGGGAGCTCTCTAGAA

linker for short-Tg vector
containing XbaI-SacI-SacII

J731_Linker 3
J732_Linker 4

Sense CTAGGAATTCTTAATTAAGGCGCGCCAGCT
Antisense GGCGCGCCTTAATTAAGAATTC

linker for short Tg vector
containing EcoRI-PacI-AscI

J761_dSalEAPdBgl
J762_dBglPAEdSal

Sense TCGAGAATTCGGCGCGCCGGTTAATTAAC
Antisense GATCGTTAATTAACCGGCGCGCCGAATTC

linker for pTol2-EPA
containing EcoRI-AscI-PacI

Genotyping of Tg mice
GFP-F
Forward GGCATCAAGGTGAACTTCAAGATCC
GFP-R3
Reverse CTTTACTTGTACAGCTCGTCCATGC

62 °C
30 secgenotype
EGFP (intermediate of transgenes)

Genotyping

J619_SacB_F
J620_SacB_R

Forward GCTGAATACAACGGCTATCACG
Reverse TCTCTCAGCGTATGGTTGTCG

62 °C
30 secgenotype
Left end of Tg-220

Genotyping

J709_BAC108L_F
J710_BAC108L_R

Forward CGTATTCAGTGTCGCTGATTTG
Reverse TTAGCGATGAGCTCGGACTTC

60 °C
30 secgenotype
Left end of deletion-Tgs

Genotyping

J621_CmR_F
J622_CmR_R

Forward GAGGCATTTCAGTCAGTTGCTC
Reverse CGGCATGATGAACCTGAATCG

62 °C
30 secgenotype
Right end of Tg-220, deletion-Tgs

Genotyping

Venus_F
Venus_R

Forward GCAAGCTGACCCTGAAGCTG
Reverse TTGCTCAGGGCGGACTGGTA

60 °C
30 secgenotype
Short transgene series

Genotyping

PrimeSTAR HS (TaKaRa)
94 °C 5 min

KOD plus ver.2 (Toyobo)
94 °C 2 min

Genotyping
94 °C 5 min[30 cycles]
98 °C 10 sec(Annealing temp.)* 5 sec
72 °C (Extension time)*[30 cycles]
98 °C 10 sec(Annealing temp.)* 30 sec
68 °C (Extension time)*[30 cycles]
94 °C 30 sec(Annealing temp.)* 30 sec
72 °C (Extension time)*

72 °C 5 min

68 °C 5 min

72 °C 5 min

Sequences of primers and oligonucleotides used in Chapter IV are summarized. They were used forconstructing homology arms, transgenes for reporter assay, and genotyping of transgenic mice.
PCRs with PrimeSTAR and KOD plus kits were basically performed according to the manufacturer's
recommendations; all PCR conditions are summarized in bottom of Table. Genotyping PCR used arecombinant Taq DNA polymerase. The annealing temperature and extension time were describedin Table.

75

ⅠV - 3Results

IV-3-1) Comparative analysis of the Tg-220 mouse genomic sequence
Transgenic analysis using the Tg-110 and Tg-250SB demonstrates that the genomic
region of 4 to 142 kb upstream the MOR42-3 transcription start site (TSS) contains a
cis-acting element necessary for the expression of the transgenic OR gene. Because thisgenomic region was included in Tg-220, I compared the mouse genomic sequence of
Tg-220 with the class I OR cluster regions in other mammals including human, rhesus
macaque, horse, dog, cow, and opossum, to estimate the position of the cis-element.
Functional genomic regions such as coding sequences, exons and intergenic regulatory
regions tend to be conserved during evolution. A nucleotide identity plots of mammals
revealed several conserved intergenic regions in addition to coding sequences of OR or
non-OR genes and exons (Figure 4-1A). Note that many OR genes have a 5´ non-coding
exon, which are not annotated in the database. Thus, I focused a 13 kb region of 16 to 29
kb upstream the MOR42-3 TSS. This region has a cluster of homology peaks and is far
from the neighboring class I OR genes, MOR42-1 and MOR31-4. No significant homology
peak was found in cow and opossum, probably due to an incomplete assembly of genomic
sequences and to a far evolutional distance.
The mouse MOR42 family is a unique OR gene subfamily that is orthologous tosome fish OR genes (Niimura, 2009). Therefore, it is conceivable that the cis-element for
MOR42-3 is conserved in fish or amphibian genome, which contains orthologous genes of
MOR42-3. To examine this possibility, I also generated a homology plot of zebrafish,
western clawed frog and fugu (human as control, Figure 4-1B). I found no significant
conservation in intergenic region, suggesting that the highly conserved regions in76

mammals might be acquired during evolution.

IV-3-2) Construction of a series of deletion mutants of the Tg-220
According to the comparative analysis of the nucleotide identity plots, I designed aseries of deletion constructs of Tg-220 at the four positions of 79 kb, 29 kb, 16 kb
upstream and 19 kb downstream the MOR42-3 TSS (Figure 4-1, 4-2). The deletion
mutagenesis was performed by replacing the targeted regions with one of antibiotic
resistance genes of tet or chloramphenicol resistance genes (cat) using the BGM vector
system (Figure 4-2A). I constructed three mutant transgenes by upstream deletion,
ΔTg-U80, ΔTg-U30 and ΔTg-U20 (Figure 4-2B). I obtained 22 colonies for ΔTg-U80, 51
colonies for ΔTg-U30 and 34 colonies for ΔTg-U20 resistant to chloramphenicol (Cm) insingle transformation. I further constructed four deletion transgenes by downstream
deletion (Figure 4-2C), and obtained 25 colonies for ΔTg-U140Δ3´, 321 colonies for
ΔTg-U80Δ3´, 339 colonies for ΔTg-U30Δ3´ and 145 colonies for ΔTg-U20Δ3´resistantto Tet. I-PpoI/CHEF analyses of each three representative clones of these constructs
showed bands correspond to the expected deletion transgene inserts (Figure 4-2D). The
deletion of BGM insert was previously described using a cI-spc cassette (Kaneko et al.,2003). Several plasmids enable the simultaneous deletion of two regions, for example,
upstream and downstream of the targeted region.

IV-3-3) The 13 kb region is necessary for transgenic MOR42-3 expression
Using the deletion constructs, I generated transgenic mice carrying Tg-220,
ΔTg-U80, ΔTg-U30 or ΔTg-U20 (Figure 4-3A). Briefly, transgenes were prepared from77BGM clones with same method in Chapter III and were microinjected into fertilized mouse
eggs. Newborn mice were genotyped by PCR using the same primer sets for Tg-110 andTg-250SB transgenic mice (Table 4-1). I obtained four Tg-220 founders carrying the intact
transgene, and one transgenic mouse carrying the partial transgene, which was PCR
positive for right end sequence only. For ΔTg-U80, five pups carried the intact transgene,and one lacked the left end sequence. For ΔTg-U30, three pups carried the intact transgene,and two carried only right end sequence. For ΔTg-U20, one pup carried the intact
transgene.
I analyzed expression of the EGFP reporter gene in transgenic founders or lines. Allfour Tg-220 transgenic lines showed EGFP fluorescence (Figure 4-3A). In the deletionmutants, three founders of ΔTg-U80 were EGFP positive, but one line was negative. Twofounders and one line of ΔTg-U30 whose upstream region was shorten to 16 kb position
from MOR42-3 TSS were also EGFP positive. No apparent difference in expression levelsof the EGFP reporter gene and axonal projection patterns among these transgenic mice
were observed, indicating that a genomic region carried in ΔTg-U30 is sufficient for
proper expression of MOR42-3 transgene (Figure 4-3B). In contrast, a ΔTg-U20 line did
not show EGFP fluorescence. In addition, none of the transgenic mice carrying Tg-110,
which included approximately 4 kb sequence upstream the MOR42-3 TSS, displayed
labeled OSNs (in Chapter III, Figure 3-3). Because I analyzed only one founder of
ΔTg-U20, the result should be confirmed by further experiments of other ΔTg-U20
transgenic founders or lines. However, considering with the bioinformatics analysis, it ismost likely that the 13 kb region contains regulatory sequence to activate expression of thetransgenic MOR42-3 gene.

78

IV-3-4) Functional analysis of the 13 kb region by conventional transgenesis
Before performing the functional analysis of the identified 13 kb region, I
investigated the genomic structure of this region using the UCSC Genome Browser (UCSC
web page), in terms of both conservation between human and binding factors. By the
nucleotide identity plot, I found that a 1.9 kb sequence displayed high homology peaks(named as Core region) (Figure 4-4). Interestingly, the Core region was flanked by two
CTCF (CCCTC-binding factor) binding sites in several tissues (depicted as CTCF/CpG
and CTCF), which also displayed homology peaks in the nucleotide identity plot of human(Figure 4-4B, C). Note that the 5´ CTCF binding site locates in a CpG island, suggestingthat DNA methylation regulates the binding of CTCF to this site (Figure 4-4B). CTCF is atranscription factor of 11-zinc finger protein, and is known as an insulator element (Chunget al., 1993; Gaszner and Felsenfeld, 2006). Recent studies have demonstrated that CTCF
is also involved in the chromatin conformation and regulates gene expression or otherevents, such as stochastic expression of clustered protocadherin (Pcdh) genes in neurons(Hirayama et al., 2012) and V(D)J recombination in immune cells (Chaumeil and Skok,
2012).
To reveal function of each component in the 13 kb region, I conducted aconventional transgenic reporter assay (Figure 4-5). I constructed a reporter transgene,
13k-423pgV, which consists of the 13 kb region, a promoter sequence including MOR42-3
TSS, a membrane-targeted Venus reporter gene and a polyadenylation sequence, and adeletion series of 13k-423pgV; the 9.1k-423pgV contains all there components of the
CTCF/CpG, Core and CTCF; the 7.9k-423pgV contains the Core and CTCF; the3.9k-423pgV lacks the CTCF/CpG and Core; the Core (Nco)-423pgV contains only the79Core; the ΔCore-423pgV lacks the Core region but contains the CTCF/CpG and CTCF
(Figure 4-5A). The transgenic mice carrying 7.9k or 3.9k transgene were generated by theconventional pronuclear-microinjection of a linearized transgene, whereas the 13k, 9.1k,
Core (Nco) or ΔCore transgenic mice were generated by the Tol2 transposon mediatedcytoplasmic injection method because of high efficient transgenesis (Sumiyama et al.,2010) (Figure 4-5A).
I analyzed expression of the Venus reporter gene in transgenic founders or lines(Figure 4-6). Note that these transgene constructs have no OR coding sequence. Thus, it
was expected that certain populations of OSN were labeled with Venus fluorescence; thesignals are broadly observed in multiple glomeruli not in a specific subset in the olfactory
bulb (OB). The 13k, 9.1k and Core (Nco) transgenic mice showed similar Venus
expression patterns; Venus fluorescence was observed in the most dorsal and dorsomedial
OB, and the dorsal MOE, suggesting expression of the Venus in class I OR-expressing
OSNs. Some signals were also observed in the ventral MOE and the ventromedial OB in
the 9.1k or Core (Nco) transgenic mice. These labeled OSNs are considered to express
some class II OR genes. It is notable that the Core (Nco) fragment can mainly drive
expression in the class I OR-expressing OSN population. On the other hand, transgenic
mice carrying the 7.9k fragment, which lacks the CTCF/CpG element, showed a different
pattern compared to other Venus-positive transgenic constructs; strong signals wereobserved in the dorsal and ventral MOE and the ventromedial region of OB. Additionally,bright Venus fluorescence was observed in the vomeronasal sensory nerves in two
founders and one line. Different expression pattern between 7.9k and other reporter
positive transgenes indicates that the CTCF/CpG sequence or the combination of80

CTCF/CpG and CTCF sequences defines the region of transgene expression in the MOE.
In contrast, all the 3.9k and majority of the ΔCore transgenic mice, both lacked the Core
region, did not show Venus fluorescence. One ΔCore transgenic founder showed strong
Venus expression in the most dorsal and dorsomedial OB, in addition to moderate
expression in the dorsolateral OB, suggesting a possible positional effect oftransgene-integration locus. The numbers of reporter positive founders or lines are
summarized in Figure 4-5A.

IV-3-5) The Core region is conserved in 13 placental mammals
The OR family, one of chemosensory receptor families, is conserved in many species
including mammals, birds, amphibians, fishes and insects (Nei et al., 2008). In alarge-scale phylogenetic analysis, mammalian OR genes were categorized into threegroups; class I genes corresponding to group α or β, whereas all class II genes
corresponding to group γ (Niimura and Nei, 2005). The group β OR genes including
mouse MOR42-3 are conserved in many mammals, though this group includes only a few
class I OR genes (Niimura, 2009). Therefore, it is conceivable that the Core region isconserved in other mammalian genomes. In fact, nucleotide identity plots revealed that theCore region was conserved in human, rhesus macaque, horse and dog genomes (Figure
4-1A).
To analyze the conservation of the Core region in detail, I performed a large-scalebioinformatics approach based on a sequence comparison analysis, and I found that theCore region consisted of two conserved fragments, designated as core1 and core2, whichwere conserved in 13 placental mammalian species (Figure 4-7). In mouse, the core1 (0.981kb) and core2 (1.3 kb) were detected within the 2.3 kb genomic region (Figure 4-7 inset),
which is wider than the 1.9 kb Core region; 355 bp larger in upstream and 133 bp larger indownstream. Accordingly, the highly conservation of the Core region was confirmed by
the alternative bioinformatics approach.
In addition, I found that the group β OR genes (OGG1-60 and OGG1-105) areconsistently present at the end of the class I OR gene cluster in all tested 13 mammals.
However, the OGG1-105 has been pseudogenized, and the OGG1-60 has been lost in
primate genome including human, chimpanzee, orangutan and macaque. Among analyzed
13 mammals, the core1 and core2 located at the intergenic region between group α andβ OR genes, with the exception of cow. The cow class I cluster region is divided into two
contigs because of possible assemble error, but the JH122518 contig including two coresequences and a group β OR gene is likely to be inserted into between OGG1-105 andOGG1-109 (group α). Therefore, the bioinformatics analysis demonstrated that the
genomic organization was also conserved in 13 placental mammals in addition to thepresence of the Core region.

82

ⅠV - 4

Discussion

In this chapter, I performed the deletion mutagenesis and the bioinformatics analyses
and to identify the cis-element for the class I OR gene, MOR42-3. Starting from the 140 kb
region containing the cis-element for MOR42-3 existed (Chapter III), I shortened theputative cis-acting element into the 13 kb genomic region by deletion mutagenesis.
Further functional analyses of the 13 kb region demonstrated that the Core (Nco) (3.8
kb NcoI fragment) was capable of activating expression the reporter transgene in the class
I OR expressing region in the MOE (Figure 4-5, 4-6). This 3.8 kb region locatesapproximately 20 kb upstream the MOR42-3 TSS between MOR42-1 and MOR31-4(Figure 4-5A). Because some cis-elements for class II OR genes activate the expression ofmultiple class II OR genes among the gene cluster in a monogenic manner (Serizawa et al.,
2003; Fuss et al., 2007; Nishizumi et al., 2007; Khan et al., 2011), the identified region
may regulate not only MOR42-3 but also other two MOR42 subfamily and/or other class I
OR genes. Interestingly, loss of the upstream CTCF binding site (CTCF/CpG) affects theexpression pattern of the reporter transgene, suggesting the involvement in regulation of
the spatial expression pattern. These results suggest that the 3.8 kb region activates theclass I OR gene expression, and the CTCF/CpG alone or the combination of CTCF/CpG
and CTCF regulates the spatial and temporal expression patterns of class I OR genes. To
examine these possibilities, further experiments are required, such as transgenic analysis ofother class I OR gene-tagged transgene like Tg-220 and loss-of-function analyses of the
non-coding regions of the Core, and the CTCF/CpG and CTCF.
The large-scale comparative analysis demonstrated the Core region was conserved in
all tested 13 placental mammals (Figure 4-7), supporting the idea of an important role of83

the Core region as a functional non-coding element. The group β class I OR genes, MOR42
family, were widely conserved in vertebrates from fish to mammals, whereas the group αclass I OR genes present only in mammals (Niimura, 2009). Four tested primates including
human, chimpanzee, macaque and orangutan have only one group β class I OR genes(OGG1-105). Although all of them are pseudogenized, the Core region is still conserved inthese species (Figure 4-7), suggesting the possible role of the Core region to control bothgroups of class I OR, α and β class I OR genes. In contrast, although non-mammalianvertebrates, zebrafish, western clawed frog and fugu, had intact group β OR genes, these
species did not have the Core region (Figure 4-1B). Note that the genome sequencing andassembly are incomplete in those species, however, it is possible to speculate that the Core
region has been acquired in mammals during evolution to regulate mammalian class I OR
gene expression.

84

A

5142

Tg5220

526 516
25 28

ΔTg5U80

516 516579
29 16

ΔTg5U30 ΔTg5U20

6
13 546 529536
31 315 31516

516 2536 2526
42
4 4Distance6from6MOR42&3(TSS6
+78

Rrm1

SPm1

1006%

Human
506%

MacaqueHorseDog

Cow

Opossum

B5142

Tg5220

526 516
25 28

ΔTg5U80

579
516 516
29 16

ΔTg5U30 ΔTg5U20

6
13 546 529536
31 315 31516

516 2536 2526
42
4 4

+78

Rrm1

SPm1

1006%

Human
506%

Zebraﬁsh*Western6
clawed6frog*
Fugu*
*6CalculaPon6window:6306bp6(high6sensiPve)6

Figure 4-1. Comparative analysis of a Tg-220 mouse genomic sequence with class I OR lociin other species.
(A) Genomic organization of class I OR genes were analyzed by nucleotide identity plots of human,rhesus macaque, horse, dog, cow and opossum with the mouse genomic sequence of Tg-220 using
VISTA program. Highly conserved regions (> 70% identity) in intergenic region and coding region
are shown in pink and blue, respectively. Triangles represent class I OR genes. An upstream region
from -29 to -16 kb positions showed a significant conservation among intergenic regions. (B)
Nucleotide identity plots of human, zebrafish, western clawed frog and fugu with the mouse
genomic sequence. No significant conservations in intergenic region were detected even in the a 30
bp-calculate window for high-sensitive detection.

85

A

I

I
G

tet

cat

B
Insert size

7J


ǻ7J8


ǻ7J8


ǻ7J8


0 kb156 kb 

c

10 kb
G

G

c

107 kb

G

c

94 kb

G

C
Insert size

7J


ǻ7J8ǻ¶

ǻ7J8ǻ¶

ǻ7J8ǻ¶

ǻ7J8ǻ¶

+19

0 kbG

161 kb

G

t

G

t

G

t

G

t

97 kb

c

 kb

c

kb

c

10 kb

D
M

[kb]

194

194
145

145

97

97

4


4

9.4

Figure 4-2. Deletion series to identify the cis-element for MOR42-3(A) Schematic illustration of a deletion mutagenesis using the BGM vector. A target region is
replaced with tet and/or cat genes by homologous recombination at particular homology regions and
BAC vector sequences. The BAC vector sequences (closed and open arrows) were approximately
3.5 kb each. The I-PpoI sites are designated as “I”. (B, C) Structures of a deletion series of theTg-220. The tet and cat genes are designated as t and c in the boxes, respectively. Distances from
the MOR42-3 TSS to insert ends are shown in parenthesis (5´: -, and 3´:+). (D) The insert sizes ofthe deletion transgenes were confirmed by I-PpoI and CHEF analyses. Representative clones ofeach deletion constructs showed the expected size of the deletion transgene (open arrowheads).
The closed arrowhead indicates the BGM vector.

86

$79

42
#1
42 $
#3
42 $
#2
$

31
$3
(
31
$1
31 3(
$4
(

$142

25
$2
(
28
$1
(
29
$1
16 (
$1
(

A

$29 $16

50(kb
+78

EGFP(
Fluorescence((posi:ve(/(total(analyzed)

4(/(4

Tg$220(
IRES&tauEGFPΔTg$U80(

3(/(4c

ΔTg$U30((

3(/(3c

ΔTg$U20(0(/(1

c

B
Tg$220(

ΔTg$U80(Tg(

ΔTg$U30(Tg(

ΔTg$U20(Tg(Figure 4-3. Deletion analysis of Tg-220 to identify the cis-element for MOR42-3.
(A) Schematic illustration of Tg-220 and its deletion series. The number of EGFP-positive founders
and lines among the total analyzed is shown in right side. Tg-220, ΔTg-U80 and ΔTg-U30
transgenic mice expressed the EGFP reporter gene, where as a ΔTg-U20 transgenic mouse did not.
(B) Whole-mount images of the medial aspect of the OB in the transgenic mice. ΔTg-U80 and
ΔTg-U30 transgenic mice showed similar axonal projection patterns and formed a glomerulus on
the dorsal portion of OB as well as Tg-220. In contrast, ΔTg-U20 whose upstream region was
truncated to -16 kb position did not show EGFP fluorescence.

87

A

1"kb"
100"%Core

CTCF/CpGCTCF

VISTA"alignment"(with"human)
50"%
""Core"region"(1.9"kb)

UCSC"
Genome"browser"

CTCF"Binding"Sites""by"ChIPAseq"from""ENCODE/LICR

B

C
Homology"peak"(100"bp)

Homology"peak"(112"bp)

Figure 4-4. The genomic structure of the identified 13 kb region for the cis-element for
MOR42-3.
(A) The nucleotide identity plot of human and UCSC Genome Browser view of mm9 assembly witha CTCF binding profile on several mouse tissues (deposited as ENCODE/LIR) in the 13 kb region.
The 13 kb region contains an approximately 1.9 kb conserved region (named as the Core region,
black bar) and two CTCF binding sites flanking the Core region. Of these, the 5´-CTCF binding site
locates on CpG island (CpG51, green bar). The CTCF binding profile shows intensities ofprocessed ChIP-sequence data of CTCF in grey scale; upper row represents “Peaks” (regions ofsignal enrichment), lower row represents “Signals” (density graph of signal enrichment). Data fromdifferent tissues are depicted in different colors. (B, C) Magnified views of CTCF/CpG and CTCF
sites. Each conserved region overlaps with CTCF-binding peak.

88

A
31#4%

929

13#kb#region 916

42#1$

42#3$

2#kb#

42#3#TSS#(0)#
Xh, Sal P  Nh Nc

CTCF/CpGVISTA#alignment#(with#human)

42#2$

Xb 
100#%

Nc 

Core

CTCF

50#%

1#kb#

Venus#
Fluorescence#

13k9423pgV

1#/#1#(Tol2)

9.1k9423pgV

6#/#6#(Tol2)

7.9k9423pgV

4#/#4#

3.9k9423pgV

0#/#5

Core(Nco)9423pgV

5#/#6#(Tol2)

ΔCore9423pgV

1#/#3#(Tol2)
423p gV pA

B

423%promoter%(904#bp)#
UTR#(135#bp)#

0.5#kb#42#3#ORF
1st#exon#(36#bp)#

intron#(3013#bp)#

CDS#(1002#bp)#

Figure 4-5. Constructs for the functional analysis of the 13 kb region.
(A) Schematic structures of a 13 kb transgene and its deletion series to refine the 13 kb region.
Schematic structure of a genomic region around 13 kb region are shown in the top of the panel.
Restriction sites used for constructing deletion series of 13 kb region are represented as P, PmlI; Nh,
NheI; Nc, NcoI; Xh, XhoI; Sal, SalI; Xb, XbaI. The 9.1k fragment has all three components of
CTCF/CpG, Core, and CTCF. The 7.9k fragment lacks the CTCF/CpG. The 3.9k fragment lacks the
CTCF/CpG and Core region. The Core (Nco) fragment contains only the Core region. The ΔCore
lacks the Core region. The 13k, 9.1k, Core (Nco) and ΔCore transgenic mice were generated by theTol2 transposon-mediated cytoplasmic injection method. 423p, 423 promoter; gV,
membrane-targeted Venus reporter (gapVenus); pA, poly adenylation sequence. The number ofVenus-positive founders and lines among the total analyzed are summarized in parentheses. (B)The structure of MOR42-3 gene (Hoppe et al., 2006). An approximately 0.9 kb fragment (green box)
consists of a non-coding exon 1 and a upstream region of TSS used as the 423 promoter.

89

Figure 4-6. Venus expression patterns of reporter transgenes in transgenic mice.
Representative confocal images of the dorsal view of the OB, and the medial view of the MOE andthe OB (upper or lower parts) of each transgenic mouse. The 13k, 9.1k and Core (Nco) transgenic
mice showed similar Venus expression patterns; Venus signals were mainly observed in the mostdorsal region and the dorsomedial of OB (left and right middle panels), and the dorsal MOE (middle
left panel, medial view). Some signals were also observed in the ventral MOE (left middle panel)and the ventromedial region of OB (right panel) in the 9.1k or Core (Nco) transgenic mice. 7.9ktransgenic mice showed a different pattern compared to other Venus-positive transgenic constructs;
strong signals were observed in the dorsal and ventral MOE and the ventromedial region of OB in
addition to the vomeronasal sensory nerves (medial views of the OB). Signals in the most dorsal or
dorsomedial region of the OB were also observed, but the intensity is lower than lateral or ventral
parts. In contrast, 3.9k and ΔCore transgenic mice did not show Venus fluorescence. Scale bar,
500 µm.

90

Figure 4-7. Conservation of the Core region in 13 placental mammalian species.
Schematic illustrations of class I OR genes and the Core region in 13 placental mammalian species.
Two orthologous gene groups (OGG), OGG1-60 and OGG1-105, belong to group β OR gene family(Niimura, 2009). Mouse MOR42-1 and MOR42-3 genes are categorized into OGG1-60, whereasmouse MOR42-2 gene is categorized into OGG1-105. The Core region (depicted as green boxes,
core1 plus core2) is conserved in all tested mammalian genomes, and is localized in intergenic
region between group α and β OR genes in the end of the class I OR gene cluster. In cow, class I
OR locus was divided into two contigs because of assemble error. Red or blue triangles indicate
intact or pseudo OR genes and their direction. The inset represents the relationship between Core
region and core1 plus core2 sequence in mouse.

91CHAPTERV

CONCLUSION

92

The BGM vector system is a unique and developing technology for propagation ofvery large fragments of heterologous DNA. To explore the potential of the BGM vector, Iapplied this system to identify the cis-element for mouse class I OR genes. Through thisstudy, I have demonstrated that the BGM vector system enables the complete genetic
modification of large genomic DNA fragments, including targeted insertion, deletion,
inversion and fusion. Using these techniques, a 252 kb transgene was reconstructed fromtwo BAC clones whose inserts were initially oriented in opposite direction with reference
to the BAC vector sequence (Chapter II). In addition, I demonstrated that this system could
be used to generate transgenic mice. Analyzing the transgenic mice, I provided anexperimental evidence of a cis-element for a class I OR gene (Chapter III). Finally, I
performed several transgenic analyses and bioinformatics analyses, and identified the firstcis-element for class I OR gene (Chapter IV).

The BGM vector system as an alternative tool for chromosome engineering
The BGM vector has several specific features that give advantages over the BAC andYAC systems. First, the megabase-scale cloning capacity of the BGM vector is greaterthan that of conventional systems. The BGM vector system is capable of cloning the entire
3.5 Mb Synechocystis genome (Itaya et al., 2005), and the upper limit of the cloning size
has not yet been determined. Second, cloned DNA inserts show high structural and genetic
stability (Kaneko et al., 2005) because of direct insertion into the single circular hostgenome. In fact, the next-generation DNA sequencing of modified and reconstructed
genomic DNA fragments confirmed the structural stability of inserts in the BGM vector
even though genome inserts included many repetitive sequences and several similar class I

93

OR genes. However, it should be noted that recombination is constitutively active in B.
subtilis like the YAC system; thus, unwanted rearrangements may occur. This issue can beovercome by introducing an inducible RecA system into the BGM vector. Third, various
accurate modification approaches are available. The cI repressor cassette-mediating
modification technique provides desired gene modifications without leaving any traces inthe DNA, enabling the repetitive modification of BGM inserts. In addition to insertion anddeletion modifications, I demonstrated the elongation of inserts via the fusion of two DNA
fragments even though they were initially oriented in opposite directions in the BAC
vector, thus providing a method for the construction of giant recombinant DNA fragments.
Considering with maximum cloning capacity of the BGM vector of 3 Mb (Itaya et al.,2005), the fusion of contiguous genomic fragments can be a powerful technique toreconstruct genomic structures (Kozzamanis et al., 2004; Kaneko et al., 2009). In addition,
my sequencing analyses confirmed that these targeted genetic modifications were accurate
and reliable. Fourth, BGM inserts can be retrieved because a single host cell contains asingle genome composed of the recombinant insert and the 4.2 Mb BGM vector. As I
demonstrated, BAC clones are easily transferred, modified and reconstructed in the BGM
vector. It is noteworthy that modified BGM inserts can be restored to a circular BAC form
(Kaneko et al., 2005) (Chapter I, Figure 1-4), enabling this “shuttle genetic modification”
of BAC clones to enhance the utility of BGM vector system (Figure 5-1).

Application of the BGM vector systemI have used mouse genomic DNA to demonstrate the utilities of the BGM system forgenetic manipulation and transgenesis. Many BAC libraries have been already established94

and/or are under construction for diverse species, including mammals, other vertebrates
and plants (Web page: Other sources of bacterial artificial chromosome libraries).
Because the BGM vector harbors BAC vector sequences for cloning BAC inserts, thissystem can be applied to other species. Moreover, the BGM vector can be designed toclone other library resources by introducing cloning vector sequences from other systems,e.g., YACs and human artificial chromosomes.
An interesting achievement in bioengineering field is gene assembly involved inbiosynthesis of a natural carotenoid, zeaxanthin, for production in E. coli (Nishizaki et al.,2007). This novel DNA fragment assembly method, named Ordered Gene Assembly in B.
subtilis (OGAB), produces a DNA in plasmid form via B. subtilis transformation (Tsuge etal., 2003). This method is applicable to make DNA cassettes with many relevant genes(Nishizaki et al., 2007; Itaya and Tsuge, 2011). Gene assembly technologies are useful for
efficient productions of natural products in heterologous cells. Here, I demonstrated thatthe BGM inserts were able to introduce into the mouse eggs to generate transgenic mice.
The combination of BGM-based gene manipulation system and mammalian transgenesis,
including transgenic animals and transfected cell lines, expands the range of application of
the system. Because the BGM vector system can provide large cloning capacity in size andvarious accurate gene manipulation approaches, the BGM vector system is an attractive
cloning tool for the manipulation of large DNA fragments, such as in the functional
analysis of genomic DNA, gene expression analysis, material production, recombinant
genomes in synthetic biology (Gilbson et al., 2008; Itaya et al., 2008) and other life
science research fields (Figure 5-1).

95

Function of the identified cis-element in class I OR genes expression
I demonstrated the existence of a cis-acting element of a mouse class I OR gene in
the Tg-220 transgene by the BGM transgenesis. The BGM-based deletion assay andfunctional analysis suggest that the 3.8 kb region is a cis-element activating class I OR
gene expression. This is supported by the large-scale comparative analysis; the Core region,
which is contained the 3.8 kb region, is conserved in all tested 13 mammalian genomes. In
addition, the functional analysis suggested the CTCF/CpG site alone or the combination of
CTCF/CpG and CTCF also regulates class I OR gene expression.
Because some identified class II cis-elements, H region and P sequence, regulates
several distal class II OR genes, the identified 3.8 kb region also regulates not only
MOR42-3 but also other MOR42 genes and/or other class I OR genes. It is known that
cis-elements for sonic hedgehog (Shh) gene are scattered within several hundred kb of
genomic region; the most distal element locates ~840 kb upstream from the Shh gene
(Sagai et al., 2005; Sagai et al., 2009). Thus, as previously predicted (Hirota et al., 2007;
Rodriguez, 2007), the 3.8 kb region may regulate all class I OR genes forming ~3 Mb
cluster. However, because the 3.8 kb region locates near the centromeric end of the class I
OR gene cluster (Figure 1-7), the distance from the 3.8 kb element to the other end class I
OR gene (MOR36-1) is approximately 3 Mb long. Thus, I cannot exclude possibility ofexistence of other cis-elements for class I OR genes.
The molecular mechanisms underlying single OR genes expression remain unclear.
Recently, the nuclear architecture and chromatin status (euchromatin/heterochromatin) of
OR gene loci in OSNs are involved in proper expression of OR genes (Magklara et al.,2011; Clowney et al., 2012; Lyons et al., 2013). The 3.8 kb region is flanked by two CTCF

96

binding sites. CTCF is known as a regulator of the chromatin structure including DNA
loops, which regulates enhancer activity (Phillips and Corces, 2009). Therefore, the 3.8 kb
region may be regulated by CTCF binding following a DNA loop formation (Figure 5-2).
To examine these possibilities, further experiments are required such as transgenic analysis
of other class I OR gene-targeted transgene like Tg-220 and loss-of-function analyses of
the non-coding regions of the Core and the CTCF/CpG and CTCF.

Physiological function of class I OR genes
The class I OR subfamily is maintained in vertebrates during evolution, suggesting aphysiological importance of the class I OR family. In addition, the group α class I OR
genes present only in mammals, whereas the group β class I OR genes are conserved not
only in mammals but also amphibians, reptiles and fishes (Niimura 2009; Wang et al.,2013). The physiological functions of class I OR family are unclear, although several
ligands have been reported (Malnic et al., 1999; Abaffy et al., 2006; Saito et al., 2009).
One report showed that a part of the dorsal OB (including class I OR domain) was
activated by 2-methylbutyric acid and pentanal, which are a pungent odorants of spoiled
foods, and suggested the dorsal OB mediates innate fear responses (Kobayakawa et al.,2007). It is conceivable that the class I OR family is involved in some innate behaviors.
Because the identified cis-element can drive transgene expression in the class I
OR-expressing OSNs, this element can be used to label or ablate class I OR-expressing
OSNs in future physiological experiments.

97

Spore#forma6on#

Genomic#DNA#(Animal,(Plant,(Microbe)(

Long9term(preserva=on(of(the(cloned(DNA(at(room(temperature((Resistance:(Hot,(Dry,(UV(etc.)

WholeDgenome#cloning#hybrid(genome(
synthe=c(biology(

100#kb#Mb
Cloning(to(B.'sub,lis(genome(

Pr9neo

BAC#Library#BGM(clone(
Linear(form(

Complete#gene#manipula6ons#
BAC(cloneBAC(cloneBAC(cloneInser=on/Removal(of(marker(genes M

Fusion(of(two(BAC(clones

M
Pr9neoPr9neo

Dele=onCircular(form((BAC(clone)(

Retrieval#of#inset#DNA#

Transgenic(animals(
DNA9Protein(interac=ons(
Transfer(to(E.coli'as(BAC9DNA(

Inversion(

MM
Pr9neoPr9neoFigure 5-1. Manipulations and applications of the BGM vector system.
The BGM vector can use both natural genomic DNA and BAC library clones. BGM inserts can be
modified by several manipulation techniques (Chapter II), and are stably stored in spores (Kaneko
et al., 2005). BGM inserts can be retrieved as linear form (Chapter III) or circular form (Kaneko et al.,2005). The BGM vector system and its application to transgenesis offer a new genetic approach for
not only systems and synthetic biology but also other life science research fields.

98

Structure%of%enhancer%region

Class%I%OR%gene%expression

[%Linear%form%]
CH3%[%OFF%]
Inac3ve%enhancerCTCF

PromoterCTCF/CpG 3.8%kb%region% CTCF
(Core)

[%Looping%form%]

Class%I%OR%gene%cluster[%ON%]
Ac3ve%
enhancerTranscrip3on%complex%

Figure 5-2. A putative regulation mechanism for class I OR gene expression.
Local structure of the identified enhancer region is regulated by CTCF binding status. When CTCF
not bind to CTCF sites (CpG island is methylated), the region is linear form. In contrast, binding of
CTCF into the sites forms a DNA loop structure. These two enhancer forms define the activity toactivate class I OR genes expression. The active enhancer chooses one OR gene from the class I
OR gene cluster and activates the transcription.

99

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