Yonago Acta medica 2011;54:21–31

Application of a Bacterial Artificial Chromosome Modification
System for a Human Artificial Chromosome Vector
Shigeyuki Yamaguchi*†, Ryosuke Niwa*†, Yasuhiro Kazuki*‡ and Tetsuya Ohbayashi†*Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate
School of Medical Sciences, †Division of Laboratory Animal Science, Research Center for Bioscience and Technology and ‡Chromosome Engineering Research Center, Tottori University, Yonago
683-8503, Japan

Exactly controlled conditional gene expressing systems are crucial for genomic functional
research, animal transgenesis and gene therapy. Bacterial artificial chromosomes (BACs)
are optimal for harboring long fragments of genomic DNA or large cDNA up to 300 kb insize. Therefore, BACs are available to produce transgenic cells and animals for the functional studies of genes. However, BAC can insert DNA randomly into the host genome,
possibly causing unpredicted expression. We previously developed a human artiﬁcial chromosome (HAC) vector from human chromosome 21 using chromosome engineering. The
HAC vector has several important characteristics desired for an ideal gene delivery vector,
including stable episomal maintenance, and the ability to carry large genomic DNA containing its own regulatory element, thus allowing physiological regulation of the transgenein a manner similar to that of the native chromosome. In this study, we develop a system
fusing BAC library and HAC technology together to allow tight control of gene expression.
This system enables BAC to be cloned into the defined locus on the HAC vector by the Cre/
loxP system. In addition, the genome in the BAC is possible to be engineered freely by the
BAC recombineering technology. This system is a highly efficient tool for the rapid generation of stringently controlled gene expression system on the HAC vector.
Key words: bacterial artificial chromosome; bacterial artificial chromosome recombineering;human artificial chromosome

The development of genomic functional studies,transgenic animal production and gene therapy
requires stringently controlled gene expression
systems. Bacterial artificial chromosomes (BACs)
have become important tools in functional genomics (Brune et al., 2000; Heintz,2001; Sparwasser
and Eberl, 2007; Tunster et al., 2011). BACs havemade genomic fragments from humans, mice andmany other species available through public genomic databases (O’Connor et al., 1989; Shizuya

et al., 1992; Osoegawa et al., 2000). pBACe3.6
is used as a backbone vector in the majority ofcurrently available BAC libraries (Frengen etal., 1999). Speciﬁc BACs are readily obtainablefrom several bioresource centers (e. g., Children’s
Hospital Oakland Research Institute). As a result, the tedious screening for genomic DNA
clones in complex libraries is reduced. Therefore,
BACs have greater convenience in handing overother large cloning vectors like P1 phage-derived

Abbreviations: AmpR, ampicillin-resistance; BAC, bacterial artificial chromosome; CmR, chloramphenicol-resistance;
HAC, human artificial chromosome; HPRT, hypoxanthine phosphoribosyl transferase; IL, interleukin; LB, lysogeny broth;
PAC, P1 phage-derived artificial chromosome; SLG, stable luciferase green; SLR, stable luciferase red; SOC, super optimal broth with catabolite regression; ZeoR, zeocin-resistance21

S. Yamaguchi et al.

artificial chromosomes (PACs) and yeast artificial
chromosomes. BACs cover average 100- to 300kb genomic DNA and so most mammalian genes
can be encompassed by a single BAC. Given theirlarge size, BACs encode the complete open reading
frame and most regulatory sequences, which recapitulate the regulation of endogenous genes muchbetter than shorter transgenes (Yang et al., 1997).
In addition, a homologus recombination system
was developed that requires only short homology
arms and enables the full range of BAC modifications to be quickly and easily engineered in Escherichia coli (E. coli) (Zhang et al., 1998; Muyrers
et al., 1999). This BAC engineering methodology
is now termed “BAC recombineering” (Yu et al.,
2000; Copeland et al., 2001) and includes a broad
range of applications including subcloning via gap
repair (Zhang et al., 2000), point mutagenesis in
BACs (Muyrers et al., 2000), high throughput DNA
engineering (Sarov et al., 2006; Poser et al., 2008)
and a variety of other, often complex, applications.
Furthormore, BAC transgenic mice expressing avariety of reporters (Gong et al., 2003; Lobo et al.,2006) or epitope tagged-proteins (Bateup et al.,2008) driven by speciﬁc promoters become widelyused in various in vivo functional applications.
Because of these advantages, BACs have been used
for transgenesis in higher model cells and organisms. To produce a target BAC transgene expression cells, random integration of foreign DNA isfrequently used. However, the expression of BAC
transgene can not only be affected by positional
effects at the insertion site of the host genome but
also by the number of copies inserted (Gong et al.,
2003; Alexander et al., 2004; Heaney and Bronson,
2006; Chandler et al., 2007; Le et al., 2010). As aresult, the expression level of genes varies greatly
in the obtained clones.
Human artificial chromosomes (HACs) haveseveral advantages over conventional gene delivery
systems (Larin and Mejia, 2002; Basu and Willard, 2006). We recently developed HAC vectors
from human chromosome 21 by deleting all of theendogenous genes (Kazuki et al., in press). Like
natural chromosomes, the HAC vector has thecapacity to replicate and segregate freely from thehost genome. Therefore, the HAC vector can avoid
the disruption of the host genome by transgene insertion (Pravtcheva and Wise, 1995). In addition,
as reported previously, the HAC vector enables
stable expression of the inserted gene, and is notaffected by position in the genome (Katoh et al.,
2004; Kakeda et al., 2005; Kazuki et al., in press).
The HAC vector is a powerful gene delivery vector
capable of carrying large genes such as the dystrophin gene (2.4 Mb) (Oshimura and Katoh, 2008;
Hoshiya et al., 2009; Kazuki et al., 2010). Conceptually and experimentally, any circular DNA
such as BACs can be inserted into the HAC vector.
Therefore, we considered that a more precise evaluation of large DNA inserts for genetic complementation should be possible by incorporating the BAC
system into the HAC vector. Site-specific insertionof BAC into the HAC vector may eliminate unwanted position effects caused by the random integration of exogenously introduced DNA. In thisstudy, we describe the generation of modified BAC
by the BAC recombineering for their introduction
into the defined locus on the HAC vector by the
Cre/loxP system. Also, the genome of interest in
the BAC is possible to be engineered by the BAC
recombineering.

Materials and Methods
Vector construction
The modification cassette vector I for engineeringof the genome on the BAC was constructed as follows. RP11-67L14 (cloned in the pBACe3.6 vector, GenBank accession number: AC079753) was
obtained from Invitrogen (Carlsbad, CA) and used
as a model for BAC modification. As a gene ofinterest, the coding sequence of human interleukin(IL)-1 was contained in the BAC. Fragment of the
genome specific homology arms to target the cassette into the start codon of the genome in the BAC
was ampliﬁed by blunt PCR from RP11-67L14 using the primers IL-1 hm F and IL-1 hm R. This
22

BAC modification system for an HAC vector

Mach1 T1R E. coli (Invitrogen) was used according
to the supplier’s instruction. After transformation,
the cell solution was diluted with super optimal
broth with catabolite regression (SOC) medium
and incubated at 37 ˚C for 1 h. The transformation
reactions were then spread onto lysogeny broth (LB)
agar plates containing 50 μg/mL kanamycin andincubated for 14 h at 37 ˚C. The sequence of thesepENTRs was confirmed by DNA sequencing. The
destination vector (pSLR-BAC hm DEST) contained the R1-ccdB-CmR-R2 cassette (Invitrogen),homology arms of BAC and a stable luciferase red(SLR) gene. pBACe3.6 (GenBank accession number: U80929) is used as a backbone vector for human RPCI-11 BAC library. Regions 4205–4793 bp
and 4946–5833 bp of pBACe3.6 were used as BAC
homology arms and cloned into the pGEM-T vector (Promega, Madison, WI), creating pBAC hm T
vector. This vector contained KpnI and XbaI cleavage sites in exchange of original loxP site locatedin the pBACe3.6 region 4831–4864 bp. The R1ccdB-CmR-R2 cassette was ligated into the pSLR
test (Toyobo), creating pSLR DEST. To construct
the destination vector containing a toxic ccdB gene,
ccdB survival competent cells (Invitrogen) wereused for propagation according to the supplier’s
instruction. After transformation, the cell solution
was diluted with SOC medium and incubated at
37˚C for 1 h. The transformation reactions werethen spread onto LB agar plates containing 12.5μg/mL chloramphenicol and incubated for 18 h at
37˚C. The R1-ccdB-CmR-R2 cassette and the SLR
gene were digested from pSLR DEST with KpnIand SacI (blunted). This fragment was cloned into
the KpnI and XbaI (blunted) sites of pBAC hm T
vector, creating a pSLR-BAC hm DEST. Sequenceand orientation of this vector was confirmed by
DNA sequencing and restriction enzyme patternanalysis. Three entry clones and pENTR L1-pLaclacZ -R5 (Invitrogen) were recombined with apSLR-BAC hm DEST to generate the modificationcassette vector II. Before this reaction, the pSLRBAC hm DEST was linearized at the BssHII site,
between a ccdB gene and a chloramphenicolresistance (CmR) gene. Around 20 ng of each entry

PCR product was cloned into the pSTblue1 blunt
vector (Novagen, Madison, WI), creating pIL-1
hm vector. There was an internal NcoI restriction
site within the IL-1 insert which is the ATG startsite of the ﬁrst exon of the IL-1 genome. The
reporter gene of interest (stable luciferase green;
SLG) and an ampicillin-resistance (AmpR) gene
were amplified by PCR from the pSLG test (Toyobo,
Osaka, Japan) using the primers SLG-Amp F and
SLG-Amp R. The PCR product was digested with
NcoI and cloned into the equivalent site of pIL-1
hm vector, creating the modification cassette vector
I. Sequence and orientation of the genome specifichomology arms, SLG gene and an AmpR gene wereconfirmed by DNA sequencing and restriction enzyme pattern analysis.
The modification cassette vector II was constructed using the Multisite Gateway kit (Invitrogen)as previously described (Sasaki et al., 2004; Sone etal., 2008). The PCR fragments for loxP-3' HPRT,a zeocin-resistance (ZeoR) gene and G3PDH promoter flanked by the appropriate gateway attB sites
were created. V901-3'HPRT-loxP (Kazuki et al.,in press) was used as a template for loxP-3' HPRT
amplification with primer pair B5- loxP-3' HPRTB4 F and B5- loxP-3' HPRT-B4 R. The ZeoR gene
was amplified from plasmid containing a ZeoR
gene using the primers B4r-zeocin-B3r F and B4rzeocin-B3r R. Human G3PDH promoter (GenBank
accession number: AY340484 region 473–1974 bp)
was amplified with primer pair B3-G3PDH-B2 F
and B3-G3PDH-B2 R. The gateway attB-flanked
PCR product was recombined with a donor vector
containing the corresponding gateway attP signals
in a BP reaction to generate an entry clone. Approximately 150 ng of PCR fragment and a donor
vector (pDONR 221 P5-P4, pDONR 221 P4r-P3r
or pDONR 221 P3-P2) were mixed with 2 μL of
BP clonase II enzyme mix, and adjusted to 10 μL
with TE buffer. The mixture was then incubated at
25˚C for 1 h to create 3 entry clones (pENTR L5loxP-3' HPRT-L4, pENTR R4-zeocin-R3, pENTR
L3-G3PDH promoter-L2). After the BP reaction, the enzymes were inactivated by treatment
with proteinase K for 10 min at 37˚C. Competent23

S. Yamaguchi et al.

techniques. The primer pairs used for confirmationof the IL-1 seque nce were IL-1 1S/IL-1 3000
AS, IL-1 2611 S/IL-1 5180 AS, IL-1 4031 S/
IL-1 7151 AS and IL-1 8051 S/IL-1 11332 AS.
The primer pairs used for confirmation of BAC
recombination of the modification cassette I wereamp2500 S and IL-1 12100 AS, along with IL-1
10930 S and slg200 AS.
The primer pairs utilized for detection of
BAC recombination of the modification cassetteII were SLR 1650 S and BACe3.6 5839 AS, alongwith BACe3.6 4168 S and LacZ 770 AS.

clone and 80 ng of destination vector were mixed
with 2 μL of LR clonase II Plus enzyme mix, andmade up to 10 μL with TE buffer. The mixture
was then incubated at 25 ˚C for 16 h. After thisreaction, the enzyme was inactivated by treatment
with proteinase K for 10 min at 37 ˚C. CompetentMach1 T1R E. coli was used for transformation.
Following transformation, the cell solution was diluted with SOC medium and incubated at 37 ˚C for
1 h. The transformation reactions were then spread
onto LB agar plates containing 50 μg/mL ampicillin and 25 μg/mL zeocin. These plates were incubated for 14 h at 37 ˚C. The modification cassette
vector II carried the homology arms of pBACe3.6
and the non-functional loxP-3' HPRT for targetingthe HAC vector. Cre recombinase-catalyzed integration required a functional HPRT gene.

Results
Construction of the modification cassette
vectors

BAC recombineering

BACs contain all of the endogenous control elements in their natural expression of the gene ofinterest and therefore almost always recapitulate
all of the endogenous control mechanisms, including alternative splicing. BAC libraries based on
pBACe3.6 have now been generated from various
species (Table 1) and used for reporter assay ofgene expression. The modification cassette vector I
was created for engineering of the genome of interest on the BAC. This vector consists of 3 regions;i) the region encoding the reporter gene such as afluorescent protein gene and a luciferase gene (SLG
gene in our study), ii) AmpR gene and iii) homology arms (A' and B') to target the cassette into thestart codon of chosen genome on the BAC (Fig. 1a).
The homology arms were obtained by PCR froma BAC containing the target genome. In our current study, we used a BAC clone RP11-67L14 as arecipient for BAC modification. As a gene of interest, the coding sequence of IL-1 was containedin the BAC. The primers were designed to target
precisely the cassette downstream of the IL-1 startcodon (Table 2).
In attempts to clone BAC into the HAC vector, the modification cassette vector II was constructed. This vector carried a loxP sequence andA homologous recombination-proﬁcient E. coli
strain (DY380) was used for the BAC recombineering. For BAC modiﬁcation, overnight cultures containing the BAC were grown from single colonies,
diluted 10-fold in LB medium, and grown to an
optical density at 600 nm of 0.4 to 0.6 at 32 ˚C. Fiftymilliliter cultures were then induced for the expression of recombineering factors by shifting the cells
to 42 ˚C for 15 min followed by chilling on ice for10 min. Cells were then centrifuged for 5 min at
5500 × g at 4 ˚C and washed with 10 mL of ice-cold
1 mM HEPES 2 times. Cells were then resuspended in 100 μL of ice-cold 1 mM HEPES and electroporated. Cell transformation was performed byelectroporation of 1 μg linear DNA into 100 μL of
ice-cold competent cells in cuvettes (0.1 cm) usinga Bio-Rad gene pulser set at 1.75 kV, 25 μF witha pulse controller set at 200 ohms. One milliliterof SOC medium was added after electroporation.
Cells were incubated at 32 ˚C for 1 h with shaking
and spread on appropriate selective agar media.
PCR analyses
PCR analyses were carried out using standard
24

BAC modification system for an HAC vector

Table 1. A list of BAC libraries constructed in the pBACe3.6
Species
DNA origin
		
		
Human
MonkeyCow
Dog
RabbitHamsterRat
Mouse

InsectWorm
Yeast

Average Coverageinsert size		(kb)

Human male
Human female
Chimpanzee (Pan troglodytes) male
Lemur (Lemur catta)
Dusky titi (Callicebus moloch)
Baboon (olive) male
Bovine male
Canine (Doberman Pinscher) maleWhite rabbit (Oryctolagus cuniculus)
Chinese hamster ovary (Cricetulus Griseus)
Rat (Brown Norway) male
Mouse (129S6/SvEvTac) female
Mouse (MSM/Ms) male
Mouse (C57BL/B6J) female
Mouse (C57BL/B6N) male
Mouse (129S7/SvEvBrd-Hprtb-m2) male
Drosophila melanogaster
Silkworm (Bombyx mori)
Ceanorabditis briggsea
Schizosacharomyces pombe

174
166
157
174
176
175
164
155
175
158
150
154
125
197
128
–
165
159
–
166

25.3X
21.8X
3.5X
6.1X
9.4X
10.4X
11.9X
8.1X
7.0X
5.4X
11.4X
10.9X
10.0X
11.2X
2.6X
–
22X
41.3X
–
–

Total
clonesSite of construction

437,034
398,590
68,721
106,156
159,667
180,004
216,439
157,255
120,804
110,592
228,313
211,953
220,000
180,396
65,280168,192
17,540147,456
36,8643,018

RPCI (RPCI-11)
RPCI (RPCI-13)
RPCI (RPCI-43)
LBNL (LBNL-2)
LBNL (LBNL-5)
RPCI (RPCI-41)
RPCI (RPCI-42)
RPCI (RPCI-81)
LBNL (LBNL-1)
RPCI (RPCI-99)
RPCI (RPCI-32)
RPCI (RPCI-22)
RIKEN BRC
RPCI (RPCI-23)
RIKEN BRC
Sanger (Sanger-bMQ)
RPCI (RPCI-98)
RPCI (RPCI-96)
RPCI (RPCI-94)
RPCI (RPCI-104)

LBNL, Lawrence Berkeley National Laboratory; RIKEN BRC, RIKEN BioResource Center; RPCI, Roswell Park Cancer Institute.
Note that this table is not an exhaustive listing of all BAC libraries and primarily lists those from the Children’s Hospital
Oakland Research Institute and others that are publicly available.

mids carrying multiple DNA sequences (Sasaki etal., 2004; Sone et al., 2008). The PCR fragments
for loxP-3'HPRT, ZeoR gene and G3PDH promoter
contained the appropriate gateway attB sites (Table2). The 3 fragments were recombined into 3 different donor vectors (pDONR221 P5-P4, pDONR221
P4r-P3r, pDONR221 P3-P2) to create 3 entry
clones (pENTR L5- loxP-3'HPRT-L4, pENTR
R4-zeocin-R3, pENTR L3-G3PDH promoter-L2)(Fig 1b). DNA sequencing showed that 95.0% ofkanamycin-resistant E. coli transformant colonies
were correctly targeted. These 3 pENTR vectors,
a pENTR L1-pLac-lacZ -R5 and a pSLR BAC hm
DEST were mixed in the presence of LR clonase
enzyme to generate the modification cassette vector
II (Fig. 1c). A recombination event would incorporate the lacZ gene and the ZeoR gene, resulting
in a blue colony which resistances to ampicillin
and zeocin. Unrecombined plasmid would result

the 3' hypoxanthine phosphoribosyl transferase(HPRT) sequence. The HAC vector used was
21HAC1 containing the 5' HPRT-loxP site (Kazuki
et al., in press). The HPRT gene expressed in the
HAC vector conferred HAT-resistance after sitespecific recombination with the Cre/loxP system.
The modification cassette vector II consists of
5 regions; i) loxP-3' HPRT region for introducing BAC into the HAC vector, ii) ZeoR gene, iii)
lacZ gene which contributed for selection in E.
coli, iv) G3PDH promoter-SLR region which isused as a marker in the mammalian cells becauseof its convenience for luciferase expression and v)
homology arms (C' and D') to target the cassette
into the pBACe3.6 region. The homology arms are
obtained by PCR from pBACe3.6 (Table 2). The
modification cassette vector II was constructed in
E. coli using the Multiple Gateway system, which
is useful in high-throughput construction of plas25

S. Yamaguchi et al.

a
A'

b
SLG

R

Amp

B'

PCR
attB5 loxP-3' HPRT attB4
product

attB4r

Zeo R attB3r

attB3 G3PDH promoter attB2

Modification cassette vector I
attP5 ccd B

CmR

attP4r ccd B

attP4

CmR

attP3r

ccd B

attP3

CmR

pDONR221 P5-P4

pDONR221 P4r-P3rpDONR221 P3-P2

KmR

KmR

KmR

R

R

attP2

R

attL5 loxP-3' HPRT attL4

attR4

attR3

attL3 G3PDH promoter attL2

pENTR L5-loxP 3' HPRT-L4

pENTR R4-Zeocin-R3

pENTR L3-G3PDH promoter -L2

KmR

KmR

KmR

Zeo R

R

R

R

c
KmR

KmR

pENTR L1-pLac- LacZα-R5pENTR L3-G3PDH promoter -L2

attL1 pLac-LacZα attR5

attL3 G3PDH promoter attL2

R

R

attL5 loxP-3' HPRT attL4

attR4

pENTR L5-loxP 3' HPRT-L4

pENTR R4-Zeocin-R3

KmR

KmR

R

C'

attR1

Zeo R

attR3
R

ccd B

R

Cm

attR2 SLRD'pSLR BAC hm DESTC'

attB1 pLac-LacZα attB5 loxP-3' HPRT attB4

Zeo R

attB3 G3PDH promoter attB2 SLR

D'

Modification cassette vector II

Fig. 1. Generation of BAC modification cassette vectors.
a: The structure of the modification cassette vector I for engineering of the
genome on the BAC. Features of the modification cassette vector I include
genome homology arms (A' and B') that are identical to sequences A and
B in the target genome for homologous recombination, and a modificationcassette (SLG-AmpR) that carries the reporter gene to be inserted into the
genome.
b: Construction of entry clones (pENTRs) from gateway attB-flanked PCR
products and donor vectors (pDONRs).
c: Construction of the modification cassette vector II from 4 entry clones anda destination vector (pSLR-BAC hm DEST). Features of the modificationcassette vector II include: pBACe3.6 homology arms (C' and D') that are
identical to sequences C and D in the pBACe3.6, and the non-functional
loxP-3' HPRT for targeting the HAC vector.

26

AmpR, ampicillin-resistance
BAC, bacterial artificial chromosome
CmR, chloramphenicol-resistance
HAC, human artificial chromosome
KmR, kanamycin-resistanceIL,
interleukin
SLG, stable luciferase green
SLR, stable luciferase red
ZeoR, zeocin-resistanceBAC modification system for an HAC vector

Table 2. The PCR primers used in this study
PrimerIL-1 hm F
IL-1 hm R
SLG-Amp F
SLG-Amp R
B5-loxP-3' HPRT-B4 F
B5-loxP-3' HPRT-B4 R
B4r-zeocin-B3r F
B4r-zeocin-B3r R
B3-G3PDH-B2 F
B3-G3PDH-B2 R
amp2500 S
IL-1 12100 AS
IL-1 10930 S
slg200 AS
SLR1650 S
BACe3.6 5839 AS
BACe3.6 4168 S
LacZ 770 AS
IL-1 1 S
IL-1 3000 AS
IL-1 2611 S
IL-1 5180 AS
IL-1 4031 S
IL-1 7151 AS
IL-1 8051 S
IL-1 11332 AS

		Sequence (5' –3' )GCC AGG TGT AAT ATA ATG CTT ATG ACT CGG
TGC AAA CAG CCT GCC TCT CAA AGC TGC CTG
CCA AAC TCA TCA ATG TAT CTT ATC ATG TCT GGA TC
GAT CCA TGG ATC TTT TCT ACG GGG TCT GAC GCT CAG TGG AAC G
GGG GAC AAC TTT GTA TAC AAA AGT TGA GAG CCT TCA ACC CAG TCA GCT CCT TC
GGG GAC AAC TTT GTA TAG AAA AGT TGG GTG GGC GCG CCA GGC TGG TTC TTT CCG CCT CAG
GGG GAC AAC TTT TCT ATA CAA AGT TTG CGC ATG CGG ACA AAC CAC AAC TAG AAT GCA GTG
GGG GAC AAC TTT ATT ATA CAA AGT TGT GTG TCA GTT AGG GTG TGG AAA GTC CCC AGG
GGG GAC AAC TTT GTA TAA TAA AGT TGG GTC AGG GAC TGG AGT CCT GTG GGT GC
GGG GAC CAC TTT GTA CAA GAA AGC TGG GTA GCC TTC AGG CCG TCC CTA GCC TCC CGG GTT TC
AGC TGA ATG AAG CCA TAC CAA ACG ACG AGCATA CCA TGG CAT CAA AGT GGC CCA GAA CTC
GAG TCT CTC TGT CTC TCT GCC TCT TTG TG
ACC TCC TCG GTG TGG GCG TCG ATC AGG GCC
CCG AAA GGC CCA ACA GGC AAG CTG ATG AGA
AAC TTC TGT GCT TAA AAC GTC ATC TGC ATCTCA CTT CGC AGT GCC GCA AGC CAA AGG CAAGCA GGC ATG CAA GCT TGG CGT AAT CAT GGT
CAC AAA GTG AGC TTG AAA TGA ATT CCC AGG
TGT GCA CCC TCA GTG CCT GGG TGG CTT ACC
TCC CTT AGG GGC AGG ATT GAC ACA TCC AAG
TCC ATT TCT GCT GGC CCA CTT CCC TGT CTC
CCA CCC ATT CCT CGT TAC AAT GTC AAT GCC
GGA ATG GGC ACT ATG ATT TTT ATC ATA TCG
CAT TGC CCC ATG GCT CCA AAA TTT CCC TCG
GGC TGC TTC AGA CAC CTG TGT AAA AAG GAG

Amp, ampicillin; BAC, bacterial artificial chromosome; HPRT, hypoxanthine phosphoribosyl transferase; IL, interleukin;
SLG, stable luciferase green; SLR, stable luciferase red.

in a white colony. As a result, almost all colonies
were blue and had the AmpR and ZeoR. Twentyfour of the resulting blue colonies were analyzed
by restriction enzyme pattern analysis, and 10 ofthese colonies had the expected recombinant bands(41.6%). Plasmid DNA extracted from 2 of thesecolonies was sequenced in the area of the recombination junction. All had the precise expectedcrossover event.

gene that expresses reporter genes in the control oftarget genome for use in genomic functional studies. RP11-67L14 was electroporated into DY380
cells, and 13 chloramphenicol-resistant colonies
were selected. To determine the transfer of the
BAC, we employed PCR analysis. Using the appropriate primers (IL-1 11041 S/IL-1 11851 AS)(Table 2), the expected amplicon was detected in 11
of 13 colonies (84.6%). The modification cassette
I was released from the modification cassette vector I by EcoRI digestion and used for BAC recombineering. DY380 cells carrying the BAC werethen shifted to 42 ˚C to induce recombination. The
cells were then electroporated with the modificationcassette I, and the ampicillin and chloramphenicolresistant colonies were selected. Approximately,
500 double resistant colonies were obtained. No

BAC modifications
We tested the activation and efﬁciency of BAC
modification system for the HAC vector. The modification cassette I was targeted to the start codon
locus of IL-1 carried on a BAC clone RP11-67L14(Fig. 2a). The purpose was to create a BAC trans-

27

S. Yamaguchi et al.

Modification cassette I

a

SLG

A'

AmpR

B'

Gene of interest
A

B

ATG

C

C'

pLac-LacZα

Cm R

BAC

AmpR, ampicillin-resistance
BAC, bacterial artificial chromosome
CmR, chloramphenicol-resistance
HAC, human artificial chromosome
HPRT, hypoxanthine phosphoribosyltransferase
HygR, hygromycin-resistanceIL,
interleukin
SLG, stable luciferase green
SLR, stable luciferase red
ZeoR, zeocin-resistanceD

loxP-3' HPRT

Zeo R

G3PDH promoter SLR

D'

Modification cassette II
BAC recombineering

SLG

AmpR

ATG

pLac-LacZαloxP-3' HPRT

Zeo R

Cm R

Modified BAC
G3PDH promoter SLR

BACe3.6 4168 S/LacZ 770 AS

SLR1650 S/BACe3.6 5839 ASb

HAC vector (21HAC1)

HygR

5' HPRT
loxP

AmpR

SLG

Cm R

Modified BAC
+ Cre

Zeo R

G3PDH promoter SLR

Cm R

3' HPRT

AmpR

5' HPRT

loxP-3' HPRT

Zeo R

G3PDH promoter SLR

HygR

SLG

pLac-LacZα

loxP

loxP

Fig. 2. Strategy of BAC modification and introduction into the HAC vector.
a: The 1st step of the process is the homologus recombination of the BAC using the modification cassette I. It occurs
by crossing over between the homology arms and the genome in the BAC. In this case, recombination between A
B and A' B' results in incorporation of the modification cassette sequences into the genome to yield the mBAC1.
The second step of the process is the homologus recombination of the mBAC1 using the modification cassette II. It
occurs by a second homologous recombination event that occurs between C D and C' D' of the mBAC1. Recombination yields the precisely modified BAC2 (mBAC2), with the modification cassette II inserted at the correct position in the BAC. This modified BAC carries loxP-3'HPRT region for introduction to the HAC vector.
b: Construction of the HAC vector containing the modified BAC using Cre/loxP system.

28

BAC modification system for an HAC vector

the genome and the loxP-3'HPRT cassette into a
specific site in the BAC. Our system will enable
insertion of large DNA fragments of interest at
predetermined locations in the HAC vector. Thissystem may thereby allow avoidance of the position
effects and actualize intrinsic expression of transgenes. Furthermore, BAC libraries have now been
generated from several species. Table 1 lists BAC
libraries that were constructed in the pBACe3.6.
These ready-made genomic BAC libraries can beloaded onto the HAC vector using our system.
Our system is based on BAC recombineering.
The recombination activity was assessed in using
the modification cassettes I and II. The results ofthese recombination assays indicated that BAC
recombineering conferred high recombination activity (cassette I; 100%, cassette II; 54.5%). This
technology may be suited for cassette exchange
reactions in the BAC. In addition, BACs can be
modified rapidly in vitro, with a construction timeof less than 2 weeks in this study. As the HAC
vector contains the 5' HPRT-loxP, the modified
BAC could be introduced into the HAC vector. By
introducing all candidate BAC clones into the same
genomic locus, site-specific integration of large
genomic clones can be used to streamline the positional cloning and transfer of genes into various
mammalian cells. This ability of BAC-HAC system will reduce the number of subclones and transgenic cells required to determine which clones contain the target gene. This system will allow generation of an isogenic series of assays that would be
expected to display comparable expression profiles
due to their placement in identical genomic andcellular backgrounds. In addition, the HAC vector
can be used for the generation of transgenic animals (trans-chromosomic mice) for the functional
analysis of a desired gene in mice (Hoshiya et al.,2009; Kazuki et al., in press). Therefore, our BACHAC system could provide significant advantages
for comparing the gene function of species at a specific locus, and allow for a better understanding of
the correlation between cell (in vitro) and animal (invivo). These types of assays are difficult to achievewith current techniques that rely on random inte-colonies were obtained from uninduced cells. PCR
analysis of 7 selected colonies using the primers(IL-1 10930 S/slg200 AS and amp2500 S/IL-1
12100 AS) (Table 2) that ﬂanked the targeted locus
indicated that all colonies were correctly targeted(100%). This recombineering efficiency was veryhigh. This means that SLG-AmpR cassette wasrecombined into the 1st ATG locus of IL-1 carriedon a RP11-67L14. This BAC was termed modified
BAC1 (mBAC1).
Next, to insert the loxP-3'HPRT region into
the mBAC1, the modification cassette II was targeted to the chosen site on the pBACe3.6 (Fig. 2a).
This modification cassette II was released from themodification cassette vector II by SacII-NdeI digestion and used for recombineering. DY380 cells
carrying the mBAC1 were then shifted to 42˚C.
The cells were then electroporated with the modification cassette II, and the ampicillin-, chloramphenicol- and zeocin-resistant colonies wereselected. As a result, 34 colonies were obtained.
These colonies were blue and had the AmpR, CmR
and ZeoR. PCR analysis of 11 selected colonies
using the primers (SLR1650 S/BACe3.6 5839 AS
and BACe3.6 4168 S/LacZ 770 AS) (Table 2) thatﬂanked the targeted locus indicated that 6 colonies
were correctly targeted (54.5%). This indicatesthat the modification cassette containing the loxP3'HPRT region was inserted into the mBAC1. This
BAC was termed modified BAC2 (mBAC2). Finally, mBAC2 had the correct insert genome as determined by PCR analysis using primers (IL-1 1
S/IL-1 3000 AS, IL-1 2611 S/IL-1 5180 AS, IL1 4031 S/IL-1 7151 AS and IL-1 8051 S/IL-1
11332 AS) (Table 2). This modified BAC carries aloxP and 3'HPRT sequence, and has the capacity ofsite-specific insertion into the HAC vector via Cre/
loxP system (Fig. 2b).

Discussion
In this study, we constructed a BAC modification
system for the HAC vector which can be used tointegrate the reporter gene into a chosen region of29

S. Yamaguchi et al.

gration of the target genes.
In summary, our system enables BAC modifications for the HAC vector to be quickly and easily
engineered. The use of BAC clones in conjunction
with the HAC vector opens the way to many additional applications, especially for investigating genefunction, gene therapy and animal transgenesis.

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Acknowledgments: The authors thank all colleagues in
our laboratory for valuable discussion and encouragement
through this study. We also thank Mr. Daigo Mori fortechnical assistance. We are grateful to Professor Mitsuo
Oshimura, Professor Masako Tada, Associate Professor
Toshiaki Inoue, Assistant Professors Mitsuhiko Osaki and
Masaharu Hiratsuka for their helpful suggestions.
This study was supported in part by the New Energy
and Industrial Technology Development Organizationfrom the Ministry of Economy, Trade and Industry of Japan. The first author was supported by Research Fellowships from the Japan Society for the Promotion of Sciencefor Young Scientists.

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Received January 25, 2011; accepted February 4, 2011
Corresponding author: Tetsuya Ohbayashi, ScD

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