A transposon-based chromosomal engineering methodto survey a large cis-regulatory landscape in miceChikara Kokubu1,2, Kyoji Horie2, Koichiro Abe3, Ryuji Ikeda2, Sumi Mizuno1, Yoshihiro Uno4,Sanae Ogiwara3, Masato Ohtsuka3, Ayako Isotani5, Masaru Okabe5, Kenji Imai3 & Junji Takeda1,2

A large cis-regulatory landscape is a common feature ofvertebrate genomes, particularly at key developmental geneloci with finely tuned expression patterns. Existing genetic toolsfor surveying large genomic regions of interest spanning overhundreds of kilobases are limited. Here we propose achromosomal engineering strategy exploiting the local hoppingtrait of the Sleeping Beauty transposon in the mouse genome.We generated embryonic stem cells with a targeted integrationof the transposon vector, carrying an enhancer-detecting lacZreporter and loxP cassette, into the developmentally criticalPax1 gene locus, followed by efficient local transpositions,nested deletion formation and derivation of embryos bytetraploid complementation. Comparative reporter expressionanalysis among different insertion/deletion embryossubstantially facilitated long-range cis-regulatory elementmapping in the genomic neighborhood and demonstrated thepotential of the transposon-based approach as a versatile toolfor exploration of defined genomic intervals of functional orclinical relevance, such as disease-associated microdeletions.

The advent of whole-genome sequencing has led to the perceptionthat individual genes should not be viewed in isolation, but be placedin the broader genomic context that includes neighboring genes andthe cis-regulatory sequences. Genomic regions that contain informa-tion for the correct spatiotemporal and quantitative regulation of aparticular gene locus often extend over hundreds of kilobases1.Emerging evidence indicates that such long-range architecture ofcis-regulation is a general feature of vertebrate genomes, particularlyat the loci of ‘developmental genes’, many of which encode trans-cription factors and other developmental regulators.

The mouse has offered distinct advantages for vertebrate functionalgenomics because of the availability of a large variety of genetic tools,which enable either genome-wide random mutagenesis by a chemicalor insertional mutagen, or pinpointing a single narrow locus (usuallyup to 30 kb) by gene targeting techniques. However, existing genetic

tools for surveying a considerable length of the genome, ranging froma few kilobases to a few megabases, are limited. Here we havedeveloped a chromosomal engineering strategy based on the SleepingBeauty (SB) transposon. SB is a binary system consisting of trans-poson DNA and the transposase enzyme, which were geneticallyreconstructed from inactive elements found in fish genomes2. Thetransposon DNA is flanked by the transposase-binding sites, so-calledinverted repeats/direct repeats and can be mobilized by the SBtransposase supplied in trans. The SB transposon has a strongtendency to reinsert during transposition at loci closely linked tothe donor site, referred to as ‘‘local hopping’’3,4. Our previous studyachieved germline saturation mutagenesis by mobilizing chromo-somally resident SB transposons in a 4-Mb region of mouse chromo-some 12, although in that instance the donor site was created byrandom integration of the transposon transgene concatemer5. In thepresent study, we exploited the local-hopping trait of a single-copy SBtransposon in a more sophisticated manner in combination with astandard gene targeting approach, thereby creating targeted andsaturated alterations over relatively large genomic distances. Thisstrategy, referred to hereafter as the Local Hopping EnhancerDetector (LHED) system, allows monitoring of enhancer activitiesalong the targeted genomic region and generation of a nested series ofdeletion mutations to examine the loss-of-function effects of thegenomic neighborhood.

In this study, we demonstrate the principle by applying this LHEDmethod to the mouse Pax1 transcription-factor gene locus, which ischromosomally located within a distinct region of evolutionaryconserved synteny along with the other key developmental genesNkx2-2 and Foxa2 (refs. 6,7). The Pax1 gene shows restricted expres-sion domains during mouse embryogenesis, including the sclerotomeand the limb buds, which contribute to the future axial skeleton andpectoral girdle, respectively. Intriguingly, the classical spontaneousmouse mutation Pax1Un-s (Undulated short-tail), which bears a122-kb chromosomal deletion encompassing the entire coding regionof the Pax1 gene, shows more severe skeletal dysmorphology than the

Received 19 May 2008; accepted 12 May 2009; published online 26 July 2009; doi:10.1038/ng.397

1Center for Advanced Science and Innovation, Osaka University, Suita, Osaka, Japan. 2Department of Social and Environmental Medicine, Graduate School ofMedicine, Osaka University, Suita, Osaka, Japan. 3Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai UniversitySchool of Medicine, Isehara, Kanagawa, Japan. 4Experimental Animal Science, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan. 5ResearchInstitute for Microbial Diseases, Osaka University, Suita, Osaka, Japan. Correspondence should be addressed to C.K. (ckokubu@mr-envi.med.osaka-u.ac.jp) orJ.T. (takeda@mr-envi.med.osaka-u.ac.jp).

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defined knockout allele Pax1null, indicatingthat the deletion affects not only Pax1 butalso additional gene(s) within or near thedeleted interval8. We showed previously thatNkx2-2 also, which resides 180 kb upstreamof the Pax1 locus and is normally expressedin the ventral neural tube, is ectopicallyactivated in the sclerotome and limb budsof Pax1Un-s embryos, both sites that normallyexpress Pax1 (Fig. 1a)9. As the sclerotomal andlimb-bud expression of Nkx2-2 is undetect-able in the Pax1null homozygote, the ectopicNkx2-2 in Pax1Un-s is not attributed to Pax1deficiency itself but rather to the cis-actingeffect of the genomic deletion. These findingssuggest that distant Pax1 enhancers remainin the Pax1Un-s genome and the 122-kb dele-tion mutation disrupts the transcriptionalinsulation between the neighboring geneloci Nkx2-2 and Pax1 (ref. 9).

From an experimental standpoint, thegenomic region encompassing Nkx2-2, Pax1and Foxa2 is an advantageous target for thiskind of long-range (possibly cross-border) cis-regulatory analysis because one can observepossible expression patterns for the threegenes in a single view at the same developmental stage (Fig. 1a andSupplementary Fig. 1). Thus, we introduced the transposon-basedLHED vector close to the mouse Pax1 locus and explored thesurrounding cis-regulatory landscape by inducing local transpositionof the reporter vector.

RESULTSDesign of the Local Hopping Enhancer Detector systemWe devised the LHED strategy, which is a triple combination ofstandard knock-in technology, transposon-based enhancer detection10

and Cre/loxP-mediated chromosomal engineering11. First, we clonedan enhancer-detector, consisting of the mouse Hspa1b (heat shockprotein 68, also known as Hsp68) minimal promoter fused to the lacZreporter12, into an SB transposon pT2 (ref. 13) (Fig. 1b). The resultingtransposon was then inserted into a selectable marker between the

pac (puromycin resistance, also called puro) gene and the mouse Pgk1(phosphoglycerate kinase-1, also called PGK) promoter. As reportedpreviously3, excision of the transposon results in reactivation of thepuromycin-resistance gene, enabling positive selection. Three loxPsites, one in the transposon and two outside, allow chromosomalengineering to generate a nested series of deletion/inversion mutationsbetween the fixed ‘knock-in’ site and the transposition site. Moreover,after transposition, the remaining vector sequence can be excised fromthe knock-in locus by Flp/FRT recombination.

Generation of a library of embryonic stem cellsWe generated embryonic stem (ES) cells with a targeted integrationof the pLHED vector into a location 60 kb upstream of the Pax1gene locus in mouse chromosome 2 (Fig. 1b). We then transientlytransfected 7 � 106 of the pLHED knock-in ES cells in vitro with

FRT FRT

FRT

FRT

FRT

FRT FRT

loxP loxP loxP

loxP loxP loxP

loxP loxP

loxP loxP

loxP

Sleeping Beautytransposase

Cre recombinase

Cre-induced deletion

Insertion-typehomologous recombination

pLHED targeting vector

Local hopping

PGKneo

49 kb

Pax1Transposon

Pgk1puro Hsp-lacZ

Pgk1

Transposon

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puroPGKneo

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Transposon

Hsp-lacZ

Nkx2-2

49 kb

5′ BP

5′ BP

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180 kb

∆1.5 kb

∆122 kb

Sclerotome

Limb bud

Neural tube

Notochord

Pax1

Wild type

Pax1null

Pax1Un-s

Chr. 2

Nkx2-2

a

b

Pax1

Pgk1BamHI

Figure 1 Experimental design. (a) Genomic

organization of the mouse Pax1 alleles and

neighboring gene expression patterns. Top:

in the wild-type allele, Pax1 and Nkx2-2 are

located 180 kb apart, in opposite transcriptional

orientations. Middle, bottom: the first two and

all exons of Pax1 are deleted in the Pax1null and

Pax1Un-s alleles, respectively. The cartoon below

each gene locus is a schematic transverse view

of E11.5 mouse embryos at the hindlimb-bud

level, illustrating the expression domains of

Pax1 (blue) and Nkx2-2 (red) from each allele.

BP, breakpoint. (b) The Local Hopping Enhancer

Detector (LHED) system. The insertion-type

targeting vector pLHED was knocked intothe 5¢ flank of the Pax1Un-s deletion interval.

SB transposase mobilizes the transposon

predominantly to closely linked sites. When

required, Cre recombinase can introduce

a chromosomal deletion between the fixed

and transposed loxP sites.

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the pCMV-SB11 transposase plasmid14 to mobilize the transposableelements. After 6 d puromycin selection, 592 resistant colonies wereisolated, whereas control pLHED knock-in ES cells (transfected withpCX-EGFP instead of pCMV-SB11) were all puromycin sensitive. Weconfirmed the excision of the transposon by PCR amplification of therecovered junction15 and never observed spontaneous puromycinresistance in the absence of transposon excision (data not shown),indicating that the frequency of transposon excision was approxi-mately 10�4 per transfected cell.

We used PCR to detect transposon insertions in 437 puromycin-resistant clones. Of these, 215 were positive for transposon insertion,which implies that another half of the excised transposons failed to landin the genome. Transposon insertion sites have so far been determinedfor 116 clones by splinkerette-adaptor mediated PCR16 (Supplemen-tary Data). Among them, 90 clones (78%) had the LHED transposoninserted somewhere in chromosome 2, and 49 clones (42%) acquiredinsertion within 1.5 Mb of the transposon donor site (Fig. 2a). Thistrait of local hopping resulted in predominant distribution of theLHED insertions along the 1-Mb region of our interest, encompassingthe Nkx2-2, Pax1 and Foxa2 loci (Fig. 2b). The ratio of the insertionorientation relative to the donor site was unbiased, with 46 insertionsin the same orientation and 44 insertions in the opposite.

Derivation of embryos from engineered ES cellsBefore starting a battery of cis-regulatory analyses in mice, weevaluated the intrinsic impact of the LHED vector on the hostphenotype by generating the knock-in mouse line. Chimeric micecarrying an LHED transposon at the J0 donor site (Fig. 2b) weregenerated by conventional diploid blastocyst injection. The J0 allelewas transmitted through the germline. Homozygous J0 mice, which

were genotyped by blot analysis of tail or yolksac DNA (Supplementary Figs. 2a and 3b,c),were healthy and fertile with no discerniblephenotype observed up to one year of age. Inaddition, whole-mount RNA in situ hybridi-zation analyses at embryonic day (E) 11.5revealed no changes in the expression patternof endogenous Pax1 in homozygous J0embryos compared with wild-type littermates(Supplementary Fig. 3d–g). These findingsindicate minimal impact of this reportersystem on Pax1 expression.

In principle, for cis-regulatory analysis, it issufficient to generate heterozygous embryoscarrying an LHED insertion. We thereforeinjected ES cell clones, chosen from the

LHED insertion library (Fig. 2b), into tetraploid blastocysts to obtainalmost entirely ES cell–derived embryos17. At E11.5, we stained wholemounts of the resultant embryos with 5-bromo-4-chloro-3-indolyl-b-D-galactoside (X-gal) under uniform staining conditions (Fig. 3). Forthe first example, the lacZ expression in J4 embryos, which carried anLHED insertion within the intron 4 of the Pax1 gene (Fig. 3a),faithfully reproduced the endogenous Pax1 expression in the scler-otome and limb buds9 (Fig. 3o,p), demonstrating that the Hspa1b-lacZ reporter carried by the SB transposon allows monitoring theinfluence of transcription-enhancing activity at each insertion site inthe native genomic context.

The vector-derived sequence, which is retained after transposonexcision at the donor site, could introduce artifacts into neighboringgenomic regulation. Nevertheless, J2/Flp embryos, which carried anLHED transposon 4 kb inside the Pax1Un-s deleted interval and hadthe donor site removed by Flp/FRT recombination (SupplementaryFigs. 2 and 4a), showed a lacZ expression pattern indistinguishablefrom the parental J2 embryos (Supplementary Fig. 4b–e). This resultsuggests that possible artifacts, which could arise from either of thetwo Pgk1 promoters within the residual vector sequence (see Fig. 1b),do not extend to the J2 site (15 kb away) at this embryonic stage.Incidentally, the enhanced lacZ expression in the ventral neural tubeof the J0 embryos (Fig. 3g,h) could be an artifact from the flankingvector sequence, which cannot be separately removed in this vectorconfiguration (Fig. 3a).

Surveillance of the region between Nkx2-2 and Pax1As deduced from Pax1 mutant phenotypes (Fig. 1a), some mechan-ism(s) of transcriptional insulation should exist between the Nkx2-2and Pax1 gene loci. In a classical picture of genomic organization,

M4-11B**

***

*

M345-12E

M4-9AM345-12A

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J(–1)

J1

J3

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Pax1 Foxa2

Pax1Un-s

(∆122 kb)

Base position

M5-4DM5-10D

Donor(J0)M4-5A

M345-5C

M5-12D

M345-6FM4-4C

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146.8 146.9 147.0 147.1 147.2 147.3 147.4 147.5 147.6 147.7 147.8 (Mb)

0

5

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2qA1 qC1.1 2qE1qC3 qE52qA3 2qB qD qE3 qH3 qH4F1 F3 H1

40 60 80 100 120 140 160 180 Distance from the centromere (Mb)

Mouse chr. 2

Donor site

a

b

Figure 2 Distribution of LHED insertions.

(a) Most insertion sites were clustered near the

donor site in mouse chromosome 2. The entire

chromosome is divided into 1-Mb windows and

the number of ES cell clones bearing an LHED

insertion in each window plotted. (b) Enlarged

1-Mb donor-site region from a, showing clustered

insertions around the Pax1 locus. J0, donor site;

J(�2), J(�1), J1–J6, LHED insertion sites

renamed for subsequent embryo production. Open

and solid red triangles represent the same and

opposite orientation of transposed loxP sites,

respectively, relative to the fixed donor loxP.

Insertions marked with asterisk reside within

either of the duplicated 8 kb homologysegments surrounding the donor site.

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specific DNA elements, called insulators, prevent inappropriate cross-activation of neighboring genes when placed between differentiallyregulated loci18. Assuming that such a model is applicable to thisgenomic region, an insulator element should reside within 49 kbupstream from the Pax1 transcription start site (TSS), which corre-sponds to the 5¢ half of the Pax1Un-s deletion interval9. However, at theother end of this hypothetical spectrum, the neighboring gene locimight be separated from each other solely by chromosomal distance.To test which of these (or other) hypotheses better explain theinsulation mechanism, we scanned the 49-kb insulator candidateregion with the LHED vector transposing from the J0 donor siteimmediately outside it. In practice, we monitored transcriptionalenhancer activity at different locations by whole-mount X-gal stainingof tetraploid rescued embryos, which had been derived from theJ1/Flp-, J2/Flp-, J3- and J4-ES cell clones (Fig. 3a). The lacZ expression

in the sclerotome and limb buds markedlydecreased as the LHED insertion movedupstream away from the Pax1 gene locus(Fig. 3k–p). Even when the LHED transposonwas inserted at the J1 site, 3 kb outside ofthe insulator candidate region, the J1/Flpembryos still showed positive, though atten-uated, X-gal staining in the sclerotome andlimb buds (Fig. 3i,j).

In the next step, we further investigatedthe candidate region by loss-of-functionexperiments in which the J3 and J4 EScells were engineered in vitro by Cre/loxP-mediated recombination to generate J3/Creand J4/Cre alleles carrying nested 45-kband 58-kb deletions, respectively (Fig. 3b).Removal of most or all of the candidateregion for an insulator did not affectlacZ expression in the sclerotome andlimb buds (Fig. 3m–p versus 3u–x). More-over, whole-mount RNA in situ hybridiza-tion of the J3/Cre and J4/Cre embryos usingthe Nkx2-2 probe showed only endogenous

expression in the ventral neural tube and no ectopic expressionelsewhere (Fig. 3q–t). Thus, removal of the insulator candidateregion in this context did not reproduce the Pax1Un-s expressionphenotype (Fig. 1a).

We also surveyed regions more upstream from Pax1, closer to theNkx2-2 locus (Fig. 3a). Whole-mount X-gal staining of J(�2)/Flpembryos, which carried an LHED insertion 41 kb from the Nkx2-2TSS, showed an expression pattern reminiscent of Nkx2-2 in theventral neural tube and no detectable expression in the sclerotomeand limb buds (Fig. 3c,d). Notably, lacZ expression in the ventralneural tube was attenuated in J(�1)/Flp embryos, in which theLHED insertion was located farther (105 kb) from the Nkx2-2 TSS(Fig. 3e,f).

Considered together, these results suggest that distance-dependentattenuation of enhancer activity underlies transcriptional insulation

hl

X-gal staining

J3/Cre

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wu

Nkx2-2 RNA in situ

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mb mb

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J3/Cre (∆45 kb)

Donor

Chr. 2J3

Nkx2-2 Pax1

J4/Cre (∆58 kb)Chr. 2J4

b

Chr. 2J(–1) J3

J4

Nkx2-2

49 kb

Pax1J(–2) J2

Pax1Un-s (∆122 kb)

Donor

J0 FRTFRT

J1

s

s

hls

hl

hlfl

shl

flm o

pn

s hlfl

shl

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s

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kJ3J1/Flp

J0J(–2)/Flp

shl

s hl

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vnt

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h

c

d

e

f

vntvnt

vnt mb mb mb

vnt

a Base position: 146.90 146.95 147.00 147.05 (Mb)

J(–1)/Flp

J2/Flp J4

J4/Cre J4/Cre

Base position: 146.90 146.95 147.00 147.05 (Mb)

Figure 3 Surveillance of the intergenic region

between Nkx2-2 and Pax1. Schematic diagrams

of: (a) LHED insertions in J(�2)-, J(�1)-, J0-,

J1-, J2-, J3- and J4-ES cells. LHED transposons,

containing a lacZ enhancer detector (blue

rectangle) and a loxP site (red open or solid

triangles), were transposed from the J0 donor site

surrounded by duplicated homology segments

(open rectangles). After transposition, J(�2)-,

J(�1)-, J1- and J2-ES cells had the remaining

donor site removed by Flp/FRT-mediated

recombination, referred to as J(�2)/Flp, J(�1)/

Flp, J1/Flp and J2/Flp, respectively. (b) J3/Cre-

and J4/Cre-alleles, in which nested deletions were

generated by Cre/loxP-mediated recombination.(c–x) Whole-mount X-gal staining (c–p,u–x) and

Nkx2-2 RNA in situ hybridization (q–t) of entirely

ES-cell-derived E11.5 embryos generated by

tetraploid complementation with engineered

ES cells: lateral views (c,e,g,i,k,m,o,q,s,u,w;

scale bars, 1 mm) and transverse views at

the level of the anterior edge of the hindlimb

buds (d,f,h,j,l,n,p,r,t,v,x; scale bars, 500 mm).

fl, forelimb; hl, hindlimb; mb, midbrain;

s, sclerotome; vnt, ventral neural tube.

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between the Nkx2-2 and Pax1 loci, which is distinct from theclassically proposed sharp insulator model9.

Surveillance of the 3¢ region of Pax1The persistence of enhancer activity in the Pax1Un-s allele (Fig. 1a)suggests that putative sclerotomal and limb-bud enhancers reside onthe distal side of the 3¢ deletion breakpoint. We therefore surveyed the3¢ distal region with LHED to map the long-range enhancers (Fig. 4).Whole-mount X-gal staining of J6 embryos (Fig. 4j,k), which carriedan LHED insertion 371 kb downstream of the Pax1 TSS (Fig. 4a),recapitulated an endogenous Pax1 expression pattern similar to thatof the J4 embryos (Fig. 3o,p). Cre/loxP-mediated removal of the423-kb J0–J6 fragment (Fig. 4a) resulted in negative X-gal stain-ing in the Pax1 expression domains except for a faint rudimentarystaining at the ventral tip of the sclerotome (Fig. 4l,m), indicatingthat the J6 site demarcates almost the distal end of the entire cis-regulatory region for Pax1. Notably, strong X-gal staining appeared inthe ventral neural tube and midbrain of J6/Cre embryos (Fig. 4l,m).As this pattern is very similar to that of endogenous Nkx2-2 as recapi-tulated in the J(�2)/Flp embryos (Fig. 3c,d), J6/Cre seems to be underNkx2-2 regulation.

We next surveyed a more proximal region toward the Pax1 locus bycomparing J5 embryos with J5/Cre embryos, which had an LHEDinsertion 217 kb downstream of the Pax1 TSS and a 270-kb deletionfrom J0 to J5, respectively (Fig. 4a). Whole-mount X-gal staining ofthe J5 embryos again recapitulated the endogenous Pax1 expressionpattern (Fig. 4b,c), but the Cre/loxP-mediated removal of the 270-kbJ0–J5 fragment resulted in substantial loss of X-gal staining in themain dorsal part of the sclerotomal domain (Fig. 4c,e versus 4g,i) andno detection in the most caudal nine somites (Fig. 4d versus 4h).

These results indicate that the genomic region between the 3¢ break-point and J5 sites is required for expression in the dorsal or entire partof the sclerotome, and that another region between the J5 andJ6 sites is required for expression in the ventral part of the sclerotome(Fig. 4a, top blocks).

To validate the above mapping information, we performed phylo-genetic footprinting analyses followed by conventional transgenicreporter assays. Cross-species conservation data of the 3¢ break-point–J5 and J5–J6 genomic intervals were inspected on the UCSCgenome browser19 (Supplementary Fig. 5). Within each interval, wefocused on conserved noncoding elements (CNE) showing the highestPhastCons conservation score19: one element (lod ¼ 874) in the 3¢breakpoint–J5 interval and another (lod ¼ 2563) in the J5–J6 interval.According to the Chain/Net tracks in the UCSC genome browser19,the former element, referred to as Xe1, is conserved across mammals,chicken and Xenopus, while the latter, referred to as Pf1, is conservedacross mammals, chicken, Xenopus and teleosts, including Tetraodon.We then cloned the Xe1 and Pf1 elements into Hspa1b-lacZ reporterconstructs12 to generate transgenic E11.5 embryos. As we expected,five independent Xe1 transgenic founder lines showed strong lacZexpression in the entire sclerotome (Fig. 4n,o), similar to that in J5(Fig. 4b–e). By contrast, four independent Pf1 transgenic founderlines showed weaker lacZ expression in the ventral part of thesclerotome (Fig. 4p,q), similar to that of J5/Cre (Fig. 4f–i). Thus,the long-range enhancer mapping by LHED ‘loss-of-function’approach was successfully validated by the transgenic ‘gain-of-function’ approach.

DISCUSSIONWe present an exploitation of the local hopping trait of the SBtransposon for mouse chromosome engineering. It is a noteworthycoincidence that the range of local hopping, from a few kilobases to afew megabases, is compatible with a typical size of chromosomal geneclusters and relevant cis-regulatory elements. Thus, the regionaldistribution of the LHED transposon insertions allowed in vivomonitoring of enhancer activities along the targeted Pax1 genelocus, and enabled the rapid generation of nested deletion muta-tions to dissect the effects of losses of function in the genomic

J6

Pf1-tgJ6/Cre

ss

s

vs

vs

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s hlfl

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k

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Xenopus sc486

Pax1Un-s (∆122 kb)

J6/Cre (∆423 kb)

J5/Cre (∆270 kb)

J0

Xe1

3′ BP

Pf1

Pax1Mouse chr. 2

Entire sclerotomeVentral sclerotome

e

i

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J5/Cre

s

shl

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a

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g

d

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Base position 147.0 147.1 147.2 147.3 147.4 (Mb)

J5 J6

Xe1-tg

Figure 4 Surveillance of the 3¢ region of Pax1. (a) Schematic diagram of the

LHED insertion alleles J5 and J6, nested deletion alleles of the spontaneous

Pax1Un-s, J5/Cre and J6/Cre, alongside the comparative genomic map

extracted from the Mouse February 2006 (mm8) assembly at the UCSC

Genome Browser19. Vertical bars, conserved DNA elements in the

corresponding Xenopus and Tetraodon chromosomes (details in

Supplementary Fig. 5); vertical yellow highlighting, Xe1 and Pf1 transgene

elements. Genomic regions responsible for the entire and ventral sclerotome

expression, based on interpretation of the embryo data below, are shown at

top. (b–q) Whole-mount X-gal staining of E11.5 embryos generated by

tetraploid complementation with engineered ES cells (b–m), and by

pronuclear injection of the Xe1-Hspa1b-lacZ (n,o) and Pf1-Hspa1b-lacZ

(p,q) transgenes: lateral views (b,f,j,l,n,p; scale bars, 1 mm) and transverse

views at the level of the anterior edge of the hindlimb buds (c,g,k,m,o,q;

scale bars 500 mm), plus drawings of transverse views of J5 (e) and J5/Cre(i) embryos showing expression pattern in the sclerotome. X-gal staining in

the most caudal nine sclerotomes (between arrowheads in d,h; scale bars,

1 mm) was completely lost in the J5/Cre embryo. Growth variation between

the tetraploid-rescued embryos (b–m) and the pronuclear-injected embryos

(n–q) fell within the period from E11.0–12.0, during which the endogenous

pattern of Pax1 expression is constant in the sclerotome. fl, forelimb;

hl, hindlimb; mb, midbrain; s, sclerotome; vnt, ventral neural tube;

vs, ventral sclerotome; open arrowheads, a faint rudimentary staining

at the ventral tip of the sclerotome.

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neighborhood, leading to the elucidation of a large cis-regulatorylandscape of this developmentally and evolutionary critical region.

Previous studies generated nested deletion series at defined loci byCre/loxP recombination, preceded by sequential delivery of two loxPsites into the same (cis) chromosome. The two loxP sites wereintroduced one by one into predetermined genomic locations byhomologous recombination, and the range of scanning was extendedin a ‘walk and delete’ manner20, which is the most controllable butgenerally labor-intensive approach. Alternatively, one of the two loxPsites was fixed at the target locus and the other was added byretrovirus-mediated random integration21. The latter occurs through-out the genome and cannot be directed to a predetermined regionnear the first loxP site. Therefore, the cis-arrangement of the secondloxP site relative to the first loxP site could be recovered only after abuilt-in selection step for appropriately deleted alleles21,22. That is, theretrovirus-based region-specific approach is not suitable for applica-tions that entail pure insertional mutagenesis, with no accompanyingdeletions being formed, such as single-gene knockouts or enhancertraps in a native genomic context. The present LHED approach iscompatible with pure insertional mutagenesis surrounding a targetlocus, as well as with nested deletion formation.

Because of the relatively low efficiency of SB transposition, theLHED system requires successive in vitro manipulation of ES cells,including a selection step for transposition-positive clones. Althoughthe in vitro manipulation offers a practical advantage in clonearchiving, further improvement of the transposition efficiencywould allow faster chromosome engineering in vivo. The feasibilityof such an in vivo approach was somewhat supported by the finding ofa recent study, in which the efficiency of a different transposon system,piggyBac (PB), allowed mutagenesis simply by animal breeding,although the PB transposon did not behave in a ‘local hopping’manner like the SB23. Meanwhile, another recent study reported apossible local hopping bias with PB, although this was detected in EScells, not in the mouse24.

A previous study noted that SB transposition from chromosomallyresident multicopy concatemers that had been introduced by conven-tional pronuclear injection could cause undesirable rearrangements,including deletions, inversions and translocations near the transposondonor site25. In that instance, a high copy number (approximately30 copies) of the transposon elements may have exacerbated thegenomic rearrangement. In contrast, our LHED system relied on asingle copy of the SB, transposing from a single-copy donor site.Furthermore, pLHED knock-in ES cells undergoing excision of thetransposable element were positively selected for puromycin resistanceconferred by rejoining the flanking sequences on both sides of thedonor site, which guarantees a breakage/rejoining region free fromundesirable rearrangements except for insertions of a few base pairs,known as the transposon ‘footprint’3. Thus, a single-copy transposon-based approach, such as our LHED system, provides the advantage ofminimizing illegitimate rearrangement of the manipulated genome.Indeed, none of the LHED-engineered alleles produced in this studyhas so far manifested any sign of unexpected rearrangement in itsgenotypes (Supplementary Fig. 2) and phenotypes (Figs. 3 and 4).Moreover, all recovered lines of LHED embryos (four to nine embryosper line) showed stable and reproducible patterns of lacZ expression,indicating that, unlike that in a previous cell culture study26, thereporter transposon does not undergo epigenetic silencing in vivo.

In this proof-of-principle study, the LHED-based genomic surveil-lance revealed that Pax1 enhancers act over a large region along thechromosome, with their influence decreasing with longer distance(Fig. 3). The enhancer activity reached even beyond the 49-kb

insulator candidate region (Fig. 3i,j), disproving our early hypothesisthat the Pax1Un-s deletion interval contains a classical ‘sharp’ insulator.This was reinforced by the RNA in situ analysis of J3/Cre and J4/Creembryos, in which Cre/loxP-mediated removal of the insulator candi-date region did not reproduce the Pax1Un-s-like ectopic expression ofNkx2-2 (Fig. 3q–t), although in such genomic layouts, the enhanceractivity from the Pax1 locus could be competitively absorbed by theintervening Hspa1b-lacZ promoter. Meanwhile, the lack of sclerotomaland limb-bud expression of Nkx2-2 in the wild type (Fig. 1a) might beattributed to the relatively weak sensitivity of the endogenous Nkx2-2promoter compared with the Hspa1b reporter promoter. This, how-ever, is unlikely because even at the J(�1) and J(�2) sites closer todistal Pax1 enhancers, the Hspa1b promoters did not respond to them(Fig. 3c–f). Furthermore, the 49-kb insulator candidate regioncontains at least three independent binding sites for the insulator-associated protein CTCF, according to the recent genome-wide studyusing chromatin immunoprecipitation coupled with high-throughputDNA sequencing (ChIP-seq) in mouse ES cells27. This suggests thatCTCF occupancy is different between ES cells and differentiatedsclerotomal tissues and/or that CTCF-binding sites do not alwaysfunction as sharply defined insulators28. Stepping out of the Pax1Un-s

interval, however, one may see a rather sharp transition of the ventralneural tube and midbrain expression between the J0 and J1 sites (Fig.3g,h versus 3i,j). There might be sharp functional boundaries corre-sponding to a distance threshold instead of a progressive attenuationof a sloppy system, even though these boundaries are not linked to thepresence of an insulator element at their position. As it depends on theinterpretation of possible artifacts to the J0 site arising from the adja-cent vector sequences, further LHED surveillance, probably startingfrom different donor site(s), should delineate an accurate ‘landscape’of this cis-regulatory region.

Our LHED approach also mapped the distant genomic regions ofenhancer activity, which drive the Pax1 gene expression in the entireor ventral parts of the mouse sclerotome (Fig. 4a, top blocks). In thedeveloping embryo, sclerotomal cells differentiate and migrate ven-tromedially from the somitic regions, which are periodically buddedoff and pushed rostrally from the caudal presomitic mesoderm29.Therefore, the ventrally restricted lacZ-positive domain in the J5/Creembryos (Fig. 4g,i) is considered to acquire the reporter expressionat later stages of development, which is consistent with the caudalloss of lacZ expression (Fig. 4h). In the teleost fish, the sclerotome isflattened ventrally and is much smaller than in mouse30. Therefore,from an evolutionary point of view, the ventrally restricted lacZexpression domain in Pf1 (Fig. 4p,q) and J5/Cre (Fig. 4f–i) mouseembryos could be reminiscent of the fish sclerotome, which is consistentwith the depth of conservation of the Pf1 element from mammals tofish. A similar correlation between evolutionary conservation depth andmorphology was recently suggested for the Tbx4 gene locus31.

Many long-range enhancers have so far been predicted throughphylogenetic footprinting and experimentally confirmed by mousetransgenic reporter assays32. This approach is not, however, a panaceafor all cis-regulatory elements. In the present study, comparison ofthe J5/Cre and J6/Cre nested deletion alleles indicated that the J5–J6region was required for limb-bud expression (Fig. 4f,g versusFig. 4l,m)—which does not immediately imply that the limb-budenhancer resides within. Indeed, our efforts with transgenic enhancerassays using phylogenetically conserved DNA fragments have notresulted in the identification of a discrete limb-bud enhancer withinthe J5–J6 region (data not shown). The enhancers in question couldbe unconstrained across evolution, which is not exceptional as sug-gested by recent findings33. Alternatively, the enhancer activity could

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consist of more than a single discrete element within or partly outsidethe J5–J6 region. The sufficiency of this 153-kb region for limb-budexpression could be attested by a bacterial artificial chromosome(BAC) transgenic approach34. Thus, loss-of-function analysis byLHED and gain-of-function analysis by BAC are mutually comple-mentary approaches.

In conclusion, our local hopping transposon vector system providesan experimental platform by region-specific insertional mutagenesis inmice, covering defined large genomic regions of functional impor-tance. Emerging high-throughput technologies have accelerated iden-tification of chromosomal copy number variations (CNVs) in thehuman genome, which could involve not only the coding sequencesof individual genes, but also cis-regulatory regions of phenotypicrelevance, such as in microdeletion syndromes35. By merely replacingthe transposon cargo with appropriately engineered DNA cassettes,the local hopping vector platform would be immediately applicable todissecting the molecular basis of such pathogenic CNVs.

METHODSMethods and any associated references are available in the onlineversion of the paper at http://www.nature.com/naturegenetics/.

Note: Supplementary information is available on the Nature Genetics website.

ACKNOWLEDGMENTSWe thank H. Hamada for supporting the initial phase of this study; R. Jaenisch(Massachusetts Institute of Technology) for providing V6.5 ES cells; P.B. Hackett(University of Minnesota) for the pT2/HB and pCMV-SB11 plasmids; andH. Sasaki (RIKEN Center for Developmental Biology) for the pASSHsp68lacZpAplasmid. We also thank M. Kouno, K. Yae, K. Yusa and V.W. Keng for advice andtechnical assistance. This work was supported by grants from the New Energyand Industrial Technology Development Organization of Japan; RIKEN, theInstitute of Physical and Chemical Research; and a grant-in-aid for scienceresearch from the Ministry of Education, Culture, Sports, Science, andTechnology of Japan.

AUTHOR CONTRIBUTIONSC.K., K.H., K.I. and J.T. designed the experiments; C.K. and J.T. performed vectorconstruction; C.K., K.H., R.I. and S.M. engineered and analyzed the mouseES cells; Y.U. generated the knock-in mouse line; A.I. and M.Okabe generatedmouse embryos by the tetraploid complementation technique; S.O. andM.Ohtsuka generated mouse embryos by pronuclear injection; C.K. andK.A. analyzed the embryos; K.I. assisted in interpretation of the embryonicdata; C.K. performed database analyses; and C.K. wrote the manuscript withassistance from K.H., K.A., M.Okabe and J.T.

Published online at http://www.nature.com/naturegenetics/.

Reprints and permissions information is available online at http://npg.nature.com/

reprintsandpermissions/.

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ONLINE METHODSTargeting vector construction. The generic insertion-type targeting vector

pLHED was constructed (Fig. 1b). All BamHI and HindIII restriction sites were

removed from the vector plasmid, so that one can choose any genomic region,

encompassing either a BamHI or a HindIII site, for the homology arm

containing the unique linearization site. For targeted integration into the

vicinity of the mouse Pax1 locus, a 7.7-kb genomic DNA fragment, which

encompasses the BamHI site 55 kb upstream from the Pax1 TSS, was inserted

into the pLHED plasmid as a homology arm. A 567-bp gap was pre-positioned

in the 7.7 kb homology, since it is considered to improve the recombination

efficiency36 (for example, as high as 40 percent in this instance). A detailed

description of the vector construction is provided in the following paragraphs.

Note that tetracycline operator (tetO) sequences were inserted within the SB

transposon of the pLHED plasmid (not depicted in Fig. 1b) and used for

different purposes in a separate research project in the laboratory15.

First, the XhoI–SacI fragment of pUHG10-3 (ref. 37), containing heptamer-

ized tet operators (tetO) was blunt-ended with T4 DNA polymerase, and

subcloned into EcoRV site of plasmid pBluescript II KS(+) (pBS, Stratagene),

resulting in pBS-tetO. The XbaI–EcoRI and HindIII–XhoI regions in the

multiple cloning sites (MCS) were deleted sequentially by digestion of the

pBS-tetO with the corresponding enzymes, followed by blunt-end self-ligation,

which resulted in pBS-tetO-DMCS. A NotI–KpnI tetO fragment from the pBS-

tetO-DMCS and a KpnI–BamHI loxP fragment from pMulti1.2 (ref. 38) were

ligated together into the NotI/BamHI-digested pBS-tetO to generate pBS-tetO-

loxP-tetO. A 4,237-bp KpnI/blunt-BamHI fragment of pASSHsp68lacZpA (ref.

39) (kindly provided by H. Sasaki) was inserted into the EcoRI/blunt and

BamHI sites between tetO and loxP in the pBS-tetO-loxP-tetO, resulting in

pBS-THLT, whose BamHI site was then removed by BamHI digestion, Klenow

fill-in and blunt ligation. The 4,965-bp NotI/blunt-HindIII fragment of the

pBS-THLT was inserted into the BglII/blunt and HindIII sites of a transposon

vector pT2/HB (ref. 13, kindly provided by P.B. Hackett), resulting in pT2/

THLT, whose HindIII site was then removed by HindIII digestion, Klenow fill-

in, and blunt ligation. pBS-PGKpuro was constructed by cloning a blunt-ended

SalI fragment of Pgk1-puro40 into the blunt-ended XbaI and SalI sites of pBS;

the orientation was such that the Pgk1 promoter was near the vector XhoI site.

The 5,578-bp BamHI fragment of the pT2/THLT was blunt-ended and inserted

into the blunt-ended HindIII site between the Pgk1 promoter and the

puromycin-resistant gene in the pBS-PGKpuro, resulting in pBS-PGK-T2/

THLT-puro, such that the inserted Hspa1b-lacZ was in the same transcriptional

orientation with respect to the Pgk1-puro in the vector.

In parallel, we generated the synthetic linker ASEFP, containing the AscI-

SfiI-EcoRI-FseI-PacI sites in that order, by annealing the oligonucleotides

ASEFP-f1 and ASEFP-r1 (Supplementary Table 1). The ASEFP linker was

ligated into the unique BamHI and EcoRI sites of the pFRT2 plasmid, resulting

in pFRT2/ASEFP. The blunt-ended HindIII–NotI fragment of the pFRT2/

ASEFP was then inserted into the blunt-ended SacI and SfiI sites of the

pMulti1.2 plasmid, from which most of the multiple cloning site (SalI to

XbaI), HindIII and BamHI sites had been destroyed in advance; the orientation

of the resulting pMulti-FRT2/ASEFP was such that the blunt-ended HindIII of

the insert was ligated to the blunt-ended SacI of the vector. A 7,313-bp ApaI/

blunt–NotI fragment of the pBS-PGK-T2/THLT-puro was inserted into the

XhoI/blunt and NotI sites of pMulti-FRT2/ASEFP, resulting in pLHED.

A 7.7-kb genomic fragment, which covers from �3 to �11 kb upstream of

the Pax1Un-s deletion9, was retrieved by the recombineering technique41 from

BAC RP23-224L16 (BACPAC Resources) into the PCR-amplified linearized

pBluescript II SK using chimeric primers a-Asc3F and b-Pac3R (Supplemen-

tary Table 1). The retrieved fragment was site-specifically inserted into the AscI

and PacI sites of the pLHED, resulting in pLHED-Pax1. The insert served as a

homology arm, in which a 567-bp gap was subsequently established by BamHI-

SfiI digestion, T4 DNA polymerase blunt-ending, and self-ligation, leading to

regeneration of a BamHI site. The resulting plasmid was named pLHED-

Pax1D, an insertion-type targeting vector for the 5¢ flank of the Pax1Un-s

deletion interval.

Cell culture and gene targeting. Culture of V6.5 mouse ES cells ((C57BL/6 �129S4Sv/Jae) F1)42 and targeted insertion of the pLHED vector into the 5¢upstream region of the Pax1 locus were described previously15. Briefly, 25 mg of

the pLHED vector was linearized at the unique BamHI site within the

homology arm and electroporated into 1 � 107 V6.5 ES cells. After a 7-d

selection with G418 (150 mg ml�1), correctly targeted ES clones were screened

by PCR and confirmed by DNA blotting as reported previously15. For the DNA

blot analysis, the 505-bp PCR fragment (3¢EP) amplified with primer pairs

3EPf4 and 3EPr4 (Supplementary Table 1) was used as an external probe, and

the 827-bp EcoRV-SacI fragment (LZ) derived from the lacZ gene was used as

an internal probe (Supplementary Fig. 2a).

In vitro mobilization of transposon. SB transposon was mobilized in vitro in

the genome of pLHED knock-in ES cells as described in detail previously15.

Briefly, the ES cells were plated at 1 � 106 cells per 10-cm feeder plate,

transiently transfected with 20 mg of pCMV-SB11 (ref. 14) (kindly provided by

P.B. Hackett) using TransFast (Promega), and cultured for 48 h, followed by 5 d

selection with puromycin (1 mg ml�1). The resistant colonies were picked into

96-well plates, grown and transferred to 96-well storage plates (Thermo Fisher

Scientific) for freezing and to replica plates for DNA analysis. Excision of

transposon was confirmed by PCR using the PuroL3 and PGKpU2 primers as

described previously15. Reinsertion of transposon was detected by PCR using

lacZ-specific primers LZ-F1 and LZ-R1 (Supplementary Table 1).

Splinkerette-adaptor mediated PCR. We recovered the sequences flanking the

inserted transposons by splinkerette-adaptor mediated PCR16. Genomic DNA

isolation was performed in 96-well plates using the DNeasy 96 Tissue Kit

(Qiagen). The isolated genomic DNA (100 ng) was digested with a 4-base

restriction enzyme (RsaI or HaeIII; New England BioLabs), followed by heat

inactivation of the restriction enzyme for 20 min at 65 1C or 80 1C, respectively,

and by subsequent ligation of the splinkerettes compatible with the resulting

blunt ends generated by annealing the oligonucleotide Spl-Top with Spl-Blt5

(Supplementary Table 1), using a DNA ligation kit version 1 (Takara). After

another heat-inactivation of the ligation reaction mixture for 10 min at 70 1C,

we performed nested PCR with first primers Spl-P1 and T/KBA (Supplemen-

tary Table 1), which are specific for the splinkerettes and the transposon vector,

respectively, by using the HotStartTaq system (Qiagen) under the following

conditions: 50 ml PCR volume; 1 cycle at 95 1C for 15 min; 30 cycles at 95 1C

for 30 s, 55 1C for 30 s and 72 1C for 1 min; and 1 cycle at 72 1C for 5 min. For

the second nested PCR, we used 1 ml of the first nested-PCR product as a

template under similar conditions, using second PCR primers Spl-P2 and T/

KB1 (Supplementary Table 1). Nested-PCR product bands were purified with

a gel extraction kit (Qiagen), followed by sequencing using the primer Spl-P2

or T/KB1. Sequence tags were processed by clipping off the vector sequence and

were subjected to BLAT genomic alignment against the UCSC Mouse February

2006 (mm8) assembly (http://genome.ucsc.edu).

In vitro Flp/FRT- or Cre/loxP-mediated recombination. For generating

deletion alleles, 2 � 105 ES cells with an LHED transposon insertion were

transiently transfected with 1 mg of the Flpe expression (pCAGGS-Flpe-IRES-

puro)43 or Cre expression (pBS185, Invitrogen) vectors using TransFast

(Promega), and cultured in a 24-well feeder plate for 48 h. The ES cells were

then plated sparsely (1 � 103 cells per 10 cm feeder dish) and grown without

selection. Individual colonies were picked into 96-well plates, screened and

confirmed by PCR and DNA blotting (see Supplementary Fig. 2).

Embryo production by tetraploid complementation. ES cell-derived embryos

were produced by tetraploid complementation as described previously44 with

the following modifications. Briefly, B6D2F1 two-cell-stage embryos were

placed in fusion buffer, and electrofusion was performed by applying 140 V

for 50 ms after aligning embryos between the electrodes. The resulting tetraploid

embryos were incubated for 2 d to obtain blastocysts. The LHED-engineered

diploid ES cells were injected into these tetraploid blastocysts, which were then

implanted into uteri of pseudopregnant females to obtain E11.5 embryos. All

animal experiments were performed in compliance with Osaka University

guidelines.

Transgenic enhancer assay. DNA fragments containing CNEs (Supplementary

Fig. 5) were PCR-amplified from the RP23 (C57BL/6J) mouse BAC clones

(BACPAC Resources), sequence-validated and transferred into the Hspa1b-lacZ

reporter vector12. The 1,689-bp Xe1 fragment (chr2:147251596–147253284)

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was amplified with primer pairs Xt1-F1 and Xt1-R1, while the 799-bp Pf1

fragment (chr2:147382852–147383650) was excised with KpnI and SphI from a

3,048-bp PCR product amplified with primer pairs pf1F26 and pf1R26

(Supplementary Table 1). Transgenic mice were generated by pronuclear

injection of linearized DNA into fertilized eggs as described previously6.

Whole-mount X-gal staining and RNA in situ hybridization. Whole-mount

X-gal staining of LHED-engineered and control mouse embryos was performed

at E11.5 as described previously6 under uniform staining conditions (30 1C, 6

h). Whole-mount in situ hybridization with Nkx2-2 or Pax1 RNA probe was

also performed on mouse embryos at E11.5 as described previously9.

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41. Liu, P., Jenkins, N.A. & Copeland, N.G. A highly efficient recombineering-basedmethod for generating conditional knockout mutations. Genome Res. 13, 476–484(2003).

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