Bacillus subtilis genome vector-based complete manipulation and reconstruction of genomic DNA for mouse transgenesis
- Tetsuo Iwata†1,
- Shinya Kaneko†2,
- Yuh Shiwa3,
- Takayuki Enomoto4,
- Hirofumi Yoshikawa3, 5 and
- Junji Hirota1, 4Email author
© Iwata et al.; licensee BioMed Central Ltd. 2013
Received: 1 February 2013
Accepted: 1 May 2013
Published: 3 May 2013
The Bacillus subtilis genome (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, stable propagation of cloned DNA and various modification strategies using RecA-mediated homologous recombination. However, genetic modifications using the BGM vector system have not been fully established, and this system has not been applied to transgenesis. In this study, we developed important additions to the genetic modification methods of the BGM vector system. To explore the potential of the BGM vector, we focused on the fish-like odorant receptor (class I OR) gene family, which consists of 158 genes and forms a single gene cluster. Although a cis-acting locus control region is expected to regulate transcription, this has not yet been determined experimentally.
Using two contiguous bacterial artificial chromosome clones containing several class I OR genes, we constructed two transgenes in the BGM vector by inserting a reporter gene cassette into one class I OR gene. Because they were oriented in opposite directions, we performed an inversion modification to align their orientation and then fused them to enlarge the genomic structure. DNA sequencing revealed that no mutations occurred during gene manipulations with the BGM vector. We further demonstrated that the modified, reconstructed genomic DNA fragments could be used to generate transgenic mice. Transgenic mice carrying the enlarged transgene recapitulated the expression and axonal projection patterns of the target class I OR gene in the main olfactory system.
We offer a complete genetic modification method for the BGM vector system, including insertion, deletion, inversion and fusion, to engineer genomic DNA fragments without any trace of modifications. In addition, we demonstrate that this system can be used for mouse transgenesis. Thus, the BGM vector system can be an alternative platform for engineering large DNA fragments in addition to conventional systems such as bacterial and yeast artificial chromosomes. Using this system, we provide the first experimental evidence of a cis-acting element for a class I OR gene.
Technological developments in chromosome engineering are essential for the manipulation and functional analysis of genomic DNA fragments. Artificial chromosomes, such as bacterial artificial chromosomes (BACs)  and yeast artificial chromosomes (YACs) , have been used for these purposes in combination with transgenesis. BAC and YAC transgenesis techniques have contributed greatly to genome research. However, there are several technological limitations in their cloning size, genetic modification and insert stability. BAC clones are easy to manipulate and retrieve due to their plasmid form and the stability of the cloned DNA. The methods for modifying BAC inserts require additional recombination components, e.g., RecA or phage-derived recombination proteins [3–6]. The BAC system can generally accommodate up to 300 kb genomic inserts. In contrast to the BAC system, genomic DNA inserts of up to 2 Mb can be used with the YAC system. Although the inserts can be easily modified by homologous recombination, YACs often suffer from insert chimerism and unwanted rearrangements due to potent and constitutive yeast recombination activity [7, 8]. Generally, the isolation of intact YACs is difficult because of their linear form and contamination with endogenous yeast chromosomes. Thus, these two systems have complementary advantages over each other in terms of cloning capacity and insert stability.
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 [9–12]. The cloning strategy for this vector system is based on unique B. subtilis features [13, 14]. B. subtilis expresses competence-related genes at the late stage of cell growth, and their products, transformation machinery molecules, are assembled in the cell membrane. The transformation machinery non-specifically binds and imports extracellular DNA fragments into the cytoplasm in single-stranded form. The recombinogenic DNA is incorporated into the B. subtilis genome by homologous recombination. Therefore, if the B. subtilis genome is engineered to have homologous cloning site sequences, a target DNA fragment flanked by homologous sequences is easily cloned into the BGM. Special handling of the BGM vector system is not required . B. subtilis can be cultivated under the same conditions as Escherichia coli in LB broth at 37°C. The competency of B. subtilis enables easy transformation procedures and efficient recombination reactions. For instance, 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 .
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 and the amenability of various modification strategies based on RecA-mediated homologous recombination [10, 16, 17]. These advantages make the BGM vector system an attractive alternative for the manipulation of large DNA. It has been used to clone genomic DNA from several species, including cyanobacteria , Arabidopsis and mouse [17, 18]. Modifications consisting of deleting and fusing cloned inserts have also been achieved [16, 17]. 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. In addition, the BGM system has not been applied to transgenesis. Thus, the BGM vector system is a developing technology with attractive potential, which includes its megabase-cloning capacity and homologous recombination-based genetic modification.
Cloning of genomic DNA fragments into the BGM vector
Targeted insertion of a reporter gene
Inversion of the insert in the BGM vector
Fusing two inserts to reconstruct genomic structures
DNA sequencing of modified and reconstructed BGM clones
Summary of the read mapping in DNA sequencing
Total reference length
Reads in pairs
Broken paired reads
Total number of reads
Total read length (Mb)
Fraction of reference covered
Generation of BGM transgenic mice
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 . Class I ORs are expressed almost 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 the olfactory bulb (OB) [28, 29]. The transgenes of the MOR42-3 locus are designed to activate the bicistronic expression of MOR42-3 and tauEGFP, 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, we analyzed 5 transgenic lines and 1 founder. None of the Tg-110 transgenic mice displayed labeled OSNs. For Tg-250SB, two transgenic lines established from two founders were EGFP positive (Figure 6B), 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 the dorsal region of the MOE and axonal projections into the dorsomedial and dorsolateral glomerulus of each OB (Figure 6C and E). To assess the copy number and integrity of the transgene, we analyzed Tg-250SB by Southern blot using MOR42-3 coding region as a probe (Figure 6D), which cross-hybridizes to highly homologous gene MOR42-1 (97% identical). Southern blot analysis showed that the specific band corresponding to the transgene 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. 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 for line#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 . Because MOR42-3 and MOR42-1, members of the MOR42 subfamily, share 97% amino acid similarity 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  in which an IRES-tauEGFP reporter was inserted downstream of MOR42-1. The glomerular positions of transgenic-MOR42-3 (Tg-MOR42-3) in Tg-250SB transgenic mice were similar to those of endogenous MOR42-1 (Figure 6C, E). We further analyzed the detailed structure of the Tg-MOR42-3 glomeruli. Glomeruli are innervated exclusively by axons from OSNs expressing the same OR . We 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 6F), suggesting that axons of OSNs expressing Tg-MOR42-3 formed glomeruli together with those of OSNs expressing endogenous MOR42-3. The projection site and converged glomerular structure of OSNs expressing the EGFP reporter suggest 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 from Tg-110 and provide the first experimental evidence of a cis-acting element for a class I OR gene.
The BGM vector system is a unique and developing technology for propagation of very large fragments of heterologous DNA. In this study, we have demonstrated that the BGM vector system enables the complete genetic modification of large genomic DNA fragments, including targeted insertion, deletion, inversion and fusion. In addition, we demonstrated the existence of a cis-acting element of a mouse class I OR gene by combining the BGM vector system with mouse transgenesis.
The BGM vector has several specific features that create advantages over the BAC and YAC systems. First, the megabase-scale cloning capacity of the BGM vector is greater than that of conventional systems. The BGM vector system is capable of cloning the entire 3.5 Mb Synechocystis genome , and the upper limit of clone size has not yet been determined. Second, cloned DNA inserts show high structural and genetic stability  because of their direct insertion into the single circular host genome. In fact, 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 OR genes. However, it should be noted that, similar to the YAC system, recombination is active in B. subtilis; thus, unwanted rearrangements may occur. This issue can be solved 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 in the DNA, enabling the repetitive modification of BGM inserts. In addition to insertion and deletion modifications, we 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 Mbp , the fusion of contiguous genomic fragments is a powerful technique for the reconstruction of gene structures surrounding intergenic regulatory elements [16, 33]. In addition, our sequencing analyses confirmed that these targeted genetic modifications were accurate and reliable. Fourth, BGM inserts can be simply retrieved by I-PpoI digestion because a single host cell contains a single genome composed of the recombinant insert and the 4.2 Mb BGM vector. As we 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 , enabling this “shuttle genetic modification” of BAC clones to enhance the utility of BGM vector system.
Finally, we have used mouse genomic DNA to demonstrate the suitability of the BGM system for genetic manipulation and transgenesis. Many BAC libraries have been already established and/or are under construction for diverse species, including mammals, other vertebrates and plants . Because the BGM vector harbors BAC vector sequences for cloning BAC inserts, this system can be applied to other species. Moreover, the BGM vector can be designed to clone other library resources by introducing cloning vector sequences from other systems, e.g., YACs and human artificial chromosomes. Because the BGM vector system can provide large cloning capacity in size and various 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 and recombinant genomes in synthetic biology [9, 35].
We demonstrated targeted insertion and inversion methods in the BGM system to add to its repertoire of genetic modification approaches. Using these techniques, a 252 kb transgene was reconstructed from two BAC clones whose inserts were initially oriented in opposite direction with reference to the BAC vector sequence. DNA sequence analysis of modified BGM clones demonstrated the genetic stability of inserts and correct modifications. Furthermore, we established and applied BGM-based mouse transgenesis. By analyzing the generated transgenic mice, a cis-acting element for a mouse class I OR gene was experimentally demonstrated for the first time. The BGM vector is a new platform that provides a complete genetic modification approach for large genomic DNA fragments without leaving selection markers or dispensable sequences. 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.
Strains and preparation of competent B. subtilis
The B. subtilis strains and the BAC-specific BGM vectors BEST310 and BEST6528 [10, 16] 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 [1, 10] and are identical with the exception that BEST6528 contains a 100 kb spacer sequence and an additional cI repressor gene cassette . 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 preparation of competent cells and transformation of B. subtilis were performed as described elsewhere . 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).
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 Research Institute. The BAC DNA was prepared by the alkaline lysis method and subsequent equilibrium centrifugation in a CsCl-ethidium bromide gradient . BGM strains were transformed with the purified BAC DNAs . BAC1 and BAC2 were cloned into BEST310 and BEST6528, respectively.
The cloned inserts in the BGM vectors were analyzed by I-PpoI digestion followed by CHEF electrophoresis. Agarose plugs containing BGM clones were prepared as described . A sliced block of the agarose plug was soaked in 200 μl of I-PpoI digestion buffer for 15 min to replace the TE buffer in the agarose plug. After discarding 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 and separated 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).
Southern blot analysis of the BGM clones
Genomic DNA from the BGM clones was prepared using the liquid isolation method . 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 and detection with NBT/BCIP were performed using a DNA labeling and detection kit (Roche).
Construction of plasmids
To construct the reporter cassette insertion plasmid, a linker 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  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 were prepared by PCR and contained sequences that were homologous to the upstream and downstream 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 . The te-erm cassette, which was obtained from a NotI-digested fragment from pBEAZ191 , was inserted into the PmlI site of this plasmid after blunt-end treatment to generate the plasmid p108Term. Similarly, the phl-et cassette, a NotI fragment from pBEAZ195 , 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, 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 right end 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 Additional file 2. 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.
Sequences of the original BAC and modified BGM clones (Tg-110CIBS, 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 the following: 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; and Tg250SB, a predicted fused mouse genomic Tg-250SB insert sequence. The mapping results are detailed in Table 1. After mapping the reads, variant calling was performed using the probabilistic variant detection function with default parameters, and variants with frequencies of at least 90% were considered. All read data have been deposited in the DDBJ Sequence Read Archive (DRA) under accession number [DRA000859].
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, as performed 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 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 estimated based on the control band intensity in the CHEF analysis. The purified transgenes were used immediately. Alternatively, 75 μM spermidine and 30 μM spermine were added for long-term storage. The stock transgenes were diluted to 0.3-1.5 ng/μl with injection buffer before use.
Production and analyses of transgenic mice
The purified transgenes were microinjected into the pronucleus of B6C3F1 (C3H/HeSlc male × C57BL/6NCrSlc female) mouse zygotes. Injected eggs were transferred to the oviducts of pseudopregnant female ICRs. The founders were screened by PCR with the following three primer sets that specifically amplified the internal and left and right ends of the transgenes: EGFP: GFP-F, 5′-GGCATCAAGGTGAACTTCAAGATCC-3′ and GFP-R, 5′-CTTTACTTGTACAGCTCGTCCATGC-3′; the left end of the BAC sequence: SacB-F, 5-GCTGAATACAACGGCTATCACG-3′ and SacB-R, 5′-TCTCTCAGCGTATGGTTGTCG-3′, or BAC108 L-F, 5′-CGTATTCAGTGTCGCTGATTTG-3′ and BAC108 L-R, 5′-TTAGCGATGAGCTCGGACTTC-3′; and for the right end of the BAC sequence: CmR-F, 5′-GAGGCATTTCAGTCAGTTGCTC-3′ and CmR-R, 5′-CGGCATGATGAACCTGAATCG-3′. 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 6.
For Southern blot analysis, 10 μg of genomic DNA extracted from a tail was digested with EcoRI (TaKaRa) and separated on a 1% agarose gel. The DNA was transferred onto a membrane filter, and hybridized 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 (ChemiDoc™ XRS, Bio-Rad). The copy numbers were estimated by comparing the intensities of transgenic and endogenous signals.
For 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 protocol . After post-fixation, permeabilization and antigen-retrieval pretreatment, sections were blocked with 10% normal horse serum and incubated with primary antibody at 4°C overnight. The following primary antibodies and dilutions were used: 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-conjugated donkey 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 a single image for display. Images were analyzed and adjusted using Photoshop CS4 (Adobe). All of the mouse studies were approved by the Institutional Animal Experiment Committee of the Tokyo Institute of Technology and performed in accordance with institutional and governmental guidelines.
Bacterial artificial chromosome
Bacillus subtilis genome
Contour-clamped homologous electric field
Enhanced green fluorescent protein
Main olfactory epithelium
Olfactory sensory neuron
Yeast artificial chromosome.
This work was supported in part by grant support from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Grants-in-Aid for Scientific Research (C) to J.H., Strategic Research Foundation Grant-aided Project for Private Universities 2008–2012 (S0801025) to Tokyo University of Agriculture from the Japan Society for the Promotion of Science, a Grant-in-Aid for Scientific Research on Innovative Areas to J.H. and support from the Kurata Memorial Hitachi Science and Technology Foundation and the Mishima Kaiun Memorial Foundation to J.H. We thank Dr. M. Itaya for the gift of the B. subtilis strains and plasmids and for critical review of the manuscript. We thank Dr. N. Kobayashi and K. Sumiyama for instruction of pronuclear-microinjection. We thank Dr. P. Mombaerts and T. Bozza for MOR42-1-iTGFP (termed S50iTG) mice. We also thank the members of the Hirota Laboratory for their continuous support.
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