extfieldsofstudy: Biology Medicine

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Multicopy integration of mini-Tn7 transposons into selected chromosomal sites of a Salmonella vaccine strain

Chromosomal integration of expression modules for transgenes is an important aspect for the development of novel Salmonella vectors. Mini-Tn7 transposons have been used for the insertion of one such module into the chromosomal site attTn7, present only once in most Gram-negative bacteria. However, integration of multiple mini-Tn7 copies might be suitable for expression of appropriate amounts of antigen or combination of different modules. Here we demonstrate that integration of a 9.6 kb mini-Tn7 harbouring the luciferase luxCDABE (lux) occurs at the natural attTn7 site and simultaneously other locations of the Salmonella chromosome, which were engineered using λ-Red recombinase to contain one or two additional artificial attTn7 sites (a-attTn7). Multicopy integration even at closely spaced attTn7 sites was unexpected in light of the previously reported distance-dependent Tn7 target immunity. Integration of multiple copies of a mini-Tn7 containing a gfp cassette resulted in increasing green fluorescence of bacteria. Stable consecutive integration of two mini-Tn7 encoding lacZ and lux was achieved by initial transposition of lacZ-mini-Tn7, subsequent chromosomal insertion of a-attTn7 and a second round of transposition with lux-mini-Tn7. Mini-Tn7 thus constitutes a versatile method for multicopy integration of expression cassettes into the chromosome of Salmonella and possibly other bacteria.

Introduction
Engineering of whole bacterial genomes has advanced rapidly in the last decade (Carr and Church, 2009;Feher et al., 2012). However, combination of multiple functions and their orchestration remains challenging. Serial modification of bacterial genomes, such as chromosomal integration of large expression modules in a rapid, targeted and stable manner is difficult to achieve, especially in bacteria other than Escherichia coli. These difficulties also hamper the development of new recombinant live-attenuated bacterial vaccines such as Salmonella enterica ssp. (Salmonella) vector vaccines and the exploration of their potential as vector platform. Current strategies for rational design of these vaccines are based on a detailed understanding of bacteria-host interaction and the specific requirements for the induction of protective immune responses against a targeted pathogen (Galen et al., 2009;Hegazy and Hensel, 2012;Roland and Brenneman, 2013;Wang et al., 2013b;Galen and Curtiss, 2014). One approach for Salmonella vaccine strain development is the removal of genes in order to attenuate virulence, reduce metabolic burden and genetically stabilize or redirect intracellular trafficking of bacteria. Another strategy is based on programming of the bacteria for the delivery of a heterologous cargo, such as proteinaceous antigens or DNA vaccines. In this case, expression modules for one or several heterologous antigens in addition to other factors, such as lysis determinants, secretion system components, regulatory factors or adjuvant molecules, have to be stably introduced into the respective candidate strain. By combining these strategies, a vaccine strain was engineered to contain 13 gene deletions and two chromosomal integrated expression cassettes in addition to the plasmid-based DNA vaccine, yielding a self-destructing Salmonella vector system for efficient DNA vaccine delivery in mice (Kong et al., 2012). Although this provides an important proof of principle for Salmonella vector development, the recombinase-based suicide vector technology used to achieve these precise deletion/deletion-insertion mutations is cumbersome (Kang et al., 2002).
Alternatively, λ-Red recombineering has become a frequently used method for targeted gene deletions and chromosomal insertions of small DNA fragments (Sawitzke et al., 2007;Sharan et al., 2009). The polymerase chain reaction (PCR)-based version of this method (Datsenko and Wanner, 2000) allows deletion of chromosomal genes by transformation of an amplified DNA fragment, containing a selectable marker flanked by ∼ 50 bp extensions homologous to the chromosomal target sequence. In the presence of λ-Red recombinase, the specified chromosomal sequence is then replaced by the selectable marker gene in the recipient strain, which is subsequently removed. However, only relatively short additional heterologous DNA sequences can be chromosomally integrated this way due to a decrease of recombination efficiency for fragments exceeding ∼ 1500 bp (Datsenko and Wanner, 2000;Kuhlman and Cox, 2010). Chromosomal integration of larger fragments can still be achieved by λ-Red when long flanking homology regions are employed (Yu et al., 2011;Dharmasena et al., 2013;Sabri et al., 2013;Wang et al., 2013a), but such fragments cannot easily be generated by PCR. This obstacle can be overcome by a two-step procedure, in which λ-Red recombineering is combined with a second method. For example, λ-Red was used in the first step for the integration of a so-called landing pad into a selected chromosomal locus, which is a DNA fragment harbouring a selection marker flanked by I-SceI recognition sites and short sequences homologous to the integration cassette (Kuhlman and Cox, 2010). Subsequently, this integration cassette is transformed into bacteria, excised by I-SceI and recombined into the I-SceI-cleaved landing pad region, allowing the targeting of a 7 kb cassette into selected loci of the E. coli chromosome.
Mobile elements, such as transposon or phage-derived systems, integrate either randomly or site specifically into the bacterial genome and therefore do not allow deliberate targeting (Choi and Kim, 2009;Akhverdyan et al., 2011;Murphy, 2012;Loeschcke et al., 2013). Tn7-derived transposons, so-called mini-Tn7, have been frequently used for site-specific, single-copy integration of large DNA fragments into the chromosome of Gram-negative bacteria including Salmonella (Bao et al., 1991;Yan and Meyer, 1996;Choi et al., 2005;Loessner et al., 2007;Kvitko et al., 2013). In few bacteria, e.g. Burkholderia spp. or Proteus mirabilis, two or three natural sites have been found, which can be targeted by mini-Tn7 2014). In its natural form, Tn7 is a 14 kb transposable element encoding genes tnsABCDE of the transposition machinery and additional antibiotic resistance genes (Lichtenstein and Brenner, 1982;Peters and Craig, 2001). Recognition of its attachment site attTn7, located downstream of gene glmS in most bacteria including Salmonella spp., is mediated by the DNA-binding protein TnsD that recruits TnsC, an ATPdependent DNA-binding protein, thereby linking target recognition with activation of transposase TnsAB (Bainton et al., 1993;Choi et al., 2014). Orientation-specific insertion of Tn7 is mediated by the asymmetric left and right transposon ends, Tn7L and Tn7R respectively. Both ends contain multiple TnsB binding sites. The presence of these sites in the chromosome as a consequence of transposon integration was described to confer so-called target immunity impeding further transposition events by a mechanism in which bound TnsB inactivates TnsC (Arciszewska et al., 1989;DeBoy and Craig, 1996;Stellwagen and Craig, 1997). This immunity effect was shown to be active at positions of the E. coli chromosome, which are located up to 190 kb apart from TnsB binding sites but integration into a site located 1900 kb away was not inhibited .
In this work, we have combined λ-Red recombineering with chromosomal integration of mini-Tn7 into the genome of the attenuated S. enterica serovar Typhimurium (S. Typhimurium) vaccine strain SL7207. We demonstrate that λ-Red-mediated integration of one or two artificial attTn7 sites (a-attTn7) into chromosomal loci of choice allows subsequent integration of multiple mini-Tn7 copies simultaneously or consecutively into the bacterial genome. Chromosomal integration of one, two or three copies of a mini-Tn7 containing a gfp expression cassette allowed us to modulate GFP expression levels. Furthermore, consecutive integration of two different mini-Tn7 harbouring lacZ and lux, respectively, gave rise to a multifunctional strain.

Results
Mini-Tn7 transposons are frequently used for single-copy integration of DNA fragments into chromosomes of Gramnegative bacteria, with few exceptions (Choi et al., 2005;Crepin et al., 2012). However, consecutive integration of multiple mini-Tn7 copies into additional attTn7 sites present in the E. coli chromosome was until now thought to be suppressed in a distance-dependent manner by Tn7 target immunity (Arciszewska et al., 1989;DeBoy and Craig, 1996;Stellwagen and Craig, 1997;Choi et al., 2014). Here we investigated the possibility of simultaneous integration of two mini-Tn7 copies into attTn7 as well as an additional a-attTn7 site inserted at genomic positions at varying distances apart from attTn7 in the S. Typhimurium vaccine strain SL7207. The a-attTn7 was derived from the S. Typhimurium strain SL1344 genomic sequence and contains 105 bp of the 3′ end of glmS, 140 bp intergenic region and 45 bp of the 3′ end of gene SL1344_3827. The glmS sequence harbours the entire TnsD binding site and the intergenic region attTn7 downstream of glmS (Mitra et al., 2010). We successively replaced chromosomal genes ara, asd, endA, recF, rha and sifA of strain SL7207 with a-attTn7 one at a time by λ-Red-mediated homologous recombination (Fig. 1A). The targeted loci were located at varying distances below or above 190 kb apart from attTn7 (Fig. 1B). We then subjected the six different mutant strains carrying attTn7 as well as one additional a-attTn7 site to transposition with lux-mini-Tn7, which contains a constitutive lux-cassette and a kanamycin marker. Bioluminescent Salmonella colonies with chromosomal insertions of luxmini-Tn7 were selected on LB plates containing streptomycin and kanamycin. Colony PCR of all mutant strains revealed that integration of lux-mini-Tn7 occurred into both integration sites (Fig. 1C). This was even the case for strains carrying a-attTn7 in the recF and the rha loci, which are less than 190 kb away from attTn7. Therefore, we did not observe immunity-related inhibition of lux-mini-Tn7 integration into either one of the two transposon integration sites present in the genome of all tested mutant strains.
To investigate simultaneous integration of lux-mini-Tn7 into additional chromosomal locations, a-attTn7 sites were integrated into ara and asd loci by two subsequent rounds of λ-Red-mediated gene replacement ( Fig. 2A). The intermediate strain harbouring three Tn7 integration sites was then again subjected to transposition with luxmini-Tn7 ( Fig. 2A). In this strain the three Tn7 integration sites were spaced more than 190 kb apart from each other. PCR analysis of colonies obtained from selective plates revealed that mini-Tn7 integration occurred consistently into all three transposon integration sites in all tested colonies (Fig. 2B). Such clones could also be ΔrecF::a-attTn7 SL7207 attTn7::lux ΔrecF::lux a-attTn7 a t t T n 7 : : l u x Δ r e c F : : l u x a t t T n 7 : : l u x Δ r h a : : l u x a t t T n 7 : : l u x Δ a s d : : l u x a t t T n 7 : : l u x Δ e n d A : : l u x a t t T n 7 : : l u x Δ a r a : : l u x a t t T n 7 : : Fig. 1. Integration of two lux-mini-Tn7 into attTn7 and in addition one a-attTn7 site inserted into selected chromosomal loci of S. Typhimurium. A. Strain SL7207 was modified in two steps for each of the indicated genes. λ-Red-mediated deletion and concurrent integration of a-attTn7 (black circle) is depicted for recF. Strains harbouring the natural attTn7 site (black square) and in addition a-attTn7 were used for transposition with lux-mini-Tn7 9.6 kb in size (white inversed triangle). B. Approximate distances between attTn7 and the newly introduced a-attTn7 sites in respective strains. C. Integration of lux-mini-Tn7 into attTn7 or a-attTn7 (indicated by combined symbols) was confirmed by the bioluminescent phenotype (data not shown) and by colony PCR with primers homologous to sequences of the right end of lux-mini-Tn7 (Tn7R) and the neighbouring genomic location (Supporting Information Table S1). The expected band sizes for lux-mini-Tn7 integration into attTn7 is 332 bp, for integrations into a-attTn7 sites in mutant strains are recF 586 bp, rha 1523 bp, asd 629 bp, endA 526 bp, ara 378 bp and sifA 543 bp respectively. Δ indicates a gene deletion and double colon indicates a chromosomal insertion. PCR data for representative clones are shown.
identified due to the bioluminescent phenotype alone by plating a dilution series of the triple mating culture on LB plates containing only streptomycin. The mean frequency of identified positive colonies from three experimental repetitions was (4.5 ± 1.7) × 10 −5 , indicating that high efficiency of multicopy integration can even be achieved with a mini-Tn7 devoid of an antibiotic resistance marker if phenotypical identification of colonies is possible.
To determine if chromosomal integration of multiple copies of a gfp-mini-Tn7 allows a stepwise, copy numberdependent increase in the expression level of a transgene, we integrated one, two or three copies of a GFP expression cassette into SL7207. Towards this, either one or two additional a-attTn7 sites were initially recombined into the ara and asd loci, and then a gfp-mini-Tn7 was used for transposition of respective strains. Colonies containing insertions within the present Tn7 integration sites were identified by colony PCR (Fig. 3A). These clones were then grown in liquid medium and green fluorescence of cells was analysed by flow cytometry upon induction of GFP synthesis by addition of L-arabinose. The level of green fluorescence intensity measured correlated with the copy number of chromosomally integrated gfp-mini-Tn7 at selected loci, while cultures grown in the absence of L-arabinose displayed only low GFP fluorescence ( Fig. 3B and Supporting Information Fig. S1).
In addition to the integration of multiple copies of the same mini-Tn7, we also investigated the consecutive integration of two different mini-Tn7 carrying lacZ and lux into strain SL7207. In a first round of transposition, either lacZ-mini-Tn7 or lux-mini-Tn7 was integrated into the native Tn7 recognition site attTn7. Thereafter, the strain harbouring lacZ-mini-Tn7 was subjected to λ-Redmediated integration of a-attTn7 into the rha locus. Transposition of lux-mini-Tn7 into this strain gave rise to bacteria harbouring both mini-Tn7 encoding lacZ and lux (Fig. 3C). Phenotypical analysis of these strains confirmed the expression of lacZ, lux or both transgenes (Fig. 3D). To assess the stability of lacZ and lux expression cassettes we evaluated colonization and foreign gene expression of the recombinant S. Typhimurium A. A SL7207 derivative with the natural attTn7 site (black square) and two additional a-attTn7 sites (black and white circles) was generated by two rounds of λ-Red-mediated recombination linked with chromosomal integration of the a-attTn7 sequence each time. B. Strain SL7207Δara::a-attTn7Δasd::a-attTn7 harbouring three sites for Tn7 transposition was used for transposition with lux-mini-Tn7 (white inversed triangle). lux-mini-Tn7 integration into the three sites was confirmed by colony PCR for 10 bioluminescent clones selected on LB plates containing streptomycin and kanamycin. The strain harbouring three empty Tn7 transposition sites was used as control template. The band size for lux-mini-Tn7 integration into attTn7 is 332 bp and for integrations into a-attTn7 are ara 378 bp and asd 629 bp, band positions are indicated by black arrow heads.
strains in BALB/c mice. Upon oral administration, tissue colonization of bioluminescent SL7207 bacteria peaks around day 7 after oral administration (Burns-Guydish et al., 2005). Groups of five mice were orally inoculated with the original strain SL7207 and derivatives attTn7::lux and attTn7::lacZΔrha::lux. The in vivo colonization course of the two bioluminescent strains was followed by non-invasive in vivo imaging for 9 days (Supporting Information Fig. S2A). Mice were then sacrificed, and Peyer's patches, mesenteric lymph nodes and spleens were harvested for determination of colonyforming units (Supporting Information Fig. S2B). We found that all tested strains readily colonized the analysed organs, even though the strains expressing additional transgenes yielded lower numbers, indicating an attenuating effect. However, all engineered bacteria retained lacZ and lux expression as confirmed by phenotypical examination of bacterial colonies recovered from organs (Supporting Information Fig. S2B), illustrating that consecutive transposition of different mini-Tn7 is a feasible approach for stable chromosomal integration of various heterologous expression cassettes into Salmonella and should also be considered for other Gramnegative bacteria. a t t T n 7 : : l a c Z lux lacZ a t t T n 7 : : l u x a t t T n 7 : : l a c Z Δ r h a : : a -a t t T n 7 a t t T n 7 : : l a c Z Δ r h a : : l u x S L 7 2 0 7 a t t T n 7 : : l a c Z a t t T n 7 : : l u x a t t T n 7 : : l a c Z Δ r h a : : a -a t t T n 7 a t t T n 7 : : l a c Z Δ r h a : : l u x D C Fig. 3. Modulation of GFP synthesis by chromosomal integration of either one, two or three copies of gfp-mini-Tn7 and consecutive chromosomal integration of two different mini-Tn7 encoding lacZ and lux into S. Typhimurium strain SL7207. A. Strain SL7207 with the natural attTn7 site (black square) or derivatives with one or two additional a-attTn7 sites integrated into ara and asd loci (black and white circles) were used for transposition with gfp-mini-Tn7 harbouring the L-arabinose inducible gfp expression cassette (grey inversed triangle). gfp-mini-Tn7 integration into each site was verified by colony PCR and expected bands were obtained at 332 bp for integration into attTn7, 378 bp for integration into a-attTn7 at the ara locus and 629 bp for integration into a-attTn7 at the asd locus. Bands are indicated by black arrow heads. B. Strains SL7207 and derivatives with one, two or three chromosomal copies of the gfp cassette were grown in the absence or presence of L-arabinose prior to flow cytometric analysis. Depicted mean fluorescence intensities (MFI) represent three experimental repetitions and error bars indicate standard deviation. C. Strain SL7207 was used for transposition with lacZ-mini-Tn7 or lux-mini-Tn7 (black and white inversed triangles). Recipient strains carry either lacZ or lux within attTn7 (black square) and are designated attTn7::lacZ and attTn7::lux respectively. Subsequently, the rha operon of strain attTn7::lacZ was replaced by a-attTn7 (white square) giving rise to strain attTn7::lacZΔrha::a-attTn7. This strain was employed for another transposition round with lux-mini-Tn7 yielding strain attTn7::lacZΔrha::lux. Colony PCR verified transposon integrations into the chromosome of each strain. Band sizes for integration into attTn7 are 332 bp and into a-attTn7 at the rha locus 1523 bp. D. lacZ expression of bacteria was observed on X-Gal plates by blue dye formation and lux expression by detection of bioluminescence from the same plate.

Discussion
Recombinant Salmonella vector vaccines stimulate humoral and cell-mediated immune responses in mucosal and systemic compartments to self-antigens as well as to heterologous antigens derived from infectious agents or malignant tissues (Galen et al., 2009;Curtiss et al., 2010;Paterson et al., 2010;Hegazy and Hensel, 2012;Roland and Brenneman, 2013;Toussaint et al., 2013;Wang et al., 2013b). The potency of such a vaccine depends on its ability to induce high-level production of the respective antigens once bacteria have reached the immune inductive sites. Therefore, the introduction of appropriately tailored antigen expression cassettes is an important step during construction of a recombinant Salmonella vector vaccine. The use of a self-replicating plasmid is one option for the introduction of such cassettes. Such plasmids are designed to assure stable retention by the recipient bacteria and to maintain colonization of immune inductive tissues (Bauer et al., 2005;Galen et al., 2010;Xin et al., 2012). However, this situation is difficult to achieve with multicopy plasmids harbouring expression cassettes for large gene clusters, but these limitations can be overcome by integrating such cassettes into the bacterial chromosome (Husseiny and Hensel, 2008;Yu et al., 2011;Dharmasena et al., 2013).
A variety of transposons have been used as gene delivery tools for bacteria and eukaryotic cells (Choi and Kim, 2009;Ivics and Izsvak, 2011). Tn7 is the hallmark transposon for site-specific single-copy integration into the chromosome of Gram-negative bacteria (Peters and Craig, 2001). Here we investigated the possibility of integrating multiple copies of a mini-Tn7 into the chromosome of the S. Typhimurium strain SL7207 containing several attTn7 sites. Towards this, we used λ-Red recombineering for the insertion of initially one a-attTn7 site into selected positions of the Salmonella chromosome located at varying distances from attTn7. Using a 9.6 kb mini-Tn7 with a constitutive luciferase expression cassette, we obtained for each position transposon insertions at both sites attTn7 and a-attTn7. Surprisingly, this was independent from the distance between the sites, which was as small as 27 kb in the case of the recF locus and as large as 2054 kb in the case of the sifA locus. Integration of three lux-mini-Tn7 copies into attTn7 and two additional a-attTn7 sites located in the asd and ara loci occurred with the same consistency. To our knowledge, this is the first report of integration of multiple mini-Tn7 copies into deliberately selected loci of the bacterial chromosome down to a distance below 190 kb between integration sites, which was previously considered inefficient due to Tn7 target immunity . Chromosomal integration of multiple mini-Tn7 copies has been reported to occur naturally in Burkholderia ssp. and P. mirabilis, which carry two or three natural integration sites in their bacterial chromosomes . For example in Burkholderia pseudomallei strain 1026b three natural attTn7 sites are present, which are located downstream of three glmS genes (Choi et al., 2008). Mini-Tn7 integration occurs in > 65% of events as single-copy integrations downstream of gene glmS2. The other two attTn7 sites downstream of genes glmS1 and glmS3 were targeted by single-copy integration of mini-Tn7 at much lower rates. Double insertions of a mini-Tn7 in one cell occur at a rate of 10-20%, but triple insertions at once were only rarely observed (Choi et al., 2008). The reason of the low double and triple mini-Tn7 insertion frequency in this strain is unknown. Tn7 target immunity would not be expected to be the obvious reason as attTn7 sites on chromosome 1 are 1,1 Mb apart from each other, and one attTn7 site is located on chromosome 2. In P. mirabilis strain HI4320,  always observed simultaneous mini-Tn7 insertion into two sites, the attTn7 site downstream of glmS and one site located in carA, when bacteria were grown in rich medium. These sites are spaced 724 kb and would also not expected to be affected by Tn7 target immunity. However, in our work with Salmonella we observed a frequency of 100% of simultaneous mini-Tn7 insertions at two or three attTn7 sites, even in the case of closely spaced attTn7 sites 27 or 188 kb apart.
Designing Salmonella vector vaccines for the immunization against heterologous pathogens has so far required a number of precise chromosomal gene deletions and module integrations in parallel. The combination of λ-Red-mediated gene deletion with mini-Tn7 integration reported here has the potential to considerably facilitate this process. As target loci for mini-Tn7 integration, we specifically selected genes, which have been previously removed from Salmonella vaccine strains as such sites should be suited for the stable integration of heterologous expression cassettes. Δasd strains were used as hosts for plasmids stabilized by the complementing essential asd gene, referred to as balanced lethal systems (Nakayama et al., 1988). In addition, the Δasd mutation is present in strains undergoing a so-called delayed type of bacterial lysis (Kong et al., 2008). Δara and Δrha mutations have been introduced into vaccine strains in order to prevent degradation of L-arabinose or L-rhamnose when such sugars are used as inductors of gene expression (Kong et al., 2008). ΔrecE strains were shown to display a reduced capacity for recombination and therefore are suited for stable maintenance of dualplasmid systems in vaccine strains (Zhang et al., 2011;Xin et al., 2012). ΔsifA strains were employed for the cytosolic delivery of heterologous antigens or DNA vaccines due to their ability to escape the phagosomal compartment inside host cells (Brumell et al., 2002). When we used a gfp-mini-Tn7, integration of one, two or three copies into chromosomal loci attTn7, Δara::a-attTn7 and Δasd::a-attTn7, respectively, correlated with a stepwise increase of GFP fluorescence demonstrating a stepwise modulation of the expression level by targeted multicopy integration of a mini-Tn7.
Live bacterial vaccine or vector construction often necessitates chromosomal integration of expression cassettes for different transgenes (Galen et al., 2009;Curtiss et al., 2010;Hegazy and Hensel, 2012;Wang et al., 2013b). Therefore, we investigated the consecutive introduction of two different mini-Tn7 harbouring reporter genes lacZ or lux into a strain carrying attTn7 and a-attTn7 respectively. The recombinant bacteria containing both modules were readily obtained by initial transposition of lacZ-mini-Tn7, followed by λ-Red-mediated replacement of the rha locus with a-attTn7 and subsequent transposition of the lux-mini-Tn7. Importantly, lacZ and lux expression was stably retained during colonization of mouse tissues upon oral administration. However, instability of such modules might potentially be caused by endogenous recombinases acting on homologous sequences such as attTn7/a-attTn7, but even more so between multiple copies of the same mini-Tn7. This problem might be circumvented by the removal of host recombinases, reduction of sequence homologies and appropriate chromosomal positioning of insertion sequences (Zhang et al., 2011). Bioluminescence in vivo imaging and subsequent recovery and quantification of bacteria from mice indicated that the strains containing mini-Tn7 insertions colonized tissues to a somewhat lower extent possibly due to an attenuating effect of constitutive expression of lacZ and lux. However, combination of our approach with strategies for the inducible expression of heterologous factors may be able to resolve such effects (Hohmann et al., 1995).
In conclusion, we consider λ-Red recombinasemediated positioning of additional Tn7 attachment sites in the bacterial genome followed by mini-Tn7 transposition a versatile method for the construction of multifunctional Salmonella vaccines and vectors. Further work should be devoted to answer questions such as (i) how many mini-Tn7 copies can be reliably integrated and (ii) will consecutive insertions in close proximity be compromised by Tn7 target immunity. The ability of researchers to construct multifunctional vector strains may help to overcome known limitations of present recombinant Salmonella vaccines and eventually allow the development of a new generation of efficacious and safe Salmonella vaccines for clinics (Galen et al., 2009;Wang et al., 2013b). Moreover, such novel Salmonella vector systems also constitute promising delivery systems for other medical interventions, such as tumour therapy (Forbes, 2010;Leschner and Weiss, 2010).

Bacterial strains and growth conditions
Salmonella enterica serovar Typhimurium strain SL7207 (ΔaroA, ΔhisG) was originally obtained from Bruce Stocker (Hoiseth and Stocker, 1981) and kindly provided by Siegfried Weiss (Helmholtz Center for Infection Research, Braunschweig). Chromosomal modification of this strain was mediated by λ-Red recombinase or transposition of a mini-Tn7 (see below). Strain derivatives are described in the Results section. Escherichia coli strains Top10 and Pir2 (Life Technologies) were used as hosts for cloning, and E. coli strain SM10λpir (Simon et al., 1983) was used for mobilization of mini-Tn7 and the Tn7 helper plasmid. Bacteria were grown on LB agar plates or in LB medium supplemented with 100 μg ml −1 ampicillin, 30 μg ml −1 kanamycin, 30 μg ml −1 streptomycin, 2 mg ml −1 L-arabinose, 50 μg ml −1 diaminopimelic acid or 30 μg ml −1 5-Bromo-4-chloro-3indolyl-β-D-galactopyranoside (X-Gal), where appropriate. LB medium base and supplements were purchased from Carl Roth.

Plasmid constructions
A 300 bp sequence harbouring the attTn7 integration site (a-attTn7) was derived from the genomic sequence of strain S. Typhimurium SL1344 obtained from NCBI GenBank (accession number FQ312003, position 4090151-4090450 bp). This sequence including flanking EcoRI restriction sites was synthesized and sequenced (Geneart, Life Technologies). To apply λ-Red recombination for chromosomal integration of a-attTn7, the template plasmid pKD4 (Datsenko and Wanner, 2000) was modified. The synthetic DNA was cleaved with EcoRI and inserted into the NdeI site of plasmid pKD4 after ends were made compatible with Klenow enzyme. The plasmid containing the insert in the same orientation as the kanamycin resistance gene was named pKR31a. This plasmid was subsequently used as template for the generation of PCR products with homology ends for λ-Red recombination. Primer pairs for the replacement of ara, asd, endA, recF, rha and sifA are listed in Supporting Information Table S1. Plasmids pKD46 (Datsenko and Wanner, 2000) and pCP20 (Cherepanov and Wackernagel, 1995) were used for the expression of λ-Red (exo, bet and gam genes) and FLP recombinase respectively. Expression cassettes for the bioluminescence operon luxCDABE (lux) derived from Photorhabdus luminescence, the green fluorescence protein mutant 2 variant (gfp) (Cormack et al., 1996) and β-galactosidase of E. coli (lacZ) were cloned into a derivative of the mini-Tn7 pUX-BF5 (Bao et al., 1991). The constitutive lux expression cassette was originally subcloned from plasmid pLite201 (Voisey and Marincs, 1998) into plasmid pHL300 (Loessner et al., 2009). This plasmid was cut with BspLUII, blunt ended and again digested with BsrGI. The 1307 bp partial lux fragment was subsequently inserted into the mini-Tn7 transposon plasmid pHL289 (Loessner et al., 2007). For this, plasmid pHL289 was opened with NheI, blunt ended and thereafter cut with BsrGI. The product was designated pHL305 (lux-mini-Tn7). gfp linked to the L-arabinose inducible promoter PBAD (Loessner et al., 2008) was cloned into a derivative of pUX-BF5 giving rise to plasmid pKR49 (gfp-mini-Tn7). lacZ was amplified from plasmid pCMVβ with lacZ forward and reverse primers (Supporting Information Table S1). The PCR fragment was digested with XbaI and HindIII and inserted into the same restriction sites of plasmid pHL222 (Loessner et al., 2006) giving rise to plasmid pHL325. lacZ in conjunction with the constitutive β-lactamase promoter of E. coli was obtained from this plasmid by cleavage with SmaI and HindIII and inserted into the mini-Tn7 plasmid pKR50 opened with SalI and MluI after ends were blunted. The construct was designated pKR61 (lacZ-mini-Tn7). Plasmid pUX-BF13 containing Tn7 transposon genes tnsABCDE was used as helper plasmid for transposition of mini-Tn7 (Bao et al., 1991). Primers used for confirmation of chromosomal integrations of mini-Tn7 are listed in Supporting Information Table S1.
λ-Red recombinase-mediated gene replacement λ-Red recombinase-mediated gene replacement was carried out as previously described (Datsenko and Wanner, 2000). Briefly, PCR products harbouring ∼ 40 bp end sequences homologous to the respective S. Typhimurium target genes, a kanamycin resistance marker flanked by FLP recombinase recognition sites, and a-attTn7, so-called Red knock out (ko) fragments, were amplified with pKR31a as template and so-called Red ko primer (Supporting Information Table S1). Strain SL7207 harbouring plasmid pKD46 was grown in liquid LB medium supplemented with ampicillin at 30°C and 200 r.p.m. agitation up to an optical density at 600 nm (OD600) of ∼ 0.4. At this time point λ-Red recombinase expression was induced by addition of L-arabinose, and 1 h later electro-competent cells were prepared by washing cells three times in ice-cold distilled water. An amount of approximately 200 ng of the Red ko fragment was electroporated into cells with the Gen Pulser Xcell System (Bio-Rad) at 2.5 kV, 400 Ohm and 25 μF using a 0.2 cm cuvette. SL7207 mutant clones were selected on media plates containing kanamycin and streptomycin at 37°C. The kanamycin resistance gene was then removed by action of FLP recombinase as previously described (Datsenko and Wanner, 2000).

Transposition of mini-Tn7 into S. Typhimurium
Transposon-mediated site-specific integration into the chromosome of strain SL7207 was carried out according to the method of Bao et al. (1991). Briefly, triple mating cultures consisting of the helper strain E. coli SM10λpir harbouring pUX-BF13, E. coli SM10λpir harbouring a mini-Tn7 plasmid and the S. Typhimurium strain SL7207 were incubated on non-selective medium plates at 30°C for 24 h. Thereafter, bacteria were recovered and plated on medium plates containing streptomycin, kanamycin or chloramphenicol respectively. Site-specific chromosomal integration was verified by colony PCR (see Supporting Information). Of note, plasmid pUX-BF13 mediates synthesis of TnsABCDE proteins. TnsABC + D proteins are sufficient for target-specific chromosomal insertion of mini-Tn7 into attTn7 (Waddell and Craig, 1988). In contrast TnsABC + E mediated mini-Tn7 insertion occurs at non-attTn7 sites, but only on conjugal plasmids (Wolkow et al., 1996). Therefore, we do not expect interference of TnsE expression with the chromosomal mini-Tn7 insertions we observe in this work.

Flow cytometry
Bacteria were grown at 37°C and 200 r.p.m. agitation up to OD600 of ∼ 0.6. Then gfp expression was induced by addition of L-arabinose and 1 h later bacteria were pelleted, suspended in phosphate-buffered saline and subsequently analysed on a BD Accuri C6 flow cytometer (Becton Dickinson) as previously described (Loessner et al., 2006). An appropriate scatter gate was used to distinguish bacteria from other particles. Data were acquired and analysed with BD CSampler software (Becton Dickinson).

Animal work and ethics statement
Experimental procedures for the work with mice are provided as Supporting Information. All animal experiments were in compliance with the German Animal Welfare Act and approved by the competent authority (Regierungspraesidium Darmstadt).

Supporting information
Additional Supporting Information may be found in the online version of this article at the publisher's web-site: Fig. S1. Modulation of GFP synthesis in S. Typhimurium strain SL7207 by chromosomal integration of either one, two or three copies of gfp-mini-Tn7 (Fig. 3A,B). Strains SL7207 and derivatives with one, two or three chromosomal copies of the gfp cassette were induced with L-arabinose and subsequently analysed by flow cytometry. The histogram shows data of one representative experiment. Fig. S2. Consecutive chromosomal integration of two different mini-Tn7 encoding lacZ and lux into S. Typhimurium and stable maintenance of both modules by bacteria during colonization of mice. A. 10 9 bacteria of strains SL7207, attTn7::lux and attTn7:lacZΔrha::lux (Fig. 3C,D) were orally administered to BALB/c mice (n = 5 each group). The colonization course of bioluminescent strains harbouring lux was followed by noninvasive in vivo imaging. Bioluminescence intensities of abdominal regions of mice are expressed as means of radiance and error bars indicate standards deviations. B. At day 9 post-infection (p.i.) Peyer's patches (PP), mesenteric lymph nodes (MLN) and spleens were harvested (n = 5 each group), and bacterial content of organs was determined by plating dilution series of tissue homogenates on X-Gal plates. Data are presented as means of cfu with error bars indicating standard deviations. Stability of lacZ and lux cassettes was judged based on the blue and bioluminescent colony phenotype. Table S1. Primers used in this study. Appendix S1. Experimental procedures.


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Isolation and cDNA characteristics of MHC-DRA genes from gayal (Bos frontalis) and gaytle (Bos frontalis × Bos taurus)

The mammalian major histocompatibility complex (MHC) plays important roles in pathogen recognition and disease resistance. In the present study, the coding sequence and the 5′- and 3′-untranslated regions of MHC class II DR alpha chain (the DRA gene) from rare gayal and gaytle were cloned and analyzed to dissect structural and functional variations. The nucleotide and amino acid sequences for the DRA genes in gayal (Bofr-DRA) and gaytle (Bofr × BoLA-DRA) were almost identical to those for cattle and yak (99%). Compared to yak, two amino acids substitutions in the signal peptide (SP) domain for gayal were found within all Bos animals. Except for only one replacement in the amino acid within the α2 domain of the DRA protein in gayal, the additional residues were highly conserved across the species investigated. The 20 peptide-binding sites (PBS) of Bofr-DRA and Bofr × BoLA-DRA were essentially reserved in the α1 domain among all species investigated. The lesser degree of substitution in Bofr-DRA is concordant with the concept that the DRA gene is highly conserved among all mammals. The very high degree of conservativity of the DRA gene among ruminants, including gayal, suggests its recent evolutionary separation.

Introduction
The immune system is a function of biological structures and processes within an organism that protects against diseases by recognizing and responding to pathogens and antigens. [1] The mammalian major histocompatibility complex (MHC) is a group of closely linked and highly variable genes and gene clusters. [2] The cattle MHC, which is called bovine leukocyte antigen (BoLA), plays a key role in the defense against infectious diseases and regulates antigen presentation by encoding proteins that take part in the innate and adaptive immune responses to intruding pathogens. [3,4] The MHC genes are associated with susceptibility or resistance to many diseases for cattle and sheep. [5,6] Thus, the MHC genes could be indicated as primary candidates for the investigation of genetic variation for the purpose of marker-assisted breeding.
The gayal or mithun (Bos frontalis) is a rare semiwild bovine species naturally inhabiting Indo-China. [7,8] It has a chromosome complement of 2n D 58, [9À11] which differs from those of their counterparts such as cattle (B. taurus, 2n D 60) and gaur (B. gaurus, 2n D 56). [12] Gayal often consumes tree and bamboo leaves, grasses, reeds and other local plants and shows a very wide range of adaptations under the harsh conditions which range from cold to tropical regions. [13,14] Moreover, the quality of meat, including tender and muscle fibre diameter, from gayal is better than in local cattle. [15,16] Therefore, the hybrid between gayal (<) and cattle (,), which is also called gaytle (gayal £ cattle), presents better meat traits than cattle and can meet the diversity demand for consumers. Due to the remoteness of their habitats, as well as socio-political and ecological factors, gayal is one of the least studied ruminants. [17À20] Until now, the MHC-DRA gene has been cloned and sequenced in European cattle [21] and zebu, [22] yak and yakow, [23,24] buffaloes, [25] goats, [26] sheep, [22,27] donkeys, [28] pigs, [29] mice, [30] cats [31] and macaques. [32] So far, the MHC-DRA from gayal and gaytle has not been deposited in the GenBank database (http://www. ncbi.nlm.nih.gov/). These native animals are more common sources of meat and milk than cattle in Indo-China. They are considered to be less susceptible than cattle to some endemic diseases. [33,34] In the present study, we have isolated and characterized full-length cDNAs from gayal and gaytle and compared those with the sequences of MHC-DRA from different animal species in order to inspect the genetic factors for disease susceptibility and resistance. *Corresponding author. Email: yangyuai2012@126.com

Materials and methods
Samples collection, RNA extraction and first-strand cDNA synthesis Liver RNA samples from three mature gayals (B. frontalis) and three mature gaytles (B. frontalis £ B. taurus) were collected at the slaughter house, Gongshan County (Yunnan Province, China). The samples were snap frozen and kept in liquid nitrogen during storage and transportation, and then stored at ¡80 C. The total RNA was isolated using a Total RNA Extraction Kit (Beijing Tiangen Biotech Co., Ltd., China). DNase I treatment of the total RNA was performed before the cDNA was constructed using RevertAid TM First Strand cDNA Synthesis Kits (Fermentas Inc., USA) following the manufacturer's instructions.

Primers and PCR amplification of the DRA gene
The MHC-DRA genes from gayal and gaytle were isolated using forward primer (5 0 -CGAGACACCGAAGAA-GAAAAT-3 0 ) and reverse primer (5 0 -GGAGGGAAAAC-CAATACAAGAA-3 0 ), which were also successfully used to amplify DRA sequences from buffalo, yak and yakow. [20,21] Polymerase chain reaction (PCR) was conducted using Bioer Life Express Thermocycler (Bioer Technology Co., Ltd., China) using 2 mL of cDNA template, 12.5 mL of 2£ PCR Power Mix (Beijing Zoman Biotechnology Co., Ltd., China), 2 mL of 10 pmol/mL of each primer and 8.5 mL of double distilled water in a total reaction volume of 25 mL. The PCR conditions started with an initial denaturation temperature of 94 C for 4 min, followed by 36 cycles of the following steps: 94 C for 1 min, 56 C for 1 min and 72 C for 1 min, with a final extension of 10 min at 72 C. Visualization of amplified products was performed on agarose gel stained with ethidium bromide. The PCR products were sequenced bi-directionally using an ABI373X DNA analyzer (Applied Biosystems Inc.) at the Sango Biotechnology Company (Shanghai, China).

Sequences analysis
The cDNA sequence prediction was conducted using the GenScan software (http://genes.mit.edu/GENSCAN. html). [35] The theoretical isoelectric point (pI) and molecular weight (Mw) of the putative DRA proteins were computed using the Compute pI/Mw Tool (http:// www.expasy.org/tools/pi_tool.html). [36] The DNAStar v5.2.2 program and MEGA v5.0 software package with the neighbour-joining method [37] were used to conduct sequences alignment and to construct a phylogenetic tree.  Table 1 presents the nucleotide sequences and amino acid alignments. The Bofr-DRA and Bofr £ BoLA-DRA genes had the closest similarity (99%) to the DRA gene of cattle, zebu, yakow and yak, with high level of similarity with the respective gene in buffalo, goat, sheep, pig, dog, macaque, human, cat and mouse. In addition to the signal peptide (SP), other functional domains, including the a1, a2, connecting peptide (CP), transmembrane (TM) and cytoplasmatic (CY) regions, are highly conserved, particularly the sites associated with biological function in these DRA genes. [25] Importantly, the SP domain has been reported to have the most variable region of the molecule across the species. [25] However, there was high similarity among Bofr-DRA, Bofr £ BoLA-DRA genes and those of other ruminants.

Sequence alignment and comparison
The entire SP, a1, a2 and CP/TM/CY domains from DRA molecules from gayal and gaytle were almost identical to those of cattle, except the mutation in position 138 in the amino acid chain within the a2 domain (P. M138L, Figure 2). However, compared with yak, there was minimal mutation of the Bofr-DRA and Bofr   [38] were highly conserved within the a1 domain ( Figure 2). Compared to other MHC class II gene families such as the DRB, DQA and DQB, which are highly variable at the PBS,[39À42] the DRA gene appears to be highly conserved for residues forming the PBS region in different mammals, including primates. It seems that selection pressure has led to conservation for these specific functional regions in the DRA gene. However, there was evidence of variability in the first domain exon (a1) of DRA of ruminants. [22,43] These results could emphasize the complexity and function of the DRA Table 1. Sequence similarity comparison at the nucleotides and amino acids (within parentheses) sections for the signal peptide (SP), a1, a2 and connecting peptide/transmembrane/cytoplasmatic (CP/TM/CY) domains of the DRA genes from different species compared to those for gayal (B. frontalis, up) and gaytle (B. frontalis £ B. taurus, down).  gene, particularly in the PBS region. The cysteine residues that build an interchain disulphide bond between positions 107 and 163 were also stable and conserved. In gayal, gaytle and other animals including cattle, zebu, yak, yakow, buffaloes, sheep, goats and macaques, the DRA molecules have only one potential N-linked glycosylation site (78À80 amino acids) in the a1 domain as opposed to two that occur in pigs, humans and mice, with the second site located in the a2 domain (118À120 amino acids marked by the reddish box in Figure 2). [21,26] The deprivation of one glycosylation site in the a2 domain for gayal and other ruminants such as cattle and yak is due to the replacement of an asparagine residue with serine. It is not known whether the glycosylation site in the a2 domain may have an important role in the immune response and the response against specific antigens, although its lack presents a specific molecular characteristic of ruminant DRA genes. There were no substitutions in other important functional sites such as T lymphocytes receptor recognition site (positions 39, 57 and 60) and CD4 moleculebinding site (positions 91À92) in gayal, gaytle, yak, Chinese yakow, zebu, cattle, buffaloes, sheep and goats.

Phylogenetic analysis based on neighbour-joining method
From the phylogenetic tree analysis (Figure 3), it appears that the DRA genes of ruminant and non-ruminant species evolved independently, since the species they originate from have split into different branches over the evolutionary time. It is also apparent that yak and cattle are most closely related species to gayal. In the present study, the loss of mutation in Bofr-DRA and Bofr £ BoLA-DRA is concordant with the DRA gene being conserved within ruminants and all mammalian species. As a result, this could be used for the identification of shared antigenic sites of similar pathogens or the recognition of very specific antigens which may be common to most of the species. Moreover, the close similarity of the DRA gene among ruminants, including gayal, may be ascribed to recent separation in evolutionary process and/ or similar selection pressure which the ruminants have suffered during evolution.

Conclusions
The DRA genes of gayal and gaytle have been isolated and characterized for the first time, thereby enlarging cognizance of the MHC-DRA in rare ruminants. It points out that the Bofr-DRA gene is highly conserved, particularly in the first domain exon as in most mammals. In the future, it would be more interesting to dissect Bofr-DRB, which probably contains most of the polymorphism within the MHC-DR molecule.


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Genome-wide analysis of DNA methylation in bovine placentas

Background DNA methylation is an important epigenetic modification that is essential for epigenetic gene regulation in development and disease. To date, the genome-wide DNA methylation maps of many organisms have been reported, but the methylation pattern of cattle remains unknown. Results We showed the genome-wide DNA methylation map in placental tissues using methylated DNA immunoprecipitation combined with high-throughput sequencing (MeDIP-seq). In cattle, the methylation levels in the gene body are relatively high, whereas the promoter remains hypomethylated. We obtained thousands of highly methylated regions (HMRs), methylated CpG islands, and methylated genes from bovine placenta. DNA methylation levels around the transcription start sites of genes are negatively correlated with the gene expression level. However, the relationship between gene-body DNA methylation and gene expression is non-monotonic. Moderately expressed genes generally have the highest levels of gene-body DNA methylation, whereas the highly, and lowly expressed genes, as well as silent genes, show moderate DNA methylation levels. Genes with the highest expression show the lowest DNA methylation levels. Conclusions We have generated the genome-wide mapping of DNA methylation in cattle for the first time, and our results can be used for future studies on epigenetic gene regulation in cattle. This study contributes to the knowledge on epigenetics in cattle.

Background DNA methylation, a major epigenetic modification of the genome found in most eukaryotes, is essential for normal development and crucial in many biological processes, such as gene expression regulation, genomic imprinting, X-chromosome inactivation, suppression of repetitive elements, and carcinogenesis. DNA methylation preferentially occurs at the 5′ position of cytosine in CpG dinucleotides, which are mostly found in clusters known as CpG islands (CGIs) [1]. DNA methylation in the promoter or the first exon of a gene [2] generally leads to transcriptional silencing [3]. Profiling DNA methylation maps across the genome is important to understand DNA methylation changes that occur during development and in disease phenotypes. The genomewide DNA methylation maps of many organisms, such as human [4], chicken [5,6], rat [7], Arabidopsis [8], rice [9], and silkworm [10] has been reported. However, the methylation pattern of cattle remains unknown.
Somatic cell nuclear transfer (SCNT) (i.e., somatic cloning) is a promising technology with numerous potential applications, including reproduction of high-value domestic or endangered mammalians, biomedical research, human xenotransplantation, transgenic research, disease models, and therapeutic cloning. However, low cloning efficiency and a high incidence of developmental abnormalities in SCNT clones markedly hinder the use of this technology. Developmental abnormalities include the large offspring syndrome, respiratory problems, placental deficiency, obesity, prolonged gestation, short life span, fetal edema, dystocia, hydramnios, and perinatal death [11][12][13]. The developmental abnormalities in SCNT clones usually involve the placenta; the placenta is central to the onset of pathologies [11,[14][15][16][17][18][19][20][21][22], and most cloned fetuses die in utero because of placental deficiency [23]. We also found that placental weight and mean placentome weight were high, but the number of placentomes was low, in deceased cloned calves compared with calves produced by normal sexual reproduction [22]. The expression and DNA methylation levels of several imprinted genes were also aberrant in the placenta of deceased cloned calves [22]. Gene expression analyses of the placenta of cloned calves show that multiple pathways are affected [24,25]. Placental abnormality may be the main cause of fetal death in clones. The principal cause of the developmental abnormalities of the fetus and placenta in cloned animals is aberrant epigenetic nuclear reprogramming of the donor somatic cell, which involves various epigenetic modifications. DNA methylation is a major epigenetic modification of the genome and is crucial in nuclear reprogramming during SCNT.
In this study, we analyzed the genome-wide DNA methylation pattern in the placentas of cattle with the use of methylated DNA immunoprecipitation combined with high-throughput sequencing (MeDIP-seq) by Illumina Genome Analyzer II. The DNA methylome distribution in the bovine genome was shown for the first time. Two placental tissues were used in this study, namely, the placental tissues of deceased cloned calves (SCNT) and normally produced calves (control).

Global mapping of DNA methylation in cattle
To study the global mapping of DNA methylation in cattle, we generated a total of 8.1 Gb MeDIP-seq data from two placental samples, including 81,632,654 (SCNT) and 83,673,470 (control) raw reads. Of the total reads, 95.29% and 95.28% were mapped to the reference genome for the SCNT and control placentas, respectively, of which 42.18% and 42.87% were mapped to specific regions in the cattle genome (Table 1).
MeDIP-seq reads were detected in most chromosomal regions (GGA1-29 and chromosome X) ( Figure 1). Genome coverage was the percentage of bases mapped by genome-wide reads. In addition to CpG, 5-methylcytosine can also be found in several eukaryotic organisms in other sequence contexts, such as CHG and CHH (with H being A, C, or T), which are named as non-CG methylation. Therefore, we analyzed the genome coverage of the CG, CHG, and CHH sites under different sequencing depths (Additional file 1). Additional file 1 shows that the genome coverage of the CG, CHG, and CHH sites negatively correlates with read depth; a large number of regions had low-depth coverage, and a small number of regions had high-depth coverage. Additional file 2 shows the distribution of MeDIP-seq reads in different CG density regions.
The read distribution on different genome regions represents the features of genome-wide methylation pattern. Analysis of the read distribution in different components of the genome showed that uniquely mapped reads were mainly present (approximately 27%) in intron regions (Additional file 3). The proportion of reads distinctly mapped to CGIs in the SCNT and control placentas was only 1.64% and 2.42%, respectively (Additional file 3). Many CpG islands were located in repetitive elements.

DNA methylation profile in bovine genes
The DNA methylation profile in the gene region was calculated by the reads that were aligned on a distinct locus in the genome. Generally, the DNA methylation level dramatically decreased in the 2 kb region upstream of the transcription start sites (TSSs) and dropped to the lowest point before the TSS. The DNA methylation level sharply increased in the 3′ direction and peaked just before the transcription termination site (TTS), and subsequently, the level dropped rapidly and remained constant at the middle level of DNA methylation after TTS ( Figure 2). The DNA methylation level of the control placentas was higher than that of the SCNT placentas in the gene region. However, MeDIP-seq technology is dependent on high sequences, such that only regions of relatively high CpG abundance are obtained. Thus, many genes with spaced CpG sites may not be detected by MeDIP-seq, even if few CpG sites present are actually methylated.

Distribution of highly methylated regions
The uniquely mapped reads were used to detect the highly methylated regions (HMRs), which are also called peaks. The HMR distribution in different genome regions was further analyzed. For the first time, we obtained 138,975 HMRs in the SCNT placenta (Additional file 4) and 145,218 HMRs in the control placenta (Additional file 5). The average length of HMRs was approximately 1,100 bp, and the HMR coverage on the genome was 5.86% to 5.89% (Table 2). Additional file 6 shows the CpG number in HMR. Most of the HMR have 5 to 25 CpG sites. Analysis of HMR distribution in the different components of the genome showed that the HMRs are mainly in the intron (approximately 27%) and the coding sequence (CDS;  approximately 9%) regions ( Figure 3). Analysis of HMR coverage in the different components showed that the genome coverage in 5′ UTR, CDS, 3′UTR, downstream 2 k, upstream 2 k, and intron was approximately 96%, 92%, 66%, 50%, 46%, and 10%, respectively (Additional file 7). Genome coverage was obtained as follows: (base number of HMRs in component/total base number of the component) × 100.

CpG islands in bovine placenta
In this study, CpG islands that overlapped with the HMRs were considered methylated CpG islands. For the first time, we identified a total of 37,191 CpG islands in cattle genome (Additional file 8). Of these CpG islands, only 14.68% (5,461) (Additional file 9), and 20.12% (7,482) (Additional file 10) were methylated in the SCNT and control placentas, respectively. These results indicated that most CpG islands were unmethylated in bovine placenta. Most of the methylated CpG islands were present in the introns and CDS regions (Table 3).

Methylated genes in bovine placenta
Methylated genes were defined in this study as genes overlapping (≥ 50%) with HMRs in the promoter or gene-body regions. A total of 7,974 methylated genes were found in the SCNT placenta, among which 302 genes were methylated only in promoters, 6,845 only in gene bodies, and 872 in both promoters and gene bodies (Additional file 11). A total of 8,466 methylated genes were found in the control placenta, among which 315 genes were methylated only in promoters, 7,212 only in gene bodies, and 939 in both promoters and gene bodies (Additional file 12).

MeDIP-seq data validation by bisulfite sequencing
We conducted bisulfite sequencing for nine selected gene regions on each unpooled samples. There were three individual samples in SCNT and control groups, respectively. The DNA methylation status of nine gene regions between SCNT and control groups were analyzed by one-way ANOVA and LSD tests using the SPSS 16.0 software. In MeDIP-seq results (Additional files 11 and 12), upstream 2 k (promoter) of IGF2 (NM_174087), TCF7 (NM_001099186), and UBE2S (NM_001076472) were methylated in the control placentas, but unmethylated in SCNT placentas. Upstream 2 k of SENP1 (NM_ 001206876), ZNF3 (NM_001046614), USP10 (NM_ 001098924), and CD44 (NM_174013) were methylated in SCNT placentas, but unmethylated in control placentas. CPT1B (NM_001034349), not expressed in placenta, was hypermethylated in both SCNT and control placentas. The bisulfite sequencing results of the eight Figure 2 DNA methylation distribution in the bovine gene region. The DNA methylation profile in the gene region was shown by the reads that were aligned on the unique locus in the genome. The gene region was defined as the regions that contained a 2 kb region upstream of the TSS, the gene body from TSS to TTS, and a 2 kb region downstream of the TTS. In the upstream and downstream 2 kb regions, the regions were split into 20 non-overlapping windows, and the average alignment depth was calculated for each window. In the gene body, each gene was split into 40 equal windows, and the average alignment depth was calculated for each window. The y-axis is the average of the normalized depth for each window. The SCNT and control indicated the samples of placental tissues from cloned and normally produced cattle, respectively. aforementioned gene regions were in accordance with the MeDIP-seq results (Additional file 13). The upstream 2 k of HSP90AA1 (NM_001012670) was methylated in both SCNT and control placentas in MeDIP-seq results, contrary to the bisulfite sequencing results, which showed that the region was unmethylated in SCNT placentas. These validations were almost in accordance with the MeDIP-seq results (except one gene region in one sample), indicating the reliability of our methylation data obtained by MeDIP-seq.

DNA methylation and gene expression level
We analyzed the gene expression in the placental tissues of deceased cloned calves (SCNT) and normally produced calves (control) using RNA-seq (Additional file 14). We categorized genes into five groups according to expression levels (Additional file 15): 0 (silent genes), 0-1 (lowly expressed genes), 1-20 (moderately expressed genes), 20-1000 (highly expressed genes), and > 1000 (genes with the highest expression, housekeeping genes). The DNA methylation profile in and around gene bodies were compared among these five gene expression levels.
A clearly negative and monotonic correlation was found between DNA methylation levels around the TSS of genes and gene expression levels. The TSS regions of highly expressed genes were relatively insufficiently DNA methylated, whereas the genes expressed at low levels were increasingly methylated ( Figure 4). However, MeDIP-seq technology is dependent on high sequence.
Thus, only regions with relatively high CpG abundance were obtained, and many genes with spaced CpG sites may not be detected. However, the relationship between gene-body DNA methylation and expression levels shows a non-monotonic correlation ( Figure 4). Moderately expressed genes generally have the highest levels of gene-body methylation, whereas the highly and lowly expressed genes, as well as the silent genes, show moderate DNA methylation levels. The housekeeping genes show the lowest DNA methylation levels.
In the downstream 2 kb regions of TSS, the silent genes and housekeeping genes show low DNA methylation levels, whereas moderately expressed genes also have the highest levels of gene-body methylation.

Online data deposition
The MeDIP-seq and RNA-seq data from this study have been deposited in NCBI Sequence Read Archive with accession numbers SRP032370 and SRP033225, respectively, (http://www.ncbi.nlm.nih.gov/sra/).

Discussion
Over the past decade, multiple methods have been developed and applied to analyze genome-wide DNA methylation profile, including MeDIP-seq, whole-genome bisulfite sequencing (WGBS), and reduced representation bisulfite sequencing (RRBS). WGBS is an excellent approach to determine the DNA methylome. The DNA methylome   uses sodium bisulfite to convert unmethylated cytosine nucleotides to uracils and leaves methylated cytosines unmodified, which can be subsequently distinguished by sequencing. WGBS has high resolution, but it is costly, time consuming, involves complex data, and lacks comprehensive analytical tools, which considerably limit its popularity and applicability [26]. In MeDIP-seq, anti-5methylcytosine antibody was first used to specifically recognize methylated cytosines, and the methylated DNA fragments were subsequently enriched. MeDIP-seq is a suitable method to analyze the DNA methylation status of heavily methylated genome regions. Many recent studies have shown that MeDIP-seq can reflect the relative genome-wide DNA methylation profile [5,6,27]. Therefore, we selected MeDIP-seq to analyze genomewide profiles of DNA methylation in cattle in this study. However, MeDIP-seq technology is dependent on high sequence dependence. Thus, only regions of relatively high CpG abundance are obtained, and many genes with spaced CpG sites may not be detected. The methylation status of sequences where CpG sites are spaced (e.g., only one may occur in any given fragment) may appear unmethylated even if the few CpG sites present are actually methylated. Therefore, this method is no longer the technology of choice. Genome-wide DNA methylation maps of many organisms have been reported, but the methylation pattern of cattle remains unknown. In this study, we report for the first time the genome-wide profiles of DNA methylation in the placentas of cattle. Cattle show analogous DNA methylation profiles to those of mammals and plants [4][5][6][8][9][10]. In cattle, the DNA methylation level sharply decreased before the TSS, dramatically increased towards the 3′ direction, and stayed at a plateau until the 3′ end of the gene body.
In bovine placentas, the methylation levels in the gene body are relatively high, whereas the promoter (around TSSs) remains hypomethylated, a finding that is consistent in human [28] and chicken [5]. Most of the promoter regions were hypomethylated, and DNA methylation in the promoter repressed gene expression [29,30]. Laurent et al. investigated the correlation of methylation profile with the expression level in human embryonic stem cells and found that the 20% of most highly expressed genes exhibited the lowest methylation levels at ±1 kb from the TSSs. The methylation levels increased. We also analyzed the relationship between DNA methylation and expression levels with decreasing gene expression. Consistent with previous reports in humans [28,31,32] and chicken [5], DNA methylation levels around TSSs at the 5′ ends of genes are negatively correlated with the gene expression level, a finding indicating that promoter methylation is a repressive epigenetic mark that downregulates gene expression. DNA methylation within gene bodies is more prevalent than in promoters, but information on the role of DNA methylation in gene bodies is insufficient. Gene-body methylation and expression levels apparently have a complex relationship. Gene-body DNA methylation is positively correlated with gene expression in humans [28,31,[33][34][35]. However, the relationship between genebody DNA methylation and gene expression levels is not monotonic but rather bell-shaped in plants, invertebrates, and even in humans; moderately expressed genes have the highest methylation levels [32,36,37]. In bovine placentas, moderately expressed genes have the highest degree of gene-body DNA methylation.
Aberrant epigenetic nuclear reprogramming during SCNT causes low cloning efficiency and developmental abnormalities in cloned animals. DNA methylation is a major epigenetic modification of the genome, and many studies have shown that abnormal DNA methylation reprogramming is found in cloned embryos and fetuses [11,22,[38][39][40][41]. Chan et al. investigated genomescale DNA methylation patterns of SCNT-reconstructed mouse embryos using RRBS for the first time and found that epigenetic reprogramming after nuclear transfer in SCNT mouse embryos does not fully recapitulate the natural DNA demethylation events that occur at fertilization, which results in aberrant methylation at some promoters and repetitive elements that may contribute to developmental failure [38]. Bovine SCNT blastocysts bear significantly higher methylation levels than in vitro-fertilized embryos at satellite I sequence [41]. In addition, the DNA methylation levels of several imprinted genes were found aberrant in various tissues of deceased SCNT calves [22,40]. However, the genomewide analysis of aberrant methylation in cloned animals has not been reported. In the present study, we screened out potentially aberrant methylated genes in the placentas of deceased cloned calves, which may have caused developmental abnormalities and ultimately resulted in the death of the cloned cattle. For example, IGF2 was found hypomethylated in placentas of deceased SCNT calves but hypermethylated in those of normal calves in this study. The placenta-specific paternally expressed imprinted gene IGF2, which is a major modulator of placental and fetal growth [41], acts in the placenta to directly control the supply of maternal nutrients to the fetus. IGF2 was overexpressed in cloned bovine embryos [42] and various tissues of deceased cloned calves [22,40]. Yang and colleagues also found that IGF2 was drastically over-expressed in the bladder, brain, heart, and lung of cloned calves suffering from the large offspring syndrome compared with normal controls [43]. IGF2 expression was also aberrantly upregulated in the heart tissues of deceased cloned calves, and IGF2 showed aberrant DNA methylation levels in the kidneys of deceased cloned calves [44]. In humans, IGF2 upregulation is important in the pathogenesis of Beckwith-Wiedemann syndrome (BWS) [45][46][47], which is characterized by somatic overgrowth that is similar to the large offspring syndrome in ruminants. Based on these results, we inferred that hypomethylated IGF2 and the upregulated IGF2 expression may promote overall fetal growth, and thus can be related to the large offspring syndrome and death of cloned animals.
For the first time, we have completed the genome-wide mapping of DNA methylation in bovine placentas. This mapping can be used as a basis for further studies on epigenetic gene regulation in cattle and other ruminants.

Conclusions
In this study, we have generated the genome-wide mapping of DNA methylation in cattle for the first time, and our results can be used for future studies on epigenetic gene regulation in cattle. This study contributes to the knowledge on epigenetics in cattle.

Ethics statement
The entire experimental procedure was approved and supervised by the Animal Care Commission of the College of Veterinary Medicine, Northwest A&F University Shaanxi, China. The bovine ovaries used in this study were purchased from Tumen abattoir and Zhongle abattoir, two local slaughterhouses located in Xi' An, China. A 40day-old female Holstein fetus was obtained for nuclear donor cell cultures, and Angus cows were used as recipients (Yangling Keyuan Cloning Co. Ltd).

Chemicals
All chemicals and reagents were obtained from Sigma-Aldrich (St. Louis, USA), unless specifically stated otherwise. Disposable, sterile plasticware was obtained from Nunclon (Roskilde, Denmark).

Production of cloned calves and tissue collection
The production of cloned calves, including nuclear donor cell preparation, oocyte collection, and in vitro maturation, somatic cell nuclear transfer, activation, fusion, culture of cloned embryos, embryo transfer, and pregnancy diagnosis, were performed as previously described [48,49]. Before SCNT, nuclear donor cells, which were established from the skin of the Holstein fetus, were cultured in serum-starved medium (0.5% fetal bovine serum, FBS) for 2 d. Cumulus-oocyte complexes (COCs) were matured in vitro for 20 h in bicarbonatebuffered tissue culture medium 199 (TCM-199, Gibco, USA) supplemented with 10% (v/v) FBS, 1 μg/mL 17 βestradiol, and 0.075 IU/mL human menopausal gonadotropin. Oocytes with an extruded first polar body were selected and stained with 10 μg/mL Hoechst 33342 for 10 min prior to enucleation. A 20 μm inner diameter glass pipette was used to aspirate the first polar body and a small amount of surrounding cytoplasm. The expelled cytoplasm was surveyed under ultraviolet radiation to confirm the removal of nuclear material. With the use of the 20 μm inner diameter glass pipette, a single disaggregated donor cell was transferred to the pre-vitelline space of the enucleated oocytes. Oocyte-cell fusion was performed by a double electrical pulse of 35 V for 10 μs using a pair of platinum electrodes connected to a micromanipulator in microdrops of Zimmermann's fusion medium. The reconstructed cloned bovine embryos were activated in 5 μM ionomycin for 4 min, followed by treatment with 1.9 mM dimethynopyridine in synthetic oviductal fluid supplemented with amino acids and bovine serum albumin for 3 h to 4 h. The activated embryos were cultured in G1.5/G2.5 sequential media (Vitrolife AB, Gothenburg, Sweden) in a humidified atmosphere of 5% CO 2 in air at 38.5°C. Fresh day 7 cloned blastocysts were nonsurgically transferred (one embryo per recipient) to the synchronized recipient uterine horn ipsilateral to the corpus luteum 7 d after standing estrus. Eight cloned calves were delivered via C-section from days 286 to 290. Two cloned calves were stillborn, and the others died within 2 d after birth. The deceased calves suffered from placental abnormalities, including placental hypertrophy and a larger placentome, but a small number of placentomes were observed. We randomly selected placental tissues of three deceased cloned calves (SCNT samples) and three female Holstein calves produced by normal sexual reproduction (control samples) to perform genome-wide DNA methylation and RNA-seq analyses. After the birth of the calves, the fetal contributions of the placentas were immediately collected, rinsed thrice with RNA-free ddH 2 O and minced into pieces. The tissues were immediately frozen in liquid nitrogen and then stored at -80°C until DNA and RNA extractions.

DNA extraction and preparation for MeDIP-seq
Genomic DNA was extracted from the placentas with the use of a TIANamp Genomic DNA Kit (Tiangen, Beijing, China), and DNA quality was evaluated by agarose gel electrophoresis and spectrophotometer. Additional file 16 shows the preparation for MeDIP-seq. Genomic DNA isolated from the placentas of three randomly chosen deceased cloned calves (SCNT) were mixed in equal amounts to generate a pooled sample as SCNT. Genomic DNA isolated from the placentas of three female Holstein calves produced by normal sexual reproduction (control) were mixed in equal amounts to generate a pooled sample as control. In detail, DNA from each placenta was measured with Fluoroskan Ascent (Thermo Labsystems, Franklin, MA) and Picogreen reagents and kits (Molecular Probes, P-7589). The selected DNAs were diluted to a standard concentration. To ensure that the same amount of each DNA sample was transferred to the pool, Hamilton ML2200 and Vivace automated pipetting stations were used to transfer each DNA sample into a single tube. Then, the pool sample was gently mixed and requantified before further dilution to a working concentration. Subsequently, these two pooled samples were sonicated to produce 100 bp to 500 bp DNA fragments. After DNA underwent end repairing, phosphorylating, and A-tailing with Paired-End DNA Sample Prep kit (Illumina, San Diego, CA, USA), it was ligated to Illumina sequencing primer adaptors. Double-stranded DNA was then denatured and immunoprecipitated by anti-5-methylcytosine mouse monoclonal antibody (Calbiochem). The following procedure was the same as in the method described by Hu [6]. Briefly, after 220 bp to 320 bp bands were excised from the gel and purified, the products were quantified on Agilent 2100 Analyzer (Agilent Technologies, Santa Clara, CA, USA) and qPCR qualification, and DNA libraries were sequenced on the Illumina Hiseq 2000 (Illumina) by the Beijing Genomics Institute (BGI, Shenzhen, China).

Bioinformatic analysis
Bioinformatic analysis was conducted according to a previously described protocol [6]. After the low-quality reads and those containing adapter reads were filtered, the MeDIP-seq data from Illumina sequencing were aligned to the UCSC cattle reference genome [50] (http://hgdownload.cse.ucsc.edu/goldenPath/bosTau6/big Zips/bosTau6.fa.gz) with SOAPaligner v 2.21 (http://soap. genomics.org.cn/) with no more than 2 bp mismatches. We analyzed the genome coverage of the CG, CHG, and CHH sites under different sequencing depths, distributions of MeDIP-Seq reads in different CG density regions, and the read distribution analysis including the distribution in cattle chromosomes and in the different genome components. The region from TSS to transcript end site was defined as the gene body region. The DNA methylation profile in the gene region was calculated by the reads that were aligned to a unique locus the genome.
Genome-wide highly methylated region (methylation peak, regions with sequencing tags more than 20, and a p value < 1 x 10 −5 ) scanning was conducted with MACS V 1.4.2 (http://liulab.dfci.harvard.edu/MACS/) [51]. The distribution of CpG in HMRs and the HMR distribution in the different components of the cattle genome (upstream 2 kb, 5′ UTR, CDS, intron, 3′ UTR, downstream 2 kb) were analyzed in our study.
CpGPlot (https://gcg.gwdg.de/emboss/cpgplot.html) was used to scan CpG islands (CGIs) with the following criteria: length > 200 bp, GC content > 50%, and observed-to-expected CpG ratio > 0.6. CpG islands that overlapped with the HMRs were considered methylated. Genes that overlapped with the HMRs in promoter or gene body regions were considered methylated genes.

Bisulfite sequencing PCR analysis
Nine gene regions were chosen to validate MeDIP-seq data with bisulfite PCR using unpooled individual samples. There were three individual samples in SCNT and control groups, respectively. First, with the use of the EZ DNA Methylation-Gold ™ Kit (Zymo Research, Orange, CA, USA), the individual DNA samples were subjected to sodium bisulfite treatment as previously described [7]. After 20 μL DNA sample (500 ng to 900 ng) were added to the 130 μL CT Conversion Reagent in a PCR tube, the sample tubes were placed in a thermal cycle for 10 min at 95°C for DNA denaturation and 2.5 h at 64°C for bisulfite conversion. Modified DNA was desalted, purified, and finally eluted with 15 μL elution buffer. Subsequently, bisulfite sequencing PCR (BS-PCR) was immediately performed with a 2 μL modified DNA per PCR run. CPT1B in 5′ terminal region (4 kb) and upstream 2 kb of IGF2, TCF7, HSP90AA1, UBE2S, SENP1, ZNF3, USP10, and CD44 were analyzed by MethPrimer software to predict the CpG islands [52]. The specific primers for BS-PCR were designed with MethPrimer software and Methyl Primer Ex-press® Software v1.0 (Applied Biosystems Inc., Foster City, CA, USA) (Additional file 17). The Hot Start DNA polymerase Zymo Taq premix (Zymo Research) was used in BS-PCR. BS-PCR was performed in 50 μL reaction mixtures that contain 25 μL Zymo Taq premix, 2 μL modified DNA, 21 μL dH 2 O and 1 μL of both forward and reverse primers with the following program: 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at different temperatures (Additional file 17) for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 7 min. PCR products were gel-purified with TIANgel Midi Purification Kit (Tiangen). Purified fragments were subcloned into pMD20-T vectors (TaKaRa). For each individual sample, about 10 clones were selected for DNA sequencing (BGI). BIQ Analyzer software [53] was used to analyze bisulfite sequencing data and C-T conversion rates. Methylation data from bisulfite sequencing were analyzed by computation of the percentage of methylated CpGs of the total number of CpGs. The DNA methylation status of nine gene regions were compared between SCNT and control groups. Outcomes were tested by one-way ANOVA and LSD tests using the SPSS 16.0 software (SPSS Inc., Chicago, IL, USA). Differences were considered significant at P < 0.05.

RNA-seq
Total RNA from bovine placentas was extracted with TRI-ZOL reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Total placental RNAs were extracted from the same placentas as in MeDIP-seq analysis. Total RNA at 30 microgram for each sample (SCNT and control) was treated with RNase-Free DNase I (NEB, UK) for 15 min at 37°C to remove DNA contamination. Agilent 2100 was used to assess the concentration and quality of RNA. Total RNA at 2 microgram from each sample was used in library construction, respectively. The mRNAs were enriched with the oligo (dT) magnetic beads. After the mRNAs were broken into short fragments (about 200 bp) with the fragmentation buffer, the first strand cDNA was synthesized by random hexamer-primer with the mRNA fragments as templates. Buffer, dNTPs, RNase H, and DNA polymerase I were added to synthesize the second strand. The double strand cDNA was purified with QiaQuick PCR extraction kit (Qiagen) and washed with elution buffer for end repair and single nucleotide "A" addition. After the sequencing adaptors were ligated to the fragments, the required fragments were purified by agarose gel electrophoresis and enriched by PCR amplification. Finally, the library products were used for sequencing analysis with Illumina HiSeq™ 2000. The sequences obtained were mapped to the RefSeq database. Sequences uniquely mapped to the RefSeq genes were used for subsequent analysis. The gene expression level was calculated with reads per kb per million (RPKM) reads method, and the formula used was as follows: where RPKM(A) is considered as the expression of gene A, C is the number of reads that uniquely aligned to gene A, N is the total number of reads that uniquely aligned to all genes, and L is the number of bases in gene A.