Abstract
Background
Tol2, a member of the hAT family of transposons, has become a useful tool for genetic manipulation of model animals, but information about its interactions with vertebrate genomes is still limited. Furthermore, published reports on Tol2 have mainly been based on random integration of the transposon system after co-injection of a plasmid DNA harboring the transposon and a transposase mRNA. It is important to understand how Tol2 would behave upon activation after integration into the genome.
Results
We performed a large-scale enhancer trap (ET) screen and generated 338 insertions of the Tol2 transposon-based ET cassette into the zebrafish genome. These insertions were generated by remobilizing the transposon from two different donor sites in two transgenic lines. We found that 39% of Tol2 insertions occurred in transcription units, mostly into introns. Analysis of the transposon target sites revealed no strict specificity at the DNA sequence level. However, Tol2 was prone to target AT-rich regions with weak palindromic consensus sequences centered at the insertion site.
Conclusion
Our systematic analysis of sequential remobilizations of the Tol2 transposon from two independent sites within a vertebrate genome has revealed properties such as a tendency to integrate into transcription units and into AT-rich palindrome-like sequences. This information will influence the development of various applications involving DNA transposons and Tol2 in particular.
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Background
The transposable element Tol2 from medaka fish is the first functional transposon identified in vertebrates [1]. It belongs to the hAT family (named for hobo, Ac and Tam3) and integrates into host DNA through a "cut-and-paste" mechanism [2]. Recently, a non-autonomous Tol2-based system has been developed as a tool for genome analysis of vertebrates and for highly efficient transgenesis [3–11]. It has been used for both gene trap and enhancer trap (ET) screens [12–14] as well as insertional mutagenesis [15, 16]. Some of these applications have recently been reviewed [17, 18].
One of the features of non-autonomous transposon-based systems, including Tol2, is that a transposon integrated into a genome can be remobilized if transposase mRNA is available. Previous applications of the transposon system have been based on random integration after co-injection of a plasmid DNA harboring Tol2 and transposase mRNA. Such random integration is attractive for a wide variety of applications ranging from gene discovery to gene therapy. However, the pattern of transposon integration upon remobilization from the donor site can be substantially different from that of plasmid-based integration. For example, the Sleeping Beauty (SB) transposon has a strong tendency to reinsert during in vivo remobilization at loci closely linked to its donor site [19]. Such local hopping could be favorable not only for region-specific mutagenesis [20] but also for region-specific probing of enhancers. However, despite the recent surge of interest in the Tol2 transposon system, its integration and/or reintegration properties have not yet been analyzed in detail.
Using Tol2, we have previously established a collection of stable transgenic zebrafish ET lines and demonstrated that a single copy of a Tol2 transposon-based ET cassette can be remobilized into a new chromosomal location [13, 21]. Here, we report the results of a genome-wide analysis of Tol2 reintegration in zebrafish initiated from two genomic sites in two different chromosomes.
Results
Design of the Tol2 transposon remobilization screen
We used two different ET lines as donors for the remobilization experiments. The first line, SqET33, was established during our pilot enhancer trap (ET) screen [13]. It carries a single insertion of a transposable element in the 3' UTR of a novel gene of the Zic family, zic6 on chromosome 14 (Figure 1A). The second line, SqET33-E20, was established after induced remobilization of a Tol2 transposon-based ET cassette from the SqET33 line [22]. It carries a single insertion located approximately 4.2 kb upstream of a putative gene zgc:66340 (similar to Axin-1 up-regulated gene 1) on chromosome 24 (Figure 1B). In both lines, stable tissue-specific GFP expression is maintained through at least four generations of breeding, indicating that it is not affected by silencing or epigenetic modification. For both donor lines we confirmed the presence of a single Tol2 transposon insertion by Southern blot hybridization (Figure 1C).
Donor sites used for the remobilization screen. A: The SqET33 donor site (Tol2 transposon insertion in the 3' UTR of zic6). B: The SqET33-E20 donor site (Tol2 transposon insertion 4.2 kb upstream of zgc:66340). The green arrow shows EGFP; krt4 is a minimal promoter; orange arrows represent the 5' and 3' ends of Tol2; coding exons are depicted as blue boxes; and UTRs are represented as open boxes. C: SqET33-E20 F0 founder fish was outcrossed to wild type and DNA isolated from GFP-negative embryos (negative control, C), GFP-positive embryos showing a SqET33 expression pattern of GFP (donor-type, D), and GFP-positive embryos showing a new expression pattern distinct from the donor one (N), were used for Southern blot hybridization. SqET33, DNA from the donor line. D: A scheme of the Tol2 remobilization screen.
In order to induce remobilization of the Tol2 transposon-based ET cassette from the donor site in SqET33 (referred to as the SqET33 donor site), we initially used mRNA containing only an open-reading frame (ORF) for Tol2 transposase. In this experiment, transgenic fish homozygous for a single Tol2 insertion were outcrossed to wild type fish. The embryos from this cross (F0 generation) were injected with transposase mRNA at the one- or two-cell stage. Most of the injected embryos only showed a "donor-type" GFP expression pattern. However, some showed mosaic expression of GFP in somatic cells, mostly in muscle or skin [13]. We did not preselect embryos on the basis of such GFP expression, but used these observations as an indication that a transposition had been triggered. Because we were only interested in heritable Tol2 re-transpositions, the F0 generation was not analyzed in respect of transposon integration into somatic cells. All injected embryos were raised to sexual maturity and crossed to wild type fish, and their progeny (F1 generation) were analyzed for changes in GFP expression. We identified injected fish as founders (F0 founder) if their progeny showed a new GFP expression pattern that differed from the donor pattern. The F0 founder fish carried the Tol2 reintegrations in the germline. In our screening scheme, only the F1 embryos that showed new GFP expression patterns were further analyzed by TAIL-PCR (Figure 1D). Using this strategy, we initially identified 21 F0 founder fish out of 282 injected fish. This corresponded to a 7% apparent germline transposition rate (1st screen in Table 1). We assumed that the transposase mRNA used at that point was not very effective in Tol2 remobilization. Therefore, in subsequent experiments, we used a modified transposase mRNA containing the 5' and 3' UTRs of the Xenopus β-globin gene [6]. As a consequence, we identified 103 F0 founder fish out of 268 injected fish in a new round of screening, a much higher apparent germline transposition rate (38%, 2nd screen in Table 1).
To test whether the donor site influences transposon remobilization, we used the SqET33-E20 line (referred to as the SqET33-E20 donor site) as a donor. We identified 84 F0 founder fish out of 175 injected fish, with an apparent germline transposition rate similar to that of the 2nd screen (48%, 3rd screen in Table 1). Because our screening was based on the appearance of new GFP expression patterns, new insertions that caused no changes in that pattern were not taken into account. Therefore, the actual germline transposition rate for both donor sites should be higher.
In most cases, when F0 fish (heterozygous for gfp) were outcrossed to wild type fish, GFP-negative and -positive embryos (regardless of their expression patterns) were segregated in an approximately 1:1 ratio. However, we observed altered GFP segregation ratio following remobilization of the Tol2 transposon (see Additional file 1). Out of 725 injected F0 fish (from three rounds of screening), 22 produced more than 50% GFP-positive progeny, suggesting an increase of Tol2 copy number. Thirty-seven injected F0 fish produced less than 50% GFP-positive progeny, indicating either partial loss of Tol2 or silencing of gfp. A similar alteration of GFP segregation has recently been described for re-transposition of the Ds element in zebrafish [23]. Germinal excision without concomitant transposon reintegration has also been reported for the Ac/Ds transposon in plants [24, 25].
Interestingly, about 40% of F0 founder fish after outcrossing with wild type produced progeny (F1) such that individual embryos within a single F1 family showed distinct new GFP expression patterns. The number of these new patterns per single F1 family varied from two to seven, suggesting multiple transposon integration events in the germline of a single F0 fish (Table 2). In most cases, TAIL-PCR analysis of individual embryos from such F1 families demonstrated single Tol2 insertions at different positions in the genome. The presence of a single insertion in F1 fish suggests that transposition occurred independently in separate germline cells, and Tol2 was transposed by a non-replicative mechanism, since an F0 fish is heterozygous for a single insert. However, in a few cases, TAIL-PCR detected two or three insertions in one F1 embryo; this was additionally confirmed by Southern blot hybridization (see Additional file 2).
About 20% of F0 founder fish produced embryos with the donor-type GFP expression pattern as well as the new GFP expression pattern in the same embryo. In such embryos, donor insertion was always detected by TAIL-PCR, indicating that the donor copy was retained after retransposition. This result suggests that in some cases remobilization probably occurs during DNA replication. Transposition during DNA replication has been well described for other "cut-and-paste" transposons, particularly for Ac/Ds [26].
Tol2 preferentially reintegrates into linked loci
By analyzing the chromosomal distribution pattern of the new insertions (see Materials and methods), we found that all chromosomes were hit by Tol2 during re-transposition (Figure 2A and Table 3). About 15% of reintegration events occurred in the donor chromosomes. Such a linked pattern of re-transposition was found for both donor sites: 23 out of 153 insertions were mapped on chromosome 14 (re-transposition from the SqET33 donor site), while 17 out of 111 were mapped on chromosome 24 (re-transposition from the SqET33-E20 donor site). We also found that about 43% (10/23) and 24% (4/17) of reintegrations occurred less than 1 Mb from the SqET33 and SqET33-E20 donor sites, respectively (Figure 2B). However, these numbers are probably underestimates, since only transpositions that cause changes in GFP expression patterns were considered.
Genome-wide distribution pattern of Tol2 integrations. A: Chromosomal distribution pattern of Tol2 insertions depending on the donor site. Asterisks show statistically significant differences (p < 0.0001 for the donor chromosomes and p < 0.05 for the others). B: Pattern of Tol2 reintegrations linked to the different donor sites (chromosomes 14 and 24). Non-donor chromosomes 8 and 25, comparable in size to the donor chromosomes, are shown for reference. Red triangles represent the donor sites.
We noticed that some chromosomes were possibly favored targets for Tol2 integration, while others appeared to be disfavored. However, with the exception of the donor chromosomes, only chromosome 11 (when the SqET33 donor site was used for the re-transposition) and chromosome 2 (when the SqET33-E20 donor site was used) were somewhat preferred targets. Chromosome 20 was found to be a potential cold spot for Tol2 integration (when the SqET33-E20 donor site was used for the re-transposition). However, hotspots cannot be adequately evaluated at the chromosomal level at a resolution of 338 events. In addition, when all Tol2 integrations were placed on the zebrafish genome map, there was no indication of significant clustering (except on the donor chromosomes). In some cases, two independent transposon insertions were mapped within 3 to 77 kb of one another (Figure 3).
Clustering of Tol2 integrations in the zebrafish genome. Only chromosomes in which two independent Tol2 insertions are mapped within less than 100-kb of one another are shown. Data for the plasmid-based derived integrations were taken from [13] and remapped.
Tol2 integration into transcription units
Sequencing of the PCR-amplified regions that flank the target sites from all founder fish confirmed the generation of novel integration events that were not present in the original donor lines (see Additional files 3 and 4). During the three rounds of screening, we isolated 338 transposon integration sites. The genomic positions of 287 of these sites were identified using BLASTN in the Ensembl genome browser (Figure 4A; see Materials and methods for details). However, we could not unambiguously map the remaining 51 integration sites because (i) multiple hits were found that were either very similar or identical to the genomic sequence (26 integration sites), (ii) integrations occurred in repetitive dinucleotide sequences (eight integration sites) or (iii) there were sequence gaps in the database (17 sites). Approximately 39% of the mapped integration sites were found within known or predicted genes annotated in the zebrafish genome. About 82% of these sites were mapped within introns and about 18% within exons. There was a bias towards introns because their cumulative size is much larger than that of exons, so they present a much larger target for transposon integration. Assuming a 5-kb interval as an arbitrary threshold for the regulatory regions at the 5' and 3' ends of genes, the frequency of integration was about 62% for known or predicted transcription units. Furthermore, about 59% (64/109) of the "intergenic" insertions were mapped within 50-kb regions upstream or downstream of known or predicted genes annotated in the zebrafish genome. We also compared the distribution pattern of insertions depending on donor site. No significant difference was found (Table 4).
We found five genes (ENSDARG00000033473, ENSDARG00000034820, zgc:110750, foxa and fgf13) that were recurrently hit by Tol2. Two insertions in ENSDARG00000033473 (334 kb) were mapped in one intron with a distance of 41 kb between the integration sites. Similarly, two insertions in zgc:110750 (196 kb) were mapped in one intron with a distance of 6 kb between the integration sites. Two insertions in ENSDARG00000034820 (67 kb) were mapped in an intron and 4 kb downstream of the gene, while insertions in fgf13 (228 kb) were mapped in two different introns. Finally, the two insertions in foxa (5 kb) were mapped 3 kb upstream and 1 kb downstream from the gene. Four of these five genes (fgf13 was the exception) were hit from both independent donor sites. Since the sizes of the targeted genes differ substantially (from 5 kb to 334 kb), the distance between two independently integrated Tol2 transposons is more important (in these cases, the distance ranges from 6 kb to 47 kb). We calculated the probability of hitting a similarly-sized locus in the zebrafish genome and then used a binomial distribution test to determine the statistical significance of each targeted locus being hit twice. The P values ranged from 3.3 × 10-6 to 2.8 × 10-3, probably indicating potential hotspots within these genes (with an exception of fgf13, since it is linked to the donor site).
Tol2 integration into endogenous repeat elements
We analyzed the distribution pattern of Tol2 insertions with respect to various genomic repeat elements (Table 5); 128 out of the 338 integration sites were found in endogenous repeat elements that are currently annotated in the zebrafish genome. Most of these targeted repeat elements belong to DNA transposons and to unclassified Dr repeats. In addition, our results showed that Tol2 was less prone to integrate into retrotransposons (17 integration events) and tandem repeats (16 integration events). Interestingly, LTR-containing retrotransposons were very seldom targeted by Tol2 (only 3 integration events). According to Repbase Update [27], 357 different families of DNA transposons and 279 of retrotransposons are currently annotated in the zebrafish genome. Retrotransposons that contain LTRs are more diversely represented in the zebrafish genome than those that do not (222 vs. 57 families). The differences in targeting of repetitive elements observed in our experiment could be explained by a difference in either the copy number or the global distribution pattern of each class of repetitive elements in the zebrafish genome.
Specificity of Tol2 integration site
Some transposable elements exhibit a high degree of integration specificity, while others display relatively little preference for a target DNA sequence [28, 29]. We analyzed the nucleotide composition over a 48-bp sequence region comprising an 8-bp target site and 20-bp flanking sequences on each side according to [30] (Figure 4B). In addition to the 329 integration sites isolated in this study, we also included 39 integration sites isolated from our previous ET screen [13]. Comparisons among all the Tol2 integration sites and flanking DNA revealed no conserved pattern in the sequences flanking the eight base pair duplication at the integration site. The only exceptions were the nucleotides at ± 3 and ± 1 bp relative to the integration site, which were 46% and 40% conserved, respectively (see Additional file 5). In addition, we detected a weak AT-rich consensus that contained a palindrome-like core sequence TNA(C/G)TTATAA(G/C)TNA centered at the insertion site (shown in bold). However, only four integration sites were actual palindromes.
Discussion
In this study, we analyzed a genome-wide reintegration of the non-autonomous transposable element Tol2 in zebrafish when it was remobilized from two different donor sites. We showed that the genomic Tol2 copy can be remobilized upon injection of transposase mRNA into the germlines of up to 48% of founders. Since we selected only those Tol2 reintegrations in germlines that caused changes in GFP expression, we in fact measured the "apparent transposition rate"; the actual germline transposition frequency during in vivo remobilization would be higher.
We analyzed Tol2 integration sites with respect of their chromosomal distribution, integration into intragenic regions and insertion site sequence specificity. Although novel integration sites were found on different chromosomes, Tol2 reintegration was not random. Almost 39% of transposon integrations were found within known or predicted genes. Most of them were found within introns, as expected in view of the high intron/exon ratio in the zebrafish genome. If we consider insertions into the regulatory regions adjacent to transcriptional initiation and termination sites, the rate of Tol2 transposition into genes was even higher. Since we used the TAIL-PCR method to isolate transposon inserts, we could not recover all possible Tol2 insertions in the genome. However, despite of using enhancer trap approach, the frequency with which Tol2 was integrated within intragenic regions was similar to that found for the SB transposon in human (39%) and mouse (31%) cells [31] and for Ac/Ds in rice (30%) [32–34] and Arabidopsis (38%) [35]. Therefore, Tol2 is as prone to integrate into transcriptional units as other DNA transposons.
Our results further demonstrated that about 15% of Tol2 reintegrations from a specific donor site were linked to the same chromosome. Such behavior was also noticed in [11], where about 18% of the mapped integration sites (6/34) were located on the donor chromosome. We found that about one third of intrachromosomal reintegrations were located within 1 Mb of the donor site. However, since we selected reintegrations on the basis of new GFP expression patterns, this number is likely to be lower than the actual number of such transpositions (for example, closely-linked transpositions may retain the GFP expression pattern of the donor). This local hopping phenomenon has been described for other DNA transposons [19, 36–40]. For example, SB is mostly re-integrated within 3 Mb of the donor site [19] and the local hopping interval of the P element is within 100 kb [36]. Local hopping was also found for the hAT family. In this case, more than half of Ac transposon reintegrations occurred within 1.7 Mb of the donor site [37]. Overall, a linked reintegration property of the Tol2 system might be beneficial for setting a region-specific saturation ET screen.
Interestingly, our analysis revealed that some chromosomes other than the donor were somewhat preferred targets for Tol2 integration; still others appeared to be disfavored. Such transposon behavior may reflect the spatial chromosomal architecture within the nucleus, if multiple non-adjacent chromosome segments are closely juxtaposed at the nuclear interior or periphery [41–43]. There are many examples of correlations among the intranuclear positions of genes, their clusters and genetic activities, whereas the relative positioning of chromosomes seems to be maintained (reviewed in [44]). Therefore, such a property of transposons may potentially be used for analyzing the spatial organization of the genome.
We found that Tol2 differentially targeted the different classes of endogenous repetitive elements. For example, it more frequently targeted DNA transposons than retrotransposons and tandem repeats. The latter tendency contrasts with the profound preference of Tc1/mariner transposons for TA-containing microsatellite DNA [31, 45]. Expansion of such repeats during replication slippage can cause repeat instability and increased recombination rates (reviewed in [46]), suggesting that these transposons may use the recombination machinery during integration. Like the SB transposon, Tol2 also avoided retrotransposons containing LTRs. The differences in targeting of endogenous repetitive elements may reflect differences in the copy numbers of each class of repeats, as well as differences among the mechanisms of integration utilized by each transposon family.
We also found that Tol2 was prone to integrate into AT-rich DNA regions and that a target site contained a weak palindrome-like consensus sequence. An AT-rich palindromic consensus has previously been found in the target sequence of Tc1/mariner transposons such as SB in human and mouse cells [31] and the Tc1 element in worms [47]. However, in contrast to the strict preference of SB and Tc1 for TA dinucleotide targets, Tol2 has no such preference at the nucleotide level. There is some evidence that distinct preferred transposon integration sites may not necessary match consensus sequences, but rather share similar structural patterns [48]. DNA structural characteristics such as bending and protein-induced deformability play an important role in directing DNA integration [28, 48, 49]. DNA bending can lead to changes in the width and depth of the major and minor grooves, affecting a protein's access [49]. AT-rich palindromes are particularly susceptible to local melting and have been experimentally shown to adopt a bendable DNA structure [50]. In addition, palindromic sequences have the potential to form cruciform configurations, which are an efficient target for RAG-mediated transposition [51]. Also, AT-rich palindromic repeats are known to be double-strand break hotspots. It has been proposed that DNA bending plays a role in the integration specificity of the hobo transposable element from the hAT family, but hobo has no strict preference for targeting at nucleotide level [39]. This suggests the likelihood that the target site selection of Tol2 is primarily determined at the level of DNA structure, not sequence.
Tol2 element transposes by "cut-and-paste" mechanism, which involves the excision and re-integration of the transposon from one site to another, creating an 8-bp duplication of the integration site [3, 13]. Previously, we found that in one third of Tol2 excision events, reparation of donor site results in different footprints [13]. In our experiments we used extremely high amounts of transposase mRNA (around 9 × 107 molecules per a single copy of transposon), therefore it may be reasonable to expect multiple "cut-and-paste" events before transposon will finally settle. Such multiple hops could generate double-strand breaks and, as a consequence, the footprints. Our analysis of DNA sequences flanking the integration/target site revealed no signs of the footprints, at least, at the vicinity (up to 900 bp) of new integration sites. All DNA sequence modifications found at these regions exhibited DNA sequence polymorphism between zebrafish strains (data not shown). However, we could not rule out the possibility that the footprints left after multiple "cut-and-paste" events may be found far away from integration sites.
Conclusion
In summary, in a large-scale ET screen we analyzed 338 insertions generated in the zebrafish genome by remobilization of a single Tol2 transposon copy from two independent sites on two different chromosomes. About 39% of Tol2 insertions occurred within genes, mostly in introns. Upon remobilization, Tol2 showed a preference to reintegrate within the chromosome containing the donor site. Sequence analysis of integration sites revealed no strict specificity at the nucleotide level, but Tol2 was prone to integrate into AT-rich regions with weak palindrome-like consensus sequences. This information should be carefully evaluated during the design of various follow-up applications that involve Tol2. Numerous ET lines with diverse GFP expression patterns have been generated in this work. They represent a large set of research tools for in vivo studies of vertebrate development, and some have already been successfully used for that purpose [22, 52, 53].
Methods
Fish lines
The ET(krt4:EGFP)SqET33 line (referred to as SqET33) was established by coinjection of in vitro synthesized transposase mRNA and Tol2 transposon-based ET construct DNA into wild type zebrafish embryos at the one- or two-cell stage [13]. The ET(krt4:EGFP)SqET33-E20 line (referred to as SqET33-E20) was created after in vivo remobilization of a single-copy of Tol2 transposon-based ET construct from the donor line SqET33 [22]. Fish were maintained according to established protocols [54] and in agreement with the IACUC regulations and rules of the IMCB zebrafish facility. Transgenic and wild type fish were AB strain.
Plasmids
Plasmid pTem03 containing the coding region for medaka Tol2 transposase was purchased from Dr. Koga (Nagoya University, Japan) as a part of the "Gene transfer system" kit. Plasmid pDB600 containing the coding region of Tol2 transposase flanked with the 5' and 3' UTRs from the Xenopus β-globin gene was kindly provided by Dr. Ekker (University of Minnesota, USA).
In vitro mRNA synthesis and in vivo transposon remobilization
Plasmids pTem03 and pDB600 were linearized with Xba I and Spe I, respectively, and used as templates for in vitro mRNA synthesis. Transposase mRNA was synthesized using mMESSAGE mMACHINE SP6 and T3 kits (Ambion, USA) and purified using an RNeasy Mini Kit (QIAGEN, Germany). For in vivo remobilization of a single-copy of Tol2 transposon-based ET construct, 50-100 pg of transposase mRNA was injected into zebrafish embryos from the donor lines at the one- or two-cell stage.
Southern blot hybridization
Genomic DNA from adult fish or embryos was phenol extracted and digested using Hin dIII (New England Biolabs), which cut the Tol2 transposon-based ET cassette at a unique site. The digested genomic DNA was fractionated by agarose gel electrophoresis, transferred to a positively charged nylon membrane (Hybond-N+, Amersham Biosciences) by capillary blotting [55], and crosslinked by UV irradiation. The DNA probe for EGFP was labeled with digoxigenin (DIG) using a PCR DIG synthesis kit (Roche Applied Science). We used DIG EasyHyb buffer, an anti-DIG alkaline phosphatase conjugate antibody and CDP-Star chemiluminescent substrate (all Roche Applied Science) to detect the hybridized probe. Hybridization and detection were carried out according to the manufacturer's instructions.
Identification and mapping of integration sites
To recover genomic sequences flanking the integrated Tol2 transposon we used thermal asymmetric interlaced PCR (TAIL-PCR). The DNA was isolated from one GFP-positive embryo after outcrossing of F0 fish with wild type fish. TAIL-PCR was performed according to Liu and Whittier [56] using the primers and cycling conditions described elsewhere [13]. The resulting PCR products were purified and directly sequenced using primers 5'-CCCCAAAATAATACTTAAGTACAG-3' and 5'-GTACTTGTACTTTCACTTGAG-3', which anneal to the 5' and 3' transposon ends, respectively. The length of the sequence reads never exceeded 900 bp. A sequence was considered to be from an authentic integration site only if it contained the Tol2 transposon sequence from the nested primer to the ends of the inverted repeats. In total, we amplified and sequenced 519 genomic regions flanking the 338 integration sites (see Additional file 4 for details). Of these, 159 sequences were amplified from either the 5' or the 3' end of the integrated transposon only. The sequence reads were then mapped to the zebrafish genome using BLASTN (the latest zebrafish whole genome assembly version 7 (Zv7, April 2007 freeze in the Ensembl genome browser) http://www.ensembl.org. In some cases, the flanking sequences were blasted against the unfinished high-throughput genomic sequences (htgs) database or trace archive at NCBI http://www.ncbi.nlm.nih.gov. We considered the sequence to be from a unique integration site if it matched to no more than one genomic locus with 95% or greater identity to the genomic sequence over the high-quality sequence region (a whole length of sequence read). On the basis of this criterion, 423 sequences reads from 275 integration sites could be unambiguously mapped to unique genomic loci. Sixty sequence reads from 38 integration sites were matched to more than one genomic locus with 95% or greater identity to the genomic sequence. In those cases, only the hits with highest identity (>99%) and score over the whole length of sequence read were considered. Thus, 20 sequence reads from 12 integration sites were unmistakably mapped to distinct genomic loci. The remaining 40 sequence reads from 26 integration sites could not be mapped unambiguously. We also had 26 sequence reads from 17 integration sites that were either matched with less than 95% of identity to the genomic sequence, or had no significant similarity to the genomic database. Half the sequence reads that had no hits in the latest version of the database (Zv7) were matched to a single genomic locus with 98% or greater identity to the genomic sequence over the sequence region in the previous zebrafish whole genome assembly, version Zv6. Nevertheless, such sequence reads were not considered to be matched. In addition, 10 sequences from 8 integration sites represented short (<100 bp) repetitive dinucleotide sequence reads that could not be mapped to any location. Ultimately, we were able to map 287 integration sites out of 338 integration events to unique genomic loci.
Bioinformatics
We defined integration as having landed in a gene only if it was within the genomic coordinates of the 21,322 protein-coding genes or transient EST gene models annotated in the zebrafish genome. The 5' and 3' ends of the genes were considered the first and last nucleotide positions according to gene coordinates in the latest zebrafish whole genome assembly, Zv7. We analyzed the base composition over a 48-bp region encompassing the Tol2 target site using the computer program WebLogo (version 3.0) http://weblogo.threeplusone.com. We also analyzed integrations in relation to various genomic repeat elements annotated in the zebrafish genome. The expected number of insertions in each chromosome (if integration events were random) was calculated as follows: (1) to calculate the expected distribution of insertions, the total number of bases in the zebrafish genome was divided by the number of mapped insertions; (2) to calculate the expected number of insertions on each chromosome, the length of each individual chromosome was divided by the expected distribution of insertions. The total number of base pairs in the zebrafish genome (reference assembly length) according to the Zv7 assembly is 1,440,582,308 base pairs. Integration into chromosomes was tested for statistical bias using a χ2 test to compare the observed number of integrations into a particular chromosome to the value expected if integration events were random.
Abbreviations
- ET:
-
enhancer trap
- GFP:
-
green fluorescent protein
- EGFP:
-
enhanced GFP
- LTR:
-
long terminal repeats
- ORF:
-
open reading frame
- SB:
-
Sleeping Beauty transposon
- TAIL-PCR:
-
thermal asymmetric interlaced PCR
- UTR:
-
untranslated region
- WT:
-
wild type
- bp:
-
base pairs.
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Acknowledgements
We thank the personnel of the IMCB fish facility for the maintenance of the fish lines, and the personnel of the IMCB DNA sequence facility for the sequencing. This work was financially supported by the Agency for Science, Technology and Research (A-STAR) of Singapore. The authors declare absence of conflicting financial interests.
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Conceived and designed the experiments: IK, AE, SP and VK. Performed the experiments: IK and MGL. Analyzed the data: IK, MGL, AE, SP and VK. Wrote the manuscript: IK, SP and VK. All authors read and approved the final manuscript.
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Additional file 1: GFP segregation in F 1 generation. Table S1 shows the number of novel expression patterns and GFP segregation ratios in the F1 generation. Transgenic embryos (F0) carrying a single heterozygous Tol2 insert in their genome (gfp+/-) were injected with transposase mRNA, raised to maturity, and outcrossed to wild-type fish. (PDF 80 KB)
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Additional file 2: Southern blot hybridization of F 1 generation. Figure S1 shows the Tol2 copy number in F1 fish. DNA was isolated from individual F1 fish that originated from the same F0 founder. The DNA samples were digested with Hin dIII and hybridized with DIG-labeled EGFP probe. (PDF 7 MB)
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Additional file 3: Integration sites and genes located near the insertions. Table S2 shows the integration loci and nearest genes targeted by Tol2 remobilized from two different donor sites. Targeted repetitive elements are also shown. (PDF 132 KB)
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Additional file 4: Flanking genomic sequences of Tol2 transposon insertions. Table S3 shows the DNA sequence reads of the regions that flank Tol2 transposon insertions. To obtain the genomic locations of Tol2 insertions, the flanking sequence reads were blasted against the latest zebrafish genome sequence database (zebrafish whole genome assembly, version 7) from the Ensembl genome browser http://www.ensembl.org. In some cases, the flanking sequence reads were blasted against the unfinished high-throughput genomic sequence (htgs) database or trace archive at NCBI http://www.ncbi.nlm.nih.gov. (PDF 265 KB)
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Additional file 5: Target site analysis for 368 genomic Tol2 insertions. Table S4 shows the base composition of an 8-bp integration site in 368 genomic Tol2 insertions. For the region flanking the integration site only the first three nucleotides are shown. (PDF 61 KB)
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Kondrychyn, I., Garcia-Lecea, M., Emelyanov, A. et al. Genome-wide analysis of Tol2 transposon reintegration in zebrafish. BMC Genomics 10, 418 (2009). https://doi.org/10.1186/1471-2164-10-418
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DOI: https://doi.org/10.1186/1471-2164-10-418