Volume 8 Supplement 1
Transposon mediated transgenesis in a marine invertebrate chordate: Ciona intestinalis
© BioMed Central Ltd 2007
Published: 31 October 2007
Achievement of transposon mediated germline transgenesis in a basal chordate, Ciona intestinalis, is discussed. A Tc1/mariner superfamily transposon, Minos, has excision and transposition activities in Ciona. Minos enables the creation of stable transgenic lines, enhancer detection, and insertional mutagenesis.
DNA transposons are powerful tools for genetic analyses. Transposons are employed for creation of stable transgenic lines, enhancer detection, gene trapping, and insertional mutagenesis. These transposon-mediated techniques have been facilitated by the discovery and reconstruction of active transposons in several organisms [1–11]. Despite their utility, transposon technologies are restricted to a few model organisms. Marine invertebrates include most of the phyla whose study is crucial to elucidating the evolutionary molecular mechanisms of diversification in metazoans. To date, transposon technologies have been introduced for only a few marine invertebrate species. Because of the scarcity of refined genetic techniques, research into gene functions in marine invertebrates has remained limited.
Recent achievement of germline transgenesis in a marine invertebrate chordate, Ciona intestinalis, has altered this situation [12, 13]. C. intestinalis has several characteristics that make it amenable for genetics research. In this basal chordate, a Tc1/mariner superfamily transposon, Minos, has the complete activity required for its transposition . Minos introduced into Ciona is excised from a plasmid vector by transposase and is integrated into TA dinucleotides of another DNA molecule. The TA dinucleotides are known as target sequences of Tc1/mariner transposons . Transposition occurs in Ciona germ cells, and Minos is inserted into the chromosomes of germ cells . The insertions are inherited stably by subsequent generations, thereby creating stable transgenic lines. Using this transformation technique, genetic techniques such as enhancer detection and insertional mutagenesis have been introduced into Ciona using Minos [13, 16, 17]. In this article, recent achievements with transposon techniques in Ciona, as well as characteristics of Ciona as a new genetic model, are discussed.
Characteristics of Ciona intestinalisas an experimental system for genetics
Ascidian larvae change their structure through metamorphosis and become sessile adults (Figure 1c) . After metamorphosis, ascidians start to take in food by filter feeding. During metamorphosis, the larval tail is lost and adult tissues grow rapidly, which include characteristic chordate structures such as pharyngeal gills and an endostyle (the endostyle is homologous to vertebrate thyroid gland [26, 27]). Metamorphosis of typical ascidians is completed within several days. Metamorphosis is a dramatic event in ascidian development and provides a good experimental system in which to uncover the mechanisms of metamorphosis and their conservation among vertebrates.
Ciona intestinalis (hereafter referred to as Ciona) is a cosmopolitan ascidian [28–31]. It is hermaphroditic and self-fertile. This characteristic represents a great advantage when performing mutant screens because creating animals that are homozygous with respect to mutation sites is possible without genotyping [32, 33]. An adult Ciona bears thousands of eggs; eggs and sperm can be collected surgically.
Surgically collected eggs can be fertilized with sperm from a different individual. They subsequently exhibit normal, synchronized development. Natural spawning can be induced by simple dark-light adjustment to facilitate self-fertilization. Moreover, sperm can be stored on ice for 1 week without loss of fertility. Cryopreservation of sperm is also established to store mutants or transgenic lines semipermanently . The easy handling of germ cells enables reduction in labor associated with mutant screening and preservation of lines.
The most striking characteristic that distinguishes Ciona from other ascidians is the availability of a draft genome sequence . The Ciona genome size is approximately 166 megabases per haploid, which contains 15,852 protein coding genes . The genome size and gene number are comparable to those of Drosophila melanogaster, and much smaller than those of most vertebrates. In addition, the Ciona genome is less redundant than those of vertebrates [37, 38], which is probably related to the twofold to threefold duplication of genomes during vertebrate evolution . Therefore, Ciona possesses the basic set of genes for a chordate body plan. Because of its compact genome, Ciona provides a simple experimental system in which to uncover genetic mechanisms that specify the chordate body plan as well as mechanisms of chordate evolution.
BLAST search of Tc1/mariner superfamily transposons in five eukaryotic genomes
Ciona intestinalis ver 1.0
Brachiostoma belcheri ver 1.0
Fugu rubripes ver 4.0
Xenopus tropicalis ver 4.1
Nematostella vectensis ver 1.0
1.1 × e-29
3.2 × e-6
8.6 × e-26
1.69 × e-39
2.9 × e-28
1.6 × e-41
In Ciona, techniques to support the practice of genetics research have been developed. The Ciona life cycle is about 2 to 3 months. An inland culture system has been established [17, 34]. Settlement after metamorphosis enables retention of several lines in the same aquarium. Introduction of DNA and RNA into eggs by microinjection or electroporation is performed routinely [41, 42]. The latter technique can introduce DNA and RNA into hundreds of eggs within 1 hour, thereby facilitating creation of transgenic lines.
There are three major obstacles to use of Ciona to conduct genetics studies. First, no inbred strain has been created; most experiments are dependent on natural populations. Creating strains had been difficult because of complications in culturing. Recent improvements of inland culture systems are expected to resolve this problem . Second, natural Ciona harbor many single nucleotide polymorphisms. The genome project reported that 1.2% of nucleotide differences were observed between alleles of the single individual . This score is 15 times higher than that in humans, and three times higher than that in pufferfish. Such highly frequent polymorphism would render it difficult to perform systematic fine mapping of point mutations. On the contrary, high polymorphism might allow retention of highly frequent natural mutants, which are a valuable resource for mutant screening. In C. intestinalis, and its related species C. savignyi, several mutants have been isolated through screening of wild populations [34, 44, 45]. The third obstacle to genetics studies is the requirement for seawater for culture. Large-scale culturing requires a considerable amount of seawater, which limits the culturing of Ciona to laboratories that are near to the sea. Recently, Ciona culture with artificial seawater has been achieved [34, 43], which will promote the spread of Ciona studies to inland laboratories.
Activity of Minos transposon in Ciona
Another convenient transgenesis technique of Ciona with Minos was achieved using electroporation . As described above, electroporation enables rapid and reproducible transgenesis of early Ciona embryos. This technique simultaneously electroporates Minos DNA and in vitro synthesized Minos transposase mRNA in Ciona embryos. The transformation frequency by electroporation mediated transgenesis is about 20% to 30%, which is lower than that by microinjection mediated transgenesis, perhaps because of a lower amount of mRNA introduced into embryos by electroporation. By microinjection, 5 to 10 ng/μl of Minos DNA and 50 to 200 ng/μl of transposase mRNA are included in the injected solution. The current electroporation method requires 60 μg of Minos DNA and 60 μg of transposase mRNA, which would correspond to 5 to 10 ng/μl of DNA and RNA in the injection solution. Nevertheless, electroporation mediated transgenesis is now the main strategy of Ciona transformation because of its convenience.
Minos exhibited constant excision and transformation activity, even when the length of insertion is sufficiently long to suppress transposition of another Tc1/mariner transposon, namely SB . So far, an insert size of up to 10 kilobases has been found to have no adverse effect on insertion efficiency (Sasakura Y, unpublished data). Such flexibility of Minos with respect to insert length allows the creation of various transposon constructs that are appropriate for experimental purposes.
Activity of other Tc1/mariner transposons in Ciona
The identification of other active transposons would make transposon technology more versatile in Ciona, because it would be useful to create 'jump starter' lines of Minos. Modifier screens of mutants generated by Minos must be done using a different transposon. Different transposons can be expected to have different insertion site preferences. Therefore, execution of large-scale mutagenesis with two transposons would be effective for saturation mutagenesis. In addition to these technical innovations, description of activity of transposons in various organisms is necessary to elucidate cross-species activity of transposons and the mechanisms that determine transposon activity in nonhost organisms. Such knowledge would be valuable for further improvement of transposon technologies. Transposon activity in marine invertebrates has not been described, except for Minos in Ciona and in a crustacean [12, 53]. Ciona is the pioneer organism of transposon technology among marine invertebrates; testing of various transposons in this organism is important.
What might restrict Mos1, SB, and FP activity in Ciona? One possibility is that co-factors that are required for transposase activity are incompatible or absent in Ciona. In fact, SB and FP are transposons derived from vertebrates [8, 11], and therefore they retain high activity in vertebrates, indicating that all sets of co-factors required for SB and FP activity are present in vertebrates. Recent studies have revealed necessary co-factors for SB transposases [65, 66]. Although Ciona contains the basic set of genes for the chordate body plan, many genes are specific to vertebrates. The supply of such co-factors may be necessary to make transposons active in Ciona if SB and FP transposases require such vertebrate-specific co-factors. An alternative possibility is that a factor is present that inhibits transposases. Inhibition of the transposase activity has been reported in the Tn5 transposon of Escherichia coli, in which an inhibitor of the transposition protein (a truncated form of Tn5 transposase that does not possess DNA-binding activity) forms a complex with Tn5 transposase and interferes with transposition . The presence of such an inhibitor has not been demonstrated in Tc1/mariner transposons, but the possibility remains that there is a Ciona protein that binds transposases and inhibits their activity.
The inefficiency of Mos1, SB and FP in Ciona implies that activity of transposons must be tested in each animal model to seek an active transposon. Identification of factors that restricts the activity of transposons is necessary to make them more valuable tools for genetics research in various organisms.
The compact genome of Ciona is a convenient feature for studying regulatory elements of gene expression [68, 69]. High density of enhancer elements is expected in the Ciona genome, facilitating efficient enhancer detection, which is necessary to identify enhancers that cannot be identified using conventional cis element analyses. This technique is also useful in creating marker lines that express reporter genes in a tissue-specific manner. In Ciona, techniques of germline transgenesis were established recently, but to date only a few marker transgenic lines are available. Enhancer detection will provide useful marker lines for future genetics studies. In addition, novel tissues or subpopulations of tissues are identifiable by enhancer detection that were previously unidentifiable by simple observation or in situ hybridization (Awazu S, Sasakura Y, unpublished data).
In Ciona, enhancer detection by microinjection mediated transgenesis has been reported . An enhancer detection line near the musashi orthologous gene (Ci-musashi ) has been identified among 21 Minos injected animals . A recent enhancer detection screen using a promoter of the Ciona thyroid peroxidase orthologous gene (Ci-TPO) yielded six enhancer detection lines from 110 injected animals . The frequency of enhancer detection in Ciona is therefore estimated at 4.7% to 5.4% per injected animal. This frequency is higher than that of SB mediated enhancer detection in zebrafish (2.5% per injected animals ), but lower than that of Tol2 mediated enhancer detection (12% ).
Thus far, the promoter of Ci-TPO is the only promoter that has been used for enhancer detection in Ciona. It includes 860 base pairs of upstream sequence from the initiation codon of the gene and exhibits weak expression in endodermal tissues . This might not be an ideal promoter for enhancer detection in all tissues. There might be enhancers to which Ci-TPO promoter could not respond, because minimal promoters exhibit different responsiveness to enhancers (Lemaire P, personal communication). In fact, most enhancer detection lines with Ci-TPO promoter showed reporter gene expression in endodermal tissues . Comparing the efficiency of enhancer detection between the Ci-TPO promoter and a basal promoter or a minimal promoter derived from a housekeeping gene may be necessary to identify an ideal promoter for enhancer detection in Ciona.
In the Ci-musashi enhancer detection line, Minos was inserted into an intron . Detailed analysis of the line revealed that expression of Ci-musashi is regulated by many enhancers located at the 5' upstream region and in introns . These enhancers have both redundant and distinct functions for gene expression. Such an enhancer complex is probably necessary to ensure the appropriate spatial and temporal expression of Ci-musashi. Enhancer identification in the context of chromosomes is necessary to understand the in vivo function of these enhancers. Enhancer detection is a viable method for this purpose.
Remobilization of Minos in Cionagenome
We created a transgenic 'mutator' line harboring a tandem array of Minos vector for enhancer detection, which contains a promoter of Ci-TPO . The tandem array in the mutator line was estimated to include as many as 255 transposons. In this study, remobilization of a few copies of Minos copies probably occurred from the concatemer. Screening enhancer detection using the remobilization technique was conducted as follows. Transposase mRNA was injected into unfertilized wild-type eggs. These eggs were fertilized with sperm from the mutator line. Because our enhancer detection vector shows GFP expression in a part of somatic cells, these transposase-introduced Ciona were selected to remain as GFP positive, transposon containing animals. These GFP positive animals were crossed with wild-type individuals; then, the GFP expression pattern in the next generation was monitored to screen families exhibiting altered GFP expression.
The results indicated that 79% of transposase-injected animals transmitted enhancer detection insertions (Figure 5b). This frequency is considerably higher than that seen in the microinjection mediated approach. Although many of the enhancer detection lines showed GFP expression in endodermal tissues, a few lines showed expression in ectodermal or mesodermal tissues. Therefore, this method could be more efficient for large-scale enhancer detection with creation of many valuable lines. The tandem array interferes with detailed analyses of insertions by Southern blot and TAIL-PCR. In fact, Southern blot was done to show the presence of novel insertions created by remobilization. However, the signal was not conspicuous in many individuals, and as a result the number of new insertions is likely to have been underestimated. Identification of new insertion sites by TAIL-PCR was performed after digestion of genomic DNA with restriction enzymes to suppress PCR amplification within the concatemer . Numerous lines have insertion sites that were unidentifiable, even after restriction enzyme treatment. The enhancer detection insertions can be segregated from the original tandem array by passing through several generations. This may result in the establishment of transgenic lines that have a single insertions of enhancer detection in their genome. Characterization of their insertion sites may increase the efficiency of identification of the causal insertions that were obtained using the remobilization technique.
Remobilization of a single Minos insertion might reduce these problems (Figure 5c). Several tests of remobilization of a single insertion have been carried out using microinjection of transposase mRNA into embryos of transgenic lines (Sasakura Y, unpublished data). In somatic cells excision events were observed (Figure 5d). However, the frequency of excision appeared to be low, and evidence of excision or transposition in the germ cells was not obtained. The primordial germ cells of ascidians are suggested to be two small cells, called B7.6, in early embryogenesis . Thus, germ cells are derived from a small number of primordial cells. Less injected transposase mRNA would be delivered to germ cells than to somatic cells. Therefore, the frequency of excision and transposition in the germ cells would be much lower than in the somatic cells. A technical innovation, such as generation of 'jump starter' lines, is necessary to achieve highly frequent jumping of a single Minos copy in germ cells .
Insertions of Minos can disrupt gene function to create mutants. Insertional mutants are distinguishable from background mutations by the fact that they segregate with the insertions. In Ciona, a small-scale mutagenesis screen was carried out using self-fertilization (Figure 3), and two insertional mutants were isolated from 120 transgenic lines, which are estimated to correspond to 240 insertions; one mutant can be isolated for every 120 insertions. The mutant frequency is lower than with insertional mutagenesis with pseudotyped retrovirus in zebrafish (one mutant per 85 insertions [76, 77]). Taking into consideration the compact genome of Ciona, which has less redundancy, it is curious that insertional mutagenesis in Ciona would be less efficient than in zebrafish. There are two possible explanations. One is that the preference of the insertion sites in the gene, such as 5' end, introns, exons, or 3' end, might be different between Minos and pseudotyped retrovirus. In the zebrafish approach, approximately 60% of the mutagenic insertions reside in the promoter, first exon, or first intron . As mentioned above, Minos is preferably inserted into TA-rich sequences such as introns and intergenic regions. The second possible explanation is that pseudotyped retrovirus would be more mutagenic than Minos. Introduction of a gene trap cassette into the pseudotyped retrovirus vector did not affect the mutation frequency [77, 78]. Pseudotyped retrovirus might interfere with splicing to produce truncated proteins, even without such a cassette. In contrast, the single Minos insertions into introns appeared to be insufficient to cause mutations (Sasakura Y, Awazu S, unpublished data). The mutant frequency would therefore reflect the difference between two vectors.
From mutant screening, one insertional mutant has been characterized in detail . In this mutant, an insertion at the promoter of a gene encoding cellulose synthase (Ci-CesA ) disrupts expression of this gene. Animals homozygous for this insertion exhibit abnormalities in the process of metamorphosis. At the larval stage, their trunks show post-metamorphosed states, although they retain tails, which would normally be lost during metamorphosis. The trunk-metamorphosed larvae continue to swim vigorously. This mutant was named swimming juvenile (sj). This mutant showed a novel function of animal cellulose synthase for the process of normal metamorphosis as well as for the biosynthesis of cellulose. As described above, a concatemer of Minos is inserted into the promoter of Ci-CesA. In another insertional mutant (Matsuoka T, Sasakura Y, unpublished data), a concatemer of Minos is inserted into an intron. Such concatemers are very long and may therefore disrupt promoters or introns. However, mutations by a single insertion are superior to concatemers; some refinement of transposon vectors, such as gene trap, is necessary to produce highly mutagenic Minos. Recently, we attempted to introduce a gene trap method into Ciona (Oogai Y, Sasakura Y, unpublished data).
Because insertional mutagenesis with Minos has been achieved, the next step will be saturation mutagenesis using this transposon. Ciona contains a smaller set of genes with less redundancy than in vertebrates. This characteristic renders this ascidian a suitable organism for saturation mutagenesis. It is necessary to estimate the frequency of essential genes for development in order to calculate the number of transgenic lines that are necessary for saturation mutagenesis. For such estimation, isolation of more mutants is necessary. In addition, several obstacles to Ciona genetics must be overcome in order to conduct saturation mutagenesis. One is the need to create a mutagenic Minos construct. Other obstacles are associated with the primitive state of Ciona genetics, resulting from its short history. Although we take only Ciona into consideration here, most of these points also pertain to other marine invertebrates.
In the mutant screen, we used a Minos construct with a GFP reporter. The expression of GFP was used to judge whether mutations are associated with insertions. However, a correlation between a mutation and GFP expression does not always indicate that the mutation is caused by a Minos insertion. A wild population of Ciona was used to create insertional mutants. Wild populations maintain frequent background mutations. Sometimes these natural mutation sites are located very close to Minos insertion sites, and therefore natural mutants, so-called associated mutants, appear to be related to the Minos insertions. These associated mutants must be discriminated from insertional mutants because transposon insertion sites in the associated mutants are close, but not identical, to the actual sites of mutations. In the recent small-scale mutagenesis studies , four mutants exhibited strong correlation with GFP expression (>90% of homozygous mutants showed GFP expression). Two of them were associated mutants and two were insertional mutants. Associated mutants are distinguishable from insertional mutants by imperfect correlation between mutations and GFP expression. Reporter gene expression is a good marker for this purpose, because hundreds of mutants can be examined through simple observation. The mutants showing perfect correlation with GFP expression are candidates for insertional mutants. Several experiments must be performed to conclude that they actually are insertional mutants. Identification of the insertion sites is primarily required. It is also necessary to demonstrate perfect homozygosity of mutants with respect to the insertions, which is evidence that recessive insertional mutants have been created. Finally, to establish a causal link, it is necessary to identify those genes that are responsible for mutants; this may be achieved through rescue experiments or knockdown of genes by microinjection of antisense morpholino oligonucleotides or dominant negative forms [17, 42].
The second disadvantage of Ciona, after its suboptimal mutation frequency, is that its embryos sometimes develop poorly compared with those of other model organisms. Typical unhealthy development includes kinked tails at the larval stage. Families showing such unhealthy development are omitted from screens. Such omission might cause the loss of mutants that would show the kinky-tail phenotype. Several insertional mutants might have been lost through this technical limitation. Therefore, the mutation frequency with Minos described above might have been underestimated. Recent improvements in culturing systems will enable continual production of healthy embryos. If a family shows unhealthy development in this setting, then the phenotype is probably derived from mutations.
Forward genetics is a powerful technique in which to identify gene function; it is possible to identify gene functions that are neglected by reverse genetics. This approach has recently been employed in Ciona with the chemical mutagen N-ethyl-N-nitrosourea and Minos transposon [17, 32, 33, 80, 81]. Causal genes have been identified in only a few mutants. Most of the mutants generated in the near future would therefore be novel ones. Insertional mutagenesis provides an ideal system in Ciona because the causal genes are identifiable in a short period of time, without time consuming fine mapping.
In this article we review recent achievements in germline transgenesis with the Minos transposon in Ciona intestinalis. These studies have revealed that Minos is a highly active transposon in this organism, as shown by establishment of techniques such as stable transgenic lines, enhancer detection, and insertional mutagenesis. These technical innovations will be of great value to future genetic analyses in C. intestinalis. Frequent enhancer detection by remobilization will provide useful transgenic lines. Insertional mutagenesis allows the identification of novel functions of genes during development, as shown by the example of cellulose synthase. Taking into consideration the advantages of Ciona as a subject of genetics research, future genetic analyses in this organism will provide unique insights into chordate gene function. In addition to these technical innovations with Minos, we describe several technical hurdles that Ciona researchers must overcome if they are to conduct large-scale mutagenesis studies.
Minos is a valuable transposon, and its activity may be the first to be tested in organisms for which no genetic approach has yet been introduced. We also provide evidence that some other Tc1/mariner superfamily transposons have excision activity in Ciona. However, these transposons have not been found to be efficient in causing germline transgenesis in Ciona. This information may be useful in elucidating the mechanisms that determine transposon activity in different organisms. Resolving these issues would make these transposons further valuable tools in Ciona genetics research.
The authors thank Kazuo Inaba, Yoshikazu Okada, Yasuyo Kasuga, Kazuko Hirayama, Aru Konno, Katsutoshi Mizuno, Yasutaka Tsuchiya, Toshihiko Sato, Hideo Shinagawa, Yoshiko Harada, and members of the Shimoda Marine Research Center, University of Tsukuba, for their kind cooperation with our study. Erik Jorgensen, Zoltán Ivics, Zsuzsanna Izsvák, and Charalambos Savakis are acknowledged for their kind provision of Mos1, SB, FP, and Minos. We thank Koichi Kawakami, Hiroshi Wada, Takeshi Kawashima, Nik Putnam, and Patrick Lemaire for useful discussions. This study was supported by Grants-in-Aid for Scientific Research from JSPS and MEXT to YS and NS. This research was also supported by JST, Japan to NS (CREST project). YS was supported by NIG Cooperative Research Program (2006-A72).
This article has been published as part of Genome Biology Volume 8, Supplement 1, 2007: Transposons in vertebrate functional genomics. The full contents of the supplement are available online at http://genomebiology.com/supplements/8/S1.
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