Transposon-based strategies for functional genomic analyses in xenopus tropicalis, a vertebrate model system for developmental and biomedical research

Deliverables

4.1. Optimisation of somatic gene transfer methods with the sleeping beauty system in Xenopus central nervous system. We have established that the somatic gene transfer techniques with DNA complexed with the polycationic polymer polyethylenimine for transfection into the CNS can be applied to the species under study: Xenopus tropicalis. Standard plasmids expressing different reporter genes (luciferase or GFP) have been injected with or without polyethylenimine (PEI), and the time course of expression and the relative yields calculated. The time course of expression in the CNS shows loss of transgene expression with time, which is one of the reasons we turned to the SB transposon system to obtain integration and long term expression of the transgene. We addressed whether the SB transposon system could be adapted to improve the time course of expression by providing integration of the transgenes in the somatic tissue targeted. To this end we designed and constructed a series of plasmids that allowed us to follow transfection, as well as transposase-dependent excision and integration. These completed constructions include a plasmid with both dsRFP and GFP coding sequences (pdsRED/T-CMV-GFP) that enables the transposase-dependent excision step to be visualised by a switch in fluorescent emission. This plasmid is ready, and has been tested and shown to be functional in somatic gene transfer in the CNS . It was also been passed on to Partner 2 to be used in WP2 (germline transformation with SB transposase/transposon system). A second plasmid contains dsRFP and the ampORI selection gene (pdsRed /T-AmpORI) that can be exploited to follow integration of the gene of interest in the tissue transfected. A third plasmid (pT-CMV-luc) can be used to quantify expression of transgenes following different transfection conditions. Each plasmid system was tested with the transposase expressed from a plamsid co-injected into the target tissue or with mRNA for the transposase. The latter method provides a more transitory expression of the enzyme and possibly avoid multiple insertions (that could be deleterious to the target cell). The results show that transposase excision occurs but not integration. Thus, the method is not really better than standard plasmid constructs. The method has been included in a paper submitted to Genesis.

Tc1-like elements (TLEs) belong to the Tc1-mariner superfamily of transposable elements that uses a cut and paste mechanism involving a DNA molecule intermediate to move. TLEs mobility properties make them potential tools for transgenesis and mutagenesis in various animal models. Native elements such as Minos or artificial elements reconstructed by molecular engineering such as Sleeping Beauty and Frog Prince are transpositionally active. Because of this possibility to use them as transgenesis tools, we wanted to have a better overview of the endogenous TC1-Mariner of the two Xenopus species we use as developmental model organisms: Xenopus laevis and Xenopus tropicalis. The other reason of such a study resides in the fact that an engineered transposon used in a living organism could be submitted to regulatory interactions from endogenous active transposons. A number of distinct TLE lineages were shown to co-exist in zebrafish, salmon and amphibian. In Xenopus laevis, two lineages, TXr and TXz, have been described and characterized. TXr is closely related to the teleost fish transposons Tzf/Tdr and TXz is not related to any TLEs described so far. In X. tropicalis, three types of transposase ORF sequences were found using degenerated PCR. However, these sequences were not representative of full-length TLE in this species. In this study, we report the first identification of several diverse TLE lineages within the genome of X. tropicalis. We have used genomic sequencing data extracted from the first assembly of the Xenopus tropicalis genome combined with a degenerated PCR approach to identify multiple lineages of Tc1 related transposable elements. Full-length elements were isolated in each lineage and characterized. Most of them exhibit the typical characteristics of Tc1-like elements (TLEs). An open reading frame (ORF) encoding a 340-350 aa transposase containing a [D, D(34)E] signature was found as well as conserved inverted terminal repeats (ITRs) at each extremities. These ITRs could vary in length, depending on the TLE lineage. These new TLEs were named Eagle, Froggy, Jumpy, Maya, Xeminos, XtTXr and XtTXz. Phylogenetic analyses indicate that their closest relatives are present in the genomes of actinopterygian and amphibian. Interestingly, Maya and Xeminos share remarkable characteristics. Maya contains a [D,D(36)E] motif but is not related to any described TLE so far. Xeminos is the first vertebrate TLE strongly related to an invertebrate lineage. Finally, we have identified for most of these TLEs, copies containing an intact transposase ORF suggesting that these elements may still be active. Mariner-like elements (MLEs) belong to the Tc1-mariner superfamily of DNA transposons, which is very widespread in animal genomes. We report here the first complete description of a MLE, Xtmar1, within the genome of a poikilotherm vertebrate, the amphibian Xenopus tropicalis. A close relative, XMLE, is also characterized within the genome of a sibling species, Xenopus laevis. The phylogenetic analysis of the relationships between MLE transposases reveals that Xtmar1 is closely related to Hsmar2 and Bytmar1 and that, together they form a second distinct lineage of the irritans subfamily. All members of this lineage are also characterized by the 36-43 bp size of their imperfectly conserved ITRs, and by the-8 bp motif located at their outer extremity. Taking into account the fact that Xtmar1, Xlmar1 and Hsmar2 are present in species located at both extremities of the vertebrate evolutionary tree, MLE relatives belonging to the same subfamily were searched for in the available sequencing projects using the amino acid consensus sequence of the Hsmar2 transposase as an in-silico probe. We found that irritans MLEs are present in chordate genomes including most craniates. This therefore suggests that these elements have been present within chordate genomes for 750 Myr and that their main mode of maintenance in these species has been via vertical transmission. The very small number of stochastic losses observed in the data available suggests that their inactivation during evolution has been very slow. Transposition events of endogenous TLEs such as Tzf transposons have been described in zebrafish genome. This transposition occurs at a frequency detectable in a single generation. The analysis of genomic sequences with intact ORFs raises the question of their activity in X. tropicalis genome. Future work will address the possibility of current active transposition of TLEs used to develop transformation tools for the genetic manipulation of X. tropicalis.

Our approach for mobilizing gene trap transposons in vivo was to generate two types of transgenic lines; one expressing the SB transposase specificically in the male germline, and the other set of lines contain the gene trap transposons in the genome of the animals. These two lines are crossed together and, in the male progeny that contain both sets of transgenes, the gene trap transposon is mobilized in the male germline and these "hops" are revealed in the next generation (i.e. his progeny). Thus double transgenic males are crossed to wild type females and the progeny are screened for novel gene trap insertions. In order to easily identify which animals contain each transgene, we generated each set of transgenic lines linked to different eye colour markers. Specifically, we generated the transgenic lines expressing the transposase linked to a transgene containing the gamma-crystallin promoter driving green fluorescent protein (GFP). These transgenic lines are easily identified by having green eyes. The lines that contain the gene trap transposons are linked to the gamma-crystallin promoter driving red fluorescent protein (RFP). These transgenic lines are easily identified by having red eyes. When the two sets of lines are crossed with each other, those that are transgenic for both transgenes will express a combination of green and red in their eyes, which is detected as yellow eyes. For the lines that we generated which express SB transposase in the male germline, we used two different promoters. One of the promoters we used was the mouse protamine promoter. The other promoter we used was the Xenopus SP4 promoter, which we cloned using genomic PCR. Using RT-PCR, we confirmed that both transgenic lines express Sleeping Beauty in the testes of males. These lines are of great benefit for any group who is interested in using Sleeping Beauty transposition to mobilize transposons in the male germline of Xenopus tropicalis.

By using a limited site-directed mutagenesis screen, we identified hyperactive versions of the SB transposase. Three different approaches were undertaken for the choice of induced mutations: - Modification of a linker region that separates the DNA-binding and catalytic domains; - Systematic replacement of acidic residues with basic amino acids; and - Substitution for amino acids that had been selected in nature. Most of the mutations that we introduced into the SB transposase resulted in a decrease in the efficiency of transposition, suggesting very little functional redundancy in the transposase sequence. Nevertheless, one of the substitutions, the D260K mutation, produced a hyperactive phenotype. The aspartic acid in position 260 is either lysine or arginine in other Tc1-like transposases, suggesting that lysine and arginine can better function in that sequence context. It is possible, that a particular version of fish Tc1-like transposases did contain K or R at position 260, but this amino acid got replaced at some point during transposase evolution, because it is functionally non-essential for the transposase. The D260K mutation acts synergistically with two other, naturally occurring mutations, R115H and R143C. The R115H/D260K and R115H/R143C double mutants exhibited about 3.7- and 3.2-fold increase in transposition activity over wild-type transposase, respectively. Importantly, hyperactivity of the R115H/D260K mutant (referred to as SB12) displayed additive effects with a hyperactive transposon vector. The collective effect of these components is an approximately 8-fold increase in transposition, compared to the first-generation SB system. We find that about 2% of cells that had taken up transposon DNA will undergo a transposition event using the first-generation SB system, whereas stable transgenesis rates are about 10-15% by using combinations of the hyperactive transposon/transposase components. Transposition can be further optimized by systematic adjustment of transposase and transposon concentrations in transfected cells, because different ratios of the transposase expression- and transposon donor plasmids can greatly influence transposition efficiencies.

Our aim was to use the transposon Sleeping Beauty with transitory expression of the transposase to insert the transgene into Xenopus genome on a somatic basis. To follow the transposition event, we constructed the pRed-pTGFP molecule where a transposon containing the transgene CMV-GFP expressing the Green Fluorescent Protein (GFP) is inserted between another CMV promoter and the Red Fluorescent Protein (RFP). Thus, after following transfection in presence of transposase activity, we observe a green fluorescence and if there is an excision event the transposon is removed placing CMV in front of the RFP gene then giving a red fluorescence. Thus, emission of red fluorescence allows one to identify cells in which excision (and possibly integration) has occurred. Two other molecules that function on the same model as the pRed-pTGFP molecule, were designed as pRed-pTAmpiORI, used to identify stable insertion of a transposon in the genome sequence of X. tropicalis, and pLUC-pTGFP, used to quantify the efficiency of excision events in different conditions. Figure (email the contact person for schema) : Schematic representation of constructs carrying the transposon Sleeping Beauty. - To follow excision/transposition, we constructed the pRed-pTGFP plasmid where a transposon containing the transgene CMV-GFP expressing the Green Fluorescent Protein (GFP) is inserted between another CMV promoter and the Red Fluorescent Protein (RFP) sequence. Following transfection in the absence of any transposase activity, transcription of the unmodified plasmid produces green fluorescence and no red signal (GFP+RFP-). However, after transfection in presence of transposase activity, if there is an excision event, the transposon is removed placing CMV in front of the RFP gene (new plasmid) resulting in transcription of both green fluorescence (GFP+) and red fluorescence (RFP+). Thus, emission of red fluorescence allows one to identify cells in which excision (and possibly integration) has occurred. - Two others constructs were designed to identify transposition events, the pRed-pTAmpiORI molecule (top) and the pLUC -pTGFP molecule (bottom). Both function on the same model as the the pRed-pTGFP molecule. The pRed-pTAmpiORI construct was used to demonstrate that stable insertion of a transposon can occur in the genome sequence of X. tropicalis, from RFP positive cells that are obtained after an excision event. Presence of ampicilin selection (Ampi) and an origin of replication (ORI) allow the use of the plasmid rescue method to clone insertional events. The pLUC -pTGFP construct was used to quantify the efficiency of excision events following transfection performed in different conditions. Removal of the transposon after an excision event places the CMV promoter in front of the LUC gene, luciferase activity thus providing an indicator of the excision/transposition efficiency. We used a combination of the transposon/transposase system using these constructs and the Somatic Gene Transfer technique to show that transposase-dependent insertion events can be detected in vivo after brain or muscle transfections. A publication on this subject (Zincelle et al) has been submitted.

In an effort to isolate potentially active transposase genes from vertebrate species, transposase coding regions were PCR-amplified from the Rana pipiens (a frog) genome, and cloned into plasmid vectors. Ten transposase genes were aligned to generate a consensus sequence. The individual genes were about 99% identical to the consensus sequence, and one of them differed only in two nucleotides from the consensus, resulting in two amino acid substitutions in its ORF. Site-specific PCR mutagenesis was used to derive the sequence of the consensus transposase gene. The inverted repeat sequences together with the consensus transposase gene constitute the components of a novel transposable element system that we named Frog Prince (FP). The initial tests for transpositional activity of the Frog Prince element were done in cultured HeLa cells, using a transposition assay established for Sleeping Beauty. A 17-fold increase in colony number was detected when pFV-FP was cotransfected with its substrate, pFP-neo. This level of activity is similar to that of SB in HeLa cells, and demonstrates that we successfully derived and engineered an active transposon system from the R. pipens genome. Taken together, the data demonstrate that the Frog Prince transposon system can significantly increase the efficiency of transgene integration from plasmid-based vectors to the human genome. Next we compared the activities of the Sleeping Beauty and Frog Prince systems in cultured cell lines derived from two mammalian, an amphibian and two fish species with the standard transposition assay. FP appeared to be slightly more active than SB in some of the cell lines tested. These data demonstrate that transposition of Frog Prince is not restricted to phylogenetically close taxa, and that it is the most active transposable element in vertebrate species described to date. High frequency, precise transposition into different genomic loci suggests that genome-wide gene trapping is feasible with FP. For this purpose, an FP-based donor plasmid (pFP/GT-neo) was constructed which contains engrailed-2 intron sequences with the SA, a glycine bridge to allow proper folding of the marker in protein fusions, an ATG-less neo gene, a zeocin resistance gene (zeo) driven by dual eukaryotic/bacterial promoters and a plasmid origin of replication. All chromosomal transposition events give rise to zeocin-resistant cells. A subset of transformant cells will be G418-resistant, if the transposon inserted into an intron of an expressed gene in the proper orientation, and if splicing occurred in-frame with neo. Based on the numbers of zeocin-resistant cell colonies, transposition efficiency of FP/GT-neo was comparable to that of FP-neo. The number of zeocin/G418 double-resistant colonies was about one third of those resistant to zeocin alone, indicating that about 30% of all transposition events occurred in introns of expressed genes and in-frame splicing took place. Five insertion sites of the FP gene trap transposons were identified. All of them mapped to introns of genes in different chromosomes, in the correct orientation. Our results suggest that FP can potentially target a large fraction of genes in the human genome.

We developed somatic gene transfer (SGT) techniques applied to brain and muscle of Xenopus tropicalis tadpoles (Rowe et al. 2002. Dev. Dyn. 224, pp 381-390). so as to follow transcriptional regulations, protein function and cell fate within the constraints of an integrated in vivo system. Stable integration should extend gene expression and allow lineage and fate studies during and after metamorphosis. To this end, we used the transposon Sleeping Beauty with transitory expression of the transposase to insert the transgene in Xenopus genomic sequence. - Visualisation of transposon/transposase events with fluorescent proteins. To follow the transposition event, we constructed a pRed-pTGFP molecule where a transposon containing the transgene CMV-GFP expressing the Green Fluorescent Protein (GFP) is inserted between another CMV promoter and the Red Fluorescent Protein (RFP). Thus, after following transfection in presence of transposase activity, we observe a green fluorescence, and if there is an excision event the transposon is removed placing CMV in front of the RFP gene. Emission of red fluorescence allows one to identify cells in which excision (and possibly integration) has occurred. Red fluorescence was never seen in absence of transposase activity. We used a combination of the transposon/transposase system using the pRed-pTGFP construct and the SGT technique to show that transposase-dependent insertion events can be detected in vivo after transfections. The following results were obtained: -- Comparison of SGT in X. laevis and X. tropicalis, shows that the efficiency of excision event in tail muscle (increase in red fluorescence) is greater for X. laevis and furthermore is more rapid being detectable after 3 days whereas 15 days were required for X. tropicalis. -- Transposase activity was introduced into the target tissue using RNA-SB (RNA transposase synthesized in vitro) or plasmid DNA (with the construct CMV-SB where the transposase is under the control of CMV promoter). In muscle, we show that excision is very efficient using RNA-SB and obtained 1 day after injection. - Molecular characterisation of excision/insertion events. In order to characterise excision and integration events during transposition, another molecule was prepared. In this construct, the pRed-pTAmpiORI molecule, the transposon unit contains the Ampicillin resistance gene (Amp) and an origin of replication (ORI). This construct can be used to clone putative insertion in the genomic sequence after transposition, using a plasmid rescue method. Using the two constructs, pRed-pTGFP and pRed-pTAmpiORI, the following results have been obtained: -- To analyse genomic insertion events at the molecular level, we first tried a plasmid rescue method using the pRed-pTAmpiORI molecule. The construct was injected in presence of two kinds of transposase (RNA-SB or expression of RNA from plamsid CMV-SB), and genomic DNAs from different samples were analysed at 15 days post-injection. DNAs were EcoRI-digested, religated and transformed in bacteria, and 36 ampicillin resistant clones were sequenced (8 clones from pRed-pTAmpiORI + CMV-SB samples; 28 clones from pRed-pTAmpiORI + RNA-SB samples). Result shows that no positive clone could be found, i.e. no transposon/transposase dependent insertion event was found, only 2 random insertions in genomic sequence have been characterised with this method. -- Then, the same DNA samples were tested with a more sensitive technique, the 5'-RACE PCR method. Genomic DNAs were EcoRI-digested, and a compatible adaptator was ligated at the free extremities. Thus, PCR and nested PCR using two couples of primer were performed to amplify specifically genomic sequence where the transposon is integrated in the Xenopus genome. However, for each sample tested no discrete band could be isolated and analysed. -- Finally, we used LAM-PCR, (Schmidt et al 2001 Hum Gene Ther 12, ), a novel direct genomic sequencing technique for identification of vector insertion sites. We applied the LAM PCR on our different samples, either the pRed-pTAmpiORI or the pRed-pTGFP constructs in presence of RNA- or CMV-transposase). However all PCR bands sequenced were negative, representing only single primer products. -- We also analysed genomic excision events at the molecular level, using PCR and nested PCR with specific primers taken outside of the transposon unit. We were unable to find the expected sequence signature of excision transposase-dependent event in our conditions. It has not been possible to demonstrate that insertion of a transposon has occurred in the genome sequence of X. tropicalis or X. laevis species using the SGT technique to express transposon/transposase Sleeping Beauty system. These results have been submitted to Genesis for publication.

Using the Sleeping beauty (SB) transposon system, we have developed a simple method for the generation of Xenopus laevis transgenic lines. The transgenesis protocol is based on the co-injection of the SB transposase mRNA and a GFP-reporter transposon into one-cell stage embryos. We have tested different transposons containing the GFP fluorescent marker, under the control of either ubiquitous promoters (pCMV), or of the muscle-specific promoter (pCar). These transposons were usually injected in the embryos as circular plasmids. We have compared the fluorescence at different developmental stages of animals injected with plasmids with or without transposase mRNA. The fluorescence is essentially mosaic and always visible in caudal muscle fibers, whatever the promoter was used. As fluorescence is also present in the control without transposase, it is not possible in the early stages to conclude for integration of the transposon in genomic DNA. However, very interesting results were obtained with a particular construct in which the GFP gene is interrupted by an intron and under the control of the chicken ßactin promoter. In addition to the mosaic and clonal patterns of expression, we got a reasonable number of "half-transgenic" animals, which are fully fluorescent on either the left or the right side of the organism. This indicates integration of the GFP gene in one of the blastomers at the 2-cell stage. Altogether, 38% of the tadpoles where highly fluorescent mosaics or half-transgenics, compared to 12% in the absence of transposase, suggesting integration by transposition. We determined an optimal ratio of transposase mRNA versus transposon-carrying plasmid DNA that enhanced the proportion of hemi-transgenic tadpoles. Although the transposase is necessary for efficient generation of transgenic animals, the analysis of excision reveals non-canonical molecular footprints. The canonical footprint is a trinucleotide C(A/T)G between two TA dinucleotide. The sequence of several plasmids indicates that excision of the transposon has occurred, but the size and the position of the borders of the excised fragment are variable. This phenomenon has also been observed in the mouse. The possible explanations for these non-canonical footprints could involve either a precise excision of the transposon followed by different plasmid repairs like those found in mice, or a non-properly excision of the transposon from the donor plasmid. These results are in agreement with previously-reported data showing that SB transposon leaves characteristically different footprints at excision sites in different cell types. Nevertheless, the important conclusion of this analysis is that the transposase is active in the embryo, since it is able to catalyse the first step of the transposition process. We investigated the molecular nature of integrations by Southern blot analysis on two F0 animals, from lines exhibiting two different levels of fluorescence, and on their offsprings. These results proved that the transgene integration did not occur though a canonical cut and paste transposition mechanism for both lines. Our results lead us to suggest that X. laevis embryos do not possess all the co-factors necessary to mediate precise integration by transposition. This could be the consequence of a non-canonical cell cycle during Xenopus early segmentation, during which DNA repair does not occur. Surprisingly, non expressing F1 siblings were also positive for hybridization with GFP and plasmid probes. These data suggest that several integrations of the transgene occurred in F0 germline, but that some of them are silenced by position effects. From the present study, we conclude that SB transposase can be used with some advantages to obtain numerous transgenics, and specifically hemi-transgenic animals, but we cannot assess if the integration of the transgene does result from imperfect transposition events or from other DNA recombination mechanisms. In summary, the SB transposon system has potential for gene delivery in Xenopus. This method simply requires classical injection of genetic materiel into fertilized eggs. SB procedure constitutes a useful complement of the REMI technique, particularly for the transgenesis of the sibling species X. tropicalis. This species with a diploid genome has become the genomic reference in amphibians and is therefore better suited for genetic approaches and transgenic studies.

Previous work in our laboratory had shown that a gene trap approach to insertional mutagenesis in Xenopus is possible using a method of transgenesis we had developed, called Restriction Enzyme Mediated Insertion (REMI). (Bronchain et al., 1999). Although we had shown that we could trap genes using this strategy, there were several reasons why it was worth exploring other means of performing insertional mutagenesis in frogs. Firstly, the transgenic technique we were using mostly likely caused damage to host chromosomes, and therefore leading to mutations unrelated to the insertion sites. Secondly, using the REMI method, integrations occurred in large concatemers, which are known to be unstable and making cloning of insertion sites difficult. Thirdly, it was difficult to consider using REMI transgenesis for a large-scale mutagenesis screen. Hence, we decided to combine to combine a gene trap approach to insertional mutagenesis with SB-mediated transposition in vivo, in order to identify mutations in genes in Xenopus tropicalis. In this strategy we would aim to use mobile DNA elements to insert gene trap vectors into different sites of the genome. Transposons move as single copies, thus eliminating the problem with concatemer insertions. Furthermore one can set up the scheme such that the transpositions occur in vivo, which means that it can easily be scaled-up. The transposon system we decided to employ in this work was Sleeping Beauty (SB). To this end we constructed many gene trap SB transposons. The first two vectors we generated contained the gene trap vectors we had previously used to trap genes in Xenopus (Bronchain et al. (1999). However based on knowledge gained during the course of this work, we continued to modify these vectors with the aim of making them more useful for our insertional mutagenesis strategies. For example, Partner 1 had shown that transposition efficiency decreases as the size of the transposon increases, and importantly, as the transposon size increases above 4000 base pairs, the efficiency of transposition decreases dramatically. Since our first generation gene trap vectors were over 4000 base pairs in length, we generated a second generation of gene trap vectors with smaller transposon size. These vectors contain around 500 base pairs of Engrailed2 splice acceptor sequences instead of the 2200 base pairs used in the first generation of SB-based gene trap vectors, thus shortening the total size of the transposons by around 1700 base pairs. Another set of experiments performed in the laboratory of Partner 1 showed that the efficiency of transposition increases if both SB elements used contain the left inverted repeats. We therefore generated a third generation of vectors, containing the left inverted repeats on both sides of the SB-based gene trap transposons. The aim of these alterations were to improve the efficiency of transposition in vivo. The gene trap transposons we have generated could be used in other model organisms, such as the mouse and zebrafish.

Our approach for mobilizing gene trap transposons in vivo was to generate two types of transgenic lines; one expressing the SB transposase specificically in the male germline, and the other set of lines contain the gene trap transposons in the genome of the animals. These two lines are crossed together and, in the male progeny that contain both sets of transgenes, the gene trap transposon is mobilized in the male germline and these "hops" are revealed in the next generation (i.e. his progeny). Thus double transgenic males are crossed to wild type females and the progeny are screened for novel gene trap insertions. In order to easily identify which animals contain each transgene, we generated each set of transgenic lines linked to different eye colour markers. Specifically, we generated the transgenic lines expressing the transposase linked to a transgene containing the gamma-crystallin promoter driving green fluorescent protein (GFP). These transgenic lines are easily identified by having green eyes. The lines that contain the gene trap transposons are linked to the gamma-crystallin promoter driving red fluorescent protein (RFP). These transgenic lines are easily identified by having red eyes. When the two sets of lines are crossed with each other, those that are transgenic for both transgenes will express a combination of green and red in their eyes, which is detected as yellow eyes. We generated many transgenic lines containing the different gene gene trap transposons, all of them marked with red eyes. Out of these we were able to show that we could mobilize these transposons, either by injecting RNA encoding the transposase or by crossing these lines with the transgenic lines expressing the SB transposase in the male germline. These gene trap transposon lines are of great benefit for all labs intersted in performing insertional mutagenesis screens in Xenopus tropicalis.

A 14kb piece of DNA, flanked by a pair of Paris elements, appears to have transposed in Drosophila virilis. We hypothesized that this kind of sandwich arrangements of two complete SB elements flanking a transgene will increase the ability of the vector to transpose larger pieces of DNA. Thus, we flanked an approximately 5 kb piece of DNA with two intact copies of SB in an inverted orientation. The vector was designed in a way that transposase was able to bind to its internal binding sites within each element but its ability to cleave DNA at those sites was abolished. Efficiency of transposition of the sandwich element was about 4-fold increased compared to an SB vector containing the same marker gene. Thus, the sandwich transposon vector can be useful to extend the cloning capacity of SB elements for the transfer of large genes whose stable integration into genomes has been problematic with current viral and nonviral vectors.

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