JP2001512690A - Vectors and methods for introducing additional nucleic acid material into a cell's genome into the cell - Google Patents

Abstract

(57) Abstract The present invention provides a novel element for improving genetic engineering techniques for producing recombinant nucleic acid molecules and / or recombinant cells. The novel elements can integrate the desired nucleic acid material into other nucleic acid materials, especially into the genome of the host cell. The novel element is derived from or based on transposons, particularly the Tc / Mariner superfamily. In particular, the essential elements of Tc1 that allow the excision and application of the desired nucleic acid material are provided, along with the corresponding transposase activity in cis or trans.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the field of genetic engineering. Specifically, the present invention provides a method for producing a recombinant vector containing a nucleic acid of interest, a method for producing a recombinant cell (particularly, a recombinant cell expressing a recombinant product), and / or Alternatively, it relates to production of a target nucleic acid.

[0002] In a further embodiment, the present invention relates to making transgenic animals, plants or other organisms for medical, scientific or economic purposes. In yet another embodiment, the invention relates to the tools required for mutagenesis and to the tools and means necessary for localizing and / or identifying a nucleic acid sequence of interest (such as a gene) in the host genome.

[0003] Many methods for introducing additional nucleic acids into cells are known in the art. Many proteins are expressed in a wide variety of cells ranging from prokaryotic bacteria and bacilli, through yeast and fungi, to plant cells, insect cells, and mammalian cells.

[0004] Often, the expression of the above-mentioned proteins has been successful, but in the field of recombinant technology, due to safety or technical problems, such as the stability of the introduced additional nucleic acid material, the expression There are some areas where has not been easily achieved. Similarly,
The same or similar problems have arisen in genetic engineering applications where the product is not a protein, for example, a nucleic acid of interest (DNA, RNA or an antisense construct). It is in this field in particular that the invention is used.

One of the problems encountered in recombinant technology is often the desire to integrate additional nucleic acid material introduced into a host cell into the genome of the host cell. Once the additional nucleic acid material has been integrated into the genome, its stability is less of an issue. In addition, the integrated material is replicated along with the genome, and is therefore present in the progeny of the recombinant cell as well. Vectors known to be capable of incorporating additional nucleic acid material into host cells suffer from several disadvantages as described below.

[0006] The present invention now provides a method by which a wide range of vectors can be varied to integrate the desired additional nucleic acid into the genome of the host cell.

[0007] This means that this capability can now be provided in vectors that cannot integrate the desired additional nucleic acid material into the genome of the host cell (efficiently).

One of the disadvantages of the prior art integrating vectors is that such vectors often cannot efficiently transduce host cells. At that time, techniques such as electroporation are needed to achieve transduction.

In some areas, such treatment of cells may be undesirable,
Or it may not be possible. One such area is, for example, gene therapy. In such a case, a vector that can efficiently transduce the host cell with the vector itself or a vector that is packaged in a recombinant virus particle and thus can infect the host cell are often used.

[0010] However, many such vectors are then unable to (efficiently) integrate the desired additional nucleic acid material into the genome of the host. Therefore, the present invention
Both worst in that they can be efficiently transduced and prepared vectors that can efficiently incorporate the desired additional nucleic acid material into host cells.
orlds). The present invention solves this problem by providing a nucleic acid that can be integrated into the genome of the host cell within a functional transposon. Although exogenous DNA is introduced into the host genome using transposons, transposons, until the present invention, were considered to be quite species-specific, so the applicability of such systems is very high. Was thought to be limited to In the best case,
It has also been shown that transposons that function in one Drosophila can be used to integrate DNA into the genome of another species of Drosophila (Loutheris et al., 1995). The present inventors have found that such transposons can be used across large phylogenetic barriers, at least for certain types of transposons. This discovery allows such transposons to be widely applied as a means to integrate DNA into a wide range of host genomes.

Thus, the present invention provides a vector that provides cells of a genus with additional nucleic acid material that integrates into their genome. The vector contains two transposase binding sites. The transposase binding sites may be the same or different, and are derived from transposons found in another genus, each of which is in close proximity to the transposase cleavage site. The additional nucleic acid material is located between the two transposase binding sites. Of course, obviously, it is highly preferred to be able to use transposons that even cross the broader phylogenetic barrier. Thus, the present invention provides a transposable-based integration system that is widely applicable.

[0012] The obvious advantages of the present system are, for example: the use of transposons is often
Leading to more efficient integration into the host cell genome; use of transposons to give complete control of the integration sequence (ends of the integration element are known); localization of the gene (or DNA) In a preferred embodiment, transposons are useful in so-called mutagenesis experiments, particularly for gene function, or in "gene-trap" experiments where the expression pattern tracks the gene of interest. Na) may have the advantage of being integrated anywhere in the genome. For expression purposes, it may be desirable to develop transposons with more specific integration sites in the genome, or to develop integration methods that result in more specific integration sites.

[0013] Preferably, said transposase binding site and said cleavage site are based on corresponding sites of a transposon from the Tc1 / mariner superfamily of transposons, more preferably based on a Tc1-like transposon. A very useful combination of transposon elements is the minimal combination required by a Tc1 transposon: the 26 base pairs at the end of Tc1, and the Tc1 transposase cleavage site (TA) in close proximity.

An important advantage of the transposon according to the invention is that it is self-supporting. All that is needed is a functional transposase binding site, a transposase cleavage site, and a functional transposase activity for these sites. The required transposase activity should be limited as much as possible. A preferred type of transposon requires only a single transposase. The cause of transposon species specificity reported to date is the use of transposons that require host proteins to be incorporated. In this case, the host protein may be responsible for the species specificity of, for example, transposons or P elements of bacterial origin. The species provided by the present invention do not require any host elements and have moderate cis-trans requirements. Thus, such transposons are more suitable for integrating nucleic acids of interest into the genomes of a wide range of hosts.

In a preferred embodiment where a Tc1-based element is used, such a transposon binding site appears to be at least the terminal 26 base pairs. Presumably, not all of this base pair is essential for the function of this binding site. Thus, some (conservative) changes in such binding sites may be possible. A sequence of about 100 bp appears to be the most efficient transposase recognition site.

For the present invention, a transposase recognition site must be functional for the corresponding transposase activity. Such transposases can also, of course, be modified, mutated, shortened or lengthened when compared to the original transposase, as long as they have the relevant transposase activity.
The transposase activity can be encoded as part of a transposon together with additional nucleic acid material of interest, or in the same vector as another transposon element (in cis). However, the transposase activity can very advantageously be effected (in trans) by another vector according to the invention or in a cell which can be transduced by another vector.

[0017] If the transposase activity is provided in trans, and preferably is encoded by a sequence under the control of an inducible and / or repressible promoter, it can initiate and stop the "jumping" of the transposon. This can be very useful once integration into the host genome has occurred.

A vector for the purposes of the present invention is any nucleic acid vehicle capable of carrying the desired additional nucleic acid material and preferably capable of transducing and / or replicating a host cell.

[0019] The additional nucleic acid material desired may encode a protein and may therefore consist of genes and regulatory elements for expression. The protein expressed will depend on the purpose of the transduction. Many proteins useful for many applications have been identified as good candidates for recombinant expression. All of these can be expressed using the present invention.

Similarly, additional nucleic acid material (DNA) can be transcribed (once transduced) into RNA molecules that block the transcription or translation of host cell (or viral) nucleic acids. Such additional nucleic acid material can be, for example, an antisense RNA molecule.

The use of the invention in the field of gene therapy is particularly advantageous, as will be clear from the above. Viral vectors (such as adenoviruses, retroviruses or adeno-associated viruses), which are often considered for gene therapy, do not efficiently integrate the desired nucleic acid material into the genome of infected cells, but this time, Can be provided. Of course, other vectors that are not based on a virus but are provided by means of delivering the vector to a target cell population can also conveniently be provided with an integration system according to the invention. In particular, the use of liposomes or polymers as targeting systems is included.

As noted above, the present invention provides an efficient means by which various vectors can be used to integrate exogenous nucleic acid material into the genome of a host cell. Since the exogenous nucleic acid material is integrated into the genome of the host cell, the progeny of the resulting recombinant host cell will have the same recombinant potential as its parent. Thus, when stem cells are generated according to the present invention, this results in a very efficient gene therapy regime. The vectors (or recombinant systems) of the present invention can, of course, also be used to produce transgenic animals, plants or other organisms for scientific, economic or medical purposes. Useful applications of transgenesis are well known in the art.

Thus, methods for transducing cells or animals, plants and the like using the vectors according to the invention are also disclosed, as well as cells, animals or plants etc. obtainable by such methods. Secondly, it is a part of the present invention. The use of the present invention in mutagenesis studies and similar studies is another preferred embodiment, as described below. The invention will now be described in more detail, using Tc1 as a non-limiting example.

DETAILED DESCRIPTION Tc1 belongs to the transposons of the Tc1 / mariner superfamily found in nematodes, arthropods and chordates (Henikoff, 1992; Raddice et al., 1
994; Robertson, 1995; Plasterk, 1995). Both vertical and horizontal dislocations
They contribute to the dissemination of these elements throughout the animal kingdom (Robertson, 1993; Radice et al., 1994; Robertson and Lampe, 1995). The widespread appearance of transposons of the Tc1 / mariner superfamily is acceptable as an indication of the absence of species-specific host factors that limit translocation between different species. Thus, the Tc1 / mariner element is an attractive candidate for developing gene delivery vectors.

The Tc1-like element is close to 1.7 kb in length, has a short inverted terminal repeat sequence flanking the transposase gene, and is flanked by TA representing the target site, a conserved sequence that undergoes replication upon integration. It has CAGT at its end (Van Luenen et al., 1994). The protein encoded by this element has a catalytically active domain homologous to bacterial transposase and retroviral integrase (Doak et al., 1994).
. Tc1 from C. elegans is a transposon 1616 bp in length with a 54 bp inverted repeat flanking the gene encoding the 343 amino acid transposase (Emmons et al., 1983; Rosenzweig 1983; Vos et al., 1993) and binds to inverted repeats (Vos et al., 1993; Vos and Plasterk, 1994). The conserved hexanucleotide sequence TACAGT (SEQ ID NO-), located at the very end of the element, is not part of the transposase binding site, but is thought to play a role in catalyzing the transposition reaction (Vos and Plasterk, 1994).

Here, we describe the in vitro excision and translocation of Tc1 using extracts prepared from transgenic nematodes. It reveals a minimal cis requirement for transposition, and target site selection in vitro is compared to target site selection in vivo. Furthermore, we demonstrate that recombinant transposase purified from E. coli can support transposition. This indicates that other factors are not essential for Tc1 translocation in vitro.

[0027] In addition, the inventors show that Tc1 transposase supports the jumping of the Tc1-derived transposon carrying the neomycin resistance gene into the genome of human cells. This demonstrates that transposon-derived vectors can be constructed that can provide integration of additional nucleic acid material into host cells across large phylogenetic barriers. This makes such vectors suitable as delivery vehicles for integrating the gene into a wide range of host genomes, including the human genome.

Results Transposition of Tc1 in vitro We have generated transgenic helminths in which the Tc1 transposase gene is under the control of a heat shock promoter. Thereby, a nuclear extract having a high level of transposase can be prepared. This proved to be very important for detecting activity. The extract was incubated with a plasmid containing the Tc1 element. Excisions were examined by physical assay. Southern blot analysis of the reaction products shows the appearance of the excised Tc1 element (FIG. 1).
, Lane 1).

In addition, cleavage at either the left or right end of Tc1 is detected when the product is digested with ScaI in the plasmid backbone before electrophoresis (lane 2). Cleavage may require a divalent cation (Mg ²⁺ or Mn ²⁺ ), ethylene glycol or
Stimulated by the presence of 5% DMSO (results not shown). Cleavage efficiency at one end of the transposon does not decrease when the substrate is linear (compare lanes 2 and 5). Similarly, deletion of either end of the transposon does not abolish cleavage at the remaining end (lanes 6 and 11). This suggests that cleavage does not require an interaction between the two ends. In contrast to cutting at one end, excision of the complete element is reduced by about a factor of two when the substrate is linear, and cooperative cutting at both ends results in supercoiling of the substrate. Suggests being stimulated by Most of the complete excision products observed with the linear substrate can be explained by uncoordinated cleavage at both ends.

To determine the location of double-strand breaks at the nucleotide level, PCR-based primer extension was performed using end-labeled oligonucleotides specific for each strand (FIG. 2). The 5 'cut is 2 bp within the transposon, while the 3' cut is located at the end of the transposon as based on the longest PCR product observed. This confirms a model based on in vivo studies on the related nematode transposon Tc3. In this regard, excision was shown to result in a 3 'overhang shifted by 2 bp (Van Luenen et al., 1994). Complete excision of Tc1 reveals that it occurs through a cut-and-paste method: the result is the genetic data in double-strand break repair of donor DNA molecules in excision of Tc1 (Plasterk, 1991).

The inventors have devised a sensitive assay to detect integration events. We have found that in gene assays, transposons are used (transp
oson-borne) The antibiotic resistance gene was selected for jumping from the supercoiled donor plasmid to the target plasmid (FIG. 3). Reaction to the appropriate E. coli strain Electroporation of the product resulted in the detection of many transposition events (Table 1). From non-transgenic N2 worms or from the so-called high-hopper strain TR679
(Collins et al., 1987), an extract prepared from (which has a high frequency of germline transposition) does not produce detectable levels of translocation product in this assay. As a result of linearizing the donor plasmid, the transposition efficiency was reduced to about 20 times. Transposition requires two inverted repeats, since deletion of one transposon end did not result in integration. In addition, transposition levels are not increased by the addition of ATP, CTP or dNTPs (results not shown). This indicates that the method is neutral in energy consumption and does not depend on cofactors. Analyzing by sequencing about 90 independent in vitro Tc1 incorporations, such incorporation was found in TA dinucleotides replicated in this manner.
Two strange integration events where Tc1 was incorporated into the sequence of TTG (SEQ ID NO :) or CCT (SEQ ID NO :-) were detected. In both cases, we found a 3 bp duplication of the target site.

Selection of target sites Previously, hundreds of in vitro Tc1 and Tc3 incorporations in the 1 kb region of the gpa-2 gene were analyzed (Van Luenen and Plasterk, 1994). This showed that a limited set of TA dinucleotides was selectively used as targets for integration, and showed significant differences in preference between Tc1 and Tc3. To determine if chromatin structure plays a role in the choice of integration site, we used the same target region previously assayed in vivo to generate DNA in vitro to the naked DNA. The embedding pattern was determined. Therefore, we included the gpa-2 region in our target plasmid. It is clear that the same integration pattern is seen overall (Figure 4). Sites that are active in vivo appear to be active in vitro, and sites that are less active in vitro are less active in vivo. This indicates that, at least in this region of the genome, the in vivo transcriptional status of the genome, chromatin structure or DNA is not a major determinant of target selection.

Transposition by Recombinant Transbosses Nematodes are not a suitable material for obtaining proteins for purifying transbosase in large quantities. Therefore, this protein was expressed in heterologous biological systems. Expression using Baculovirus and Sf9 cells (data not shown)
Alternatively, transbos- ses were obtained that could aid in translocation of Tc1 with expression in E. coli. Recombinant transbosase was almost homogenously purified from inclusion bodies (Fig.
Five). Table 1 shows the translocation frequency of the helminth extract and the purified protein when the transbosase was used at the same amount. Sequence analysis of nine separate integrations using the recombinant protein showed that transposition to the TA target sequence was duplicated, so it can be concluded that genuine transposition has occurred. Thus, the inventors concluded that Tc1 transposition required only Tc1 transposase. The difference in efficiency between nematode-derived transposase and bacterial-derived lancebosase requires further investigation. Bacterial transbosase denatured and refolded during the purification process is folded
g) may be involved. Alternatively, nematode extracts may involve the presence of host factors with a facilitating role.

Minimum cis requirement We construct an all-transbosase binding site adjacent to the TA target site
We examined the possibility that the 26 base pairs at the end of Tc1 were sufficient to form an artificial transposon. Even elements consisting of these Tc1-specific sequences alone can transpose in vitro, albeit at low frequency (Table 1). We sequenced some integrations and found them to be correct.

Further, the importance of the conserved 6-base sequence TACAGT (SEQ ID NO :-) was examined. A mutagenesis was introduced at one end of mini-Tc1, containing only the adjacent TA dinucleotide and the terminal 26 bp. The excision of the element having two wild-type ends can be easily detected by a physical assay, but the element cannot be excised due to a mutation in the transbosase binding site which is a TA sequence adjacent to the end (FIG. 6). ). Double-strand breaks at the wild-type end were not affected by mutations in the other transposon end. Analysis of the cleavage by primer extension by PCR revealed that only the CA to TG mutation resulted in single-strand breaks at the 5 'end of the transposon (data not shown).

The inventors have developed a cell-free Tc1 translocation system. Excision occurs by double-strand breaks at the ends of the transposon, resulting in a 2 bp staggered 3 'overhang. The dislocation cut-and-paste mechanism uses Tc1
It seems that it is also applied to (Fig. 7). This mechanism has already been proposed based on genetic data (Plasterk, 1991), as well as analysis of transposition products in vivo (Plasterk, 1991).
Van Luenen et al., 1994). Non-replicative transpositions are carried out by the Drosophila P-factor (Kaufman and Rio, 1992), as well as the bacterial transposon Tn7 (Bai
nton et al., 1991, 1993) and Tn10 (Bender and Kleckner, 1986). In contrast, the Mu and Tn3 transposition elements transpose by a replication mechanism (Grindley and S
herratt, 1978; Shapiro, 1979; Mizuuchi, 1992). P element is GT as a cofactor
Using P (Kaufman and Rio, 1992) and Tn7 using ATP (Bainton et al., 1993), it is believed that translocation of Tc1 is independent of the addition of nucleotide cofactors.

A salient feature of the Tc1 / mariner family is the use of TA dinucleotides as target sites. Extensive studies on target site selection in vivo have shown that only some of the available TA dinucleotides are used, and that the two related transposons Tc1 and Tc3 in C. elegans show significant differences in target selection. Was found (Van Luenen and Plasterk 1994). We have found in vitro the same overall integration pattern as seen in vivo. This suggests that at least in the analyzed genomic region, the DNA context on the chromosome does not affect target selection. Thus, although strong consensus sequences have not been identified, we believe that the translocation complex primarily selects target sites based on the primary DNA sequence adjacent to the TA (Van Luenen and Plasterk). , 1994). Chromatin structure has been shown to clearly affect retroviral integration (Pryciak and Varmus, 1992; Mueller and Varmus, 1994). These studies have shown selectivity for regions within nucleosomal DNA, probably due to DNA bending. We cannot exclude the possibility that DNA binding proteins influence the selection of the region for Tc1 integration. Because nothing is known about the chromosomal structure of the gpa-2 gene, it would be interesting to compare the integration sites using reconstructed nucleosomal DNA in vitro.

In vitro transposition requires the transposon binding site and the extreme end of the transposon to contain a conserved hexanucleotide sequence important for excision. There is a decrease in transposition efficiency between the transposition of the entire transposon sequence and the Tc1 element with only a 26 bp terminal inverted repeat, indicating that additional sequences may contribute to the transposition efficiency. Suggests. No other transbosase binding site is shown, but probably high mobility group proteins (Grosschedl et al., 1994)
A low-basic protein such as may bind and promote translocation. Alternatively, a unique AT-rich sequence found at the end of the transposon can impart auxiliary bending to DNA. The conserved hexanucleotide sequence at the very end of the transposon has been shown to be at least important for the cleavage step. The single-strand break at the 5 'end, seen in one of the mutations (CA to TG), is probably the specific order of the single-strand breaks, ie the order reported for Tc10 (Bolland and Kleckne
Contrary to (r, 1995), it may indicate that the non-translocated strand is first cut.

Transbosase purified from E. coli to near homogeneity can pop out Tc1, indicating that transbosase is the only protein required for excision and integration of Tc1. ing. The higher efficiency obtained with nematode extracts suggests that host factors increase the frequency of response. For example, it has been shown that the mammalian proteins HMG1 and HMG2 can promote recombination in prokaryotes (Paull et al., 1993). Independence from species-specific factors may explain why members of the Tc1 / mariner family are spreading across so many different phyla, possibly by horizontal movement (Robertson and Robertson). Lampe, 1995). This is in contrast to the P element, which is limited to Drosophila species only. The dislocation in the heterogeneous P element is
Not yet observed (Rio et al., 1988). A possible candidate for species-specific host factors in the P transposition is the inverted repeat binding protein IRBP (Beall et al., 199
Four). The simple cis and trans requirements for Tc1 transposition in vitro indicate that this transposable element is a good vector for gene transfer in a wide range of animals. To determine the feasibility of using Tc1 transposons in human cells, we first tested whether Tc1 transposase was toxic to human cells. PRc / CMV, an expression vector for Tc1 transbosase
.TclA or the empty vector pRc / CMV (Invitrogen) were transformed into 911 cells, a human embryonic retinal cell line transformed by the early region I of adenovirus type 5. Approximately 48 hours after transformation, cells were placed on G418 selection media. A stable cell line derived from one colony was established and screened for Tc1 transposase protein on Western blots (FIG. 8). All cell lines transformed with pRc / CMV.TclA expressed Tc1 transbosase, demonstrating that Tc1 transbosase expression was not toxic to human cells.

Next, it was examined whether or not Tc1 transbosase helps transposition of a Tc1-derived transposon in human cells. Therefore, the plasmid pRP466.SV-neo1.PGK-
tk2 was built. This plasmid contains the neomycin resistance gene expression cassette (SV-neo) under the control of the SV40 promoter flanked by Tc1 inverted repeats, and the phosphoglyceraldehyde kinase promoter outside the Tc1 inverted repeats. Below contains the herpes simplex virus thymidine kinase expression cassette (PGK-tk). Whether the SV-neo transposon was excised from pRP466.SV-neo1.PGK-tk2 was tested by an in vitro excision assay using transbosase purified from E. coli. Southern blot analysis of the reaction product shows the appearance of the excised 2.9 kb Tc1.SV-neo sequence (FIG. 9). In other words, pRP466.SV-neo1.
PGK-tk2 can be used as a donor plasmid for the Tc1.SV-neo transposon.

To determine whether transposition of the Tc1 transposon-derived vector occurs in human cells, the plasmid pRP466.TM. Is used together with the Tc1 transposase expression vector pcDNA1 / TclA or the empty vector pcDNA1 / amp (Invitrogen). SV-neo1.PGK
Cell line 911 was transformed with -tk2. About 48 hours after the transformation, the cells were selected by G418. After 18 days, the number of G418-resistant colonies was counted (Table 2). Cotransformation with pcDNA1 / TclA resulted in approximately 40% more G418-resistant colonies, which, in addition to random integration, showed that the Tc1-neo transposon jumped into the cell genome, Is formed.

The presence of the PGK-tk expression cassette outside the Tc1 inverted repeat of plasmid pRP466.SV-neo1.PGK-tk2 made it possible to distinguish between integration of the plasmid and transposition of the Tc1-neo transposon. Thymidine kinase phosphorylates the antiviral prodrug ganciclovir (GCV) and then acts as a chain terminator of DNA synthesis, thereby killing tk-expressing cells. When plasmid integration occurs, in most cases, both the SV-neo and PGK-tk expression cassettes are integrated into the 911 genome, resulting in G418 resistance and GCV sensitivity in the cell line. But SV-ne
o When transposon transposition occurs, the cell line becomes G418-resistant and GCV-insensitive because the expression cassette for PGK-tk is not integrated into the host cell genome. A total of 73 independent G418-resistant cell lines were established by the above transformation and screened for GCV. Table 3 shows that pRP466.SV-neo1.PGK-tk2 was converted to pcDNA1 / Tc
When co-transformed with lA (64-77%), co-transformation with pcDNA1 / amp (32-77%)
-44%), indicating that more tk negative cell lines were formed.

To randomly incorporate the circular plasmid DNA, first, double-stranded DNA is cut,
The DNA needs to be linearized to allow integration into the host genome. The PGK-tk expression cassette covers approximately one-third of the total plasmid DNA (excluding the neomycin SV-neo expression cassette for selecting cell lines). Assuming that this process is random and that only one copy of the plasmid is integrated, approximately 33% of the DNA breaks will occur in the PGK-tk expression cassette, thereby abolishing tk expression. Therefore, tk generated by co-transformation of pRP466.SV-neo1.PGK-tk2 with pcDNA1 / amp
The percentage of negative cell lines was as expected. pRP466.SV-neol.PGK-tk2
The higher the incidence of GCV-resistant clones when co-transformed with pcDNA1 / TclA, and the fact that the Tcl.SV-neo transposon was found in the human genome from pRP466.SV-neol.PGK-tk2 Strongly suggests that you have jumped.

The most direct way to distinguish between plasmid integration and transposition is to determine the sequence of the DNA flanking the integrated transposon in the host cell genome. After integration of the plasmid, the nearby DNA sequence was transformed into plasmid pRP466.SV-neol.PG.
It is identical to the sequence adjacent to the K-tk2 transposon. After transposition, the neighboring sequences are of human genome origin and therefore differ from the sequence of plasmid pRP466.SV-neo1.PGK-tk2. Genomic DNA was isolated from separate tk-minus / G418 resistant colonies and digested with Sau3A. An oligo cassette (“Vectore
tte (vectorette) "). This vectoret is composed of two types of oligonucleotides whose ends are complementary and the inside is not complementary. A fragment containing a transposon and an adjacent sequence is added to the end of the transposon. Using complementary oligos, combine with the complementary oligos inside the vectorette sequence and amplify by PCR to show adjacent sequences, analyze the PCR products on an agarose gel, and determine the sequence of the amplified DNA fragment So far, transposons have been found in two of the five cases, flanked by TA dinucleotides, followed by new sequences. This indicates that Tc1 can fly in human cells when Tcl transposase is supplied.

Materials and Methods Plasmid Construction pRP466 contains a Tc1 element with a 0.4 kb flanking sequence from plM40 (Mori, 1988) cloned into pUC19 as a BamHI-XbaI fragment, while pRP467 and pRP468 are ClaI-Asp718 or PstI. -A derivative of pRP466 in which one of the ApaI fragments has been deleted. pRP472 is pAC
B104 (Boyd and Sherratt, 1995) derivative, which contains Tc1 with the AvaI-HindIII fragment of pBR322 inserted between the ClaI and ApaI sites. PUC between XhoI sites
The XbaI-BamHI fragment of pRP466 with 4K (Pharmacia) HindII Kan ^R cassette was pACB
Cloning into 104 gave pRP490. pRP491 is similar to pRP490, with all internal Tc1 sequences replaced except for the terminal 26 bp.

The plasmid pRP466.SV-neo1.PGK-tk2 was constructed as follows. pRP466 was digested with XhoI and blunt-ended with Klenow and dNTP. Plasmid pRc / CMV (Invitrogen) was digested with BamHI and EcoRI and blunt-ended with Klenow and dNTP. Next, the Ba
By cloning the mHI / EcoRI SV-neo fragment into XhoI digested pRP466,
pRP466.SV-neo1 was obtained. The plasmid pRP466.SV-neo1 was digested with EcoRI and blunt-ended with Klenow and dNTP. Plasmid pPGK-tk (PCT / NL / 00195) was digested with PvuII. The PvuII PGK-tk fragment was cloned into EcoRI digested pRP466.SV-neo1 to obtain pRP466.SV-neo1.PGK-tk2.

The plasmid pRc / CMV.Tc1A contains the Tc1-transposase cDNA, which
From P470 (Vos et al., 1993), the primer 5'CCCCAAGCTTGCCACCATGGTAAAATCTGTTGGG
Amplified by TGTAAAAATC (SEQ ID NO :) and 5'GCTCTAGATGCTTAATACTTTGTCGCGTATCC (SEQ ID NO :-) using eLONGase according to the standard protocol of the manufacturer (Gibco BRL). . PCR
Amplification program: 1 cycle at 94 ° C for 1 minute on a Biometra Trio Thermoblock; 35 cycles of 94 ° C for 30 seconds + 52 ° C for 30 seconds + 68 ° C for 1 minute; 68 ° C for 1 minute. Done. Digest the PCR fragment with HindIII / XbaI,
By cloning into HindIII / XbaI digested pRc / CMV, pRc / CMV.Tc1A was obtained. The integrity of the Tc1A cDNA was confirmed by sequencing. The pRc / CMV plasmid contains a neomycin resistance gene expression cassette. Plasmid pcDNA1 / Tc1A, Hin
pRc / CMV.T cloned into dIII / XbaI digested pcDNA1 / amp (Invitrogen)
Contains the HindIII / XbaI Tc1A fragment of c1A. The pcDNA1 / amp plasmid does not contain a neomycin resistance gene expression cassette. Restriction enzymes, primers, Klenow and
All dNTPs were purchased from Gibco BRL.

Southern Blotting For FIG. 9: The products of the in vitro excision reaction were separated by electrophoresis on a 0.8% agarose 0.5 × TBE gel at 70 volts for 5 hours. The gel was then rinsed in Southern blot buffer (0.4 M NaOH, 0.6 M NaCl) for 30 minutes and the DNA was blotted overnight on Hybond-N ⁺ (Amersham). After blotting, the gel is rinsed with 5x SSPE, 50% formamide, 5x denhardt's solution, 5x SSPE, 5% dextran sulfate, 1% SDS, and 200 µg / ml haring sperm DNA for 3 hours prehybridized and ³² P-labeled by random-primed method (R
Hybridization was performed overnight at 42 ° C. with a neo-probe (NcoI fragment of pPGK-tk) using a TS Rad prime DNA labeling system, Gibco BRL. Next, the blot was washed thoroughly at 65 ° C. in 5 × SSPE / 0.1% SDS. The blot was exposed to HyperFilm (Amersham) at room temperature.

Western Blotting Cells were washed twice with PBS (NPBI), RIPA (1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS in PBS, 1 mM phenylmethylsulfonyl fluoride and 0%
.1 mg / ml with trypsin inhibitor) and scraped off. 1 on ice
After incubation for 5 minutes, the lysate was clarified by centrifugation. Protein concentration was determined by the Bio-Rad protein assay according to the manufacturer's (Bio-Rad) standard procedures. Whole cell extracts were fractionated by SDS-PAGE on a 10% gel. The protein was transferred to an Immobilon-P membrane (Millipore) and the αTc1 transposase antibody (Schukkink et al., 1).
990). The secondary antibody is horseradish peroxidase conjugated goat anti-rabbit antibody (Bio-Rad). Antibody complexes were visualized using the ECL detection system according to the manufacturer's protocol (Amersham).

Cell Culture and Transfection Cell line 911 (Fallaux et al., 1996) was incubated at 37 ° C., 5% in Dulbecco's modified Eagle's medium (Gibco BRL) supplemented with 10% FBS (Gibco BRL).
Cultured in CO ₂ . Selection of transfected cells using G418 (Gibco BRL) was performed at a concentration of 500 μg / ml. Ganciclovir (Roche)
Was performed at a concentration of 10 μg / ml. DNA was transfected using a calcium phosphate transfection system according to the manufacturer's protocol (Gibco BRL). Briefly, one day prior to transfection, 911 cells were seeded at a density of 30% in 5 cm tissue culture dishes (Greiner). The next day, cells were incubated with calcium phosphate precipitated DNA for 15 hours. Next, the cells were washed twice with PBS and fresh medium was added. Approximately 48 hours after transfection, cells were placed in G418 selection medium. G418 resistant colonies appeared around the 6th day. A single colony was picked and a cell line was established.

Sequencing of the transposon flanks 100 ng of genomic DNA was combined with 4 units of Sau3A (Boehringer Mannheim (B
oehringer Mannheim) according to the manufacturer's instructions.
. After 2 hours at 37 ° C, the enzyme was inactivated by incubation at 65 ° C for 15 minutes. Equimolar amount of vectorette oligo (503: 5'GATCCAAGGAGA
GGACGCTGTCTGTCGAAGGTAAGGAACGGACGAGAGAAGGGAGA and 504: 5'TCTCCCTTCTCGAATCGT
AACCGTTCGTACGAGAATCGCTGTCCTCTCCTTG), 10 mM Tris-HCl (pH 7.5) and 1 mM EDT
Mix in the presence of A. The mixture was heated to 80 ° C and cooled to 40 ° C. The final concentration of this vector oligo cassette is 10 pmol / μl. Use 5 units of ligase (Boehringer Mannheim) according to the manufacturer's instructions.
Thus, 15 picomoles of vectorette in a volume of 100 μl were ligated to the digested DNA at 16 ° C. overnight. In the first PCR, one unit of Taq polymerase (Gibco BRL) is used according to the manufacturer's instructions, and 3 μl of the DNA is used in a volume of 25 μl. The final concentration of oligo is 0.4 pmol. The PCR consisted of 30 cycles of 1 minute at 95 ° C, 1 minute at 58 ° C, and 1 minute at 72 ° C. To increase specificity and sensitivity
Next, a second PCR with inner (nested) primers was performed using the template obtained in the first PCR.
This was performed using 0.01 μl. The conditions for this PCR were the same as for the first PCR. The oligo used was Tc1L2 (5'-TCAAGTCAAATGGATGCTTGAG) (SEQ ID NO: 5) or Tc1R2 (5 '
-GATTTTGTGAACACTGTGGTGAAG) (SEQ ID NO: 6) and 337new (5'GTACGAGAATCGCTGTCCT
C) (SEQ ID NO: 7). The PCR products were analyzed on a 1% agarose gel. The amplified DNA fragment is excised from the gel, and the DNA is extracted with QIAEX II Gel Extraction Kit (Qiagen
agen). After isolation from the gel, the DNA was dissolved in 20 μl of water. ³² Using P-ATP and polynucleotide kinase (Boehringer Mannheim), use the transposon primer Tcl1L2 or Tc1 according to the manufacturer's instructions.
1R2 was radiolabeled. Uses 0.25 picomoles of radiolabeled primer and isolated DNA
0.5 units of Taq polymerase (Gibco BRL) and ddNTP mixture (d
dATP (10 mM Tris-HCl (pH 8), 1 mM EDTA, 160 μM ddATP and 5 μM each dNTP),
ddTTP (250 mM ddTTP and 5 μM each dNTP in 10 mM Tris-HCl (pH 8), 1 mM EDTA), ddGTP (32 mM ddGTP and 5 μM each dNTP in 10 mM Tris-HCl (pH 8), 1 mM EDTA)
Or ddCTP (10 mM Tris-HCl (pH 8), 1 mM EDTA, 160 μM ddCTP and 5 μM each d
NTP) was used for PCR. The final volume is
20 μl. Twenty cycles were performed at 95 ° C for 1 minute, 58 ° C for 1 minute, and 72 ° C for 1 minute. 1 × T
Samples were analyzed on a 6% denaturing polyacrylamide gel in BE.

Microinjection (Mell) of 150 mg / ml pRP469 and 5 mg / ml pRP465 (Vos et al., 1993), 50 mg / ml pRF4 (Kramer et al., 1990) in C. elegans transgenesis strain CB1392 (nuc-19e1392)
O et al., 1991) later obtained a transgenic Bristol strain. Stable strain NL818 (
pkls221) was produced by X-ray irradiation (Way et al., 1991).

Preparation of Extracts Stable strain NL818 was grown in liquid culture at 18 ° C. and heat-shocked at 33 ° C. for 3 hours to induce transposase expression. After growing for an additional 2 hours at 18 ° C., nuclear extracts were prepared as described (Vos et al., 1993), but with different buffers. NIB: 25 mM Tris (pH 7.5), 20 mM KCl, 0.5 M sucrose, 0.5 mM EDTA, 5 m
Mβ-mercaptoethanol, 0.1 mM PMSF. NEB: 25 mM Tris (pH 7.5), 0.1 mM ED
TA, 500 mM NaCl, 15% glycerol, 0.25% Tween-20, 0.1 mM PMSF, 1 mM DTT. The nuclear extract contains 2.5 mg / ml protein and the concentration of Tc1A is about 10 mg / mlg.

Expression and Purification of Recombinant Transposase pRP470 containing the Tc1 transposase gene (Vos
E. coli BL21 pLysS strain was transformed with E. coli BL21 pLysS, grown in 2 × YT medium, and induced at 37 ° C. for 3 hours using 0.5 mM IPTG at an OD of 0.6 at 600 nm. Inclusion bodies were purified as described in Nagai and Thogersen (1978). Dissolve the inclusion body in 8M urea, 20mM sodium phosphate (pH 6.0) and add CM Cellulose CL
-6B column (Pharmacia). Protein was eluted with a linear gradient from 0 to 500 mM NaCl. Sephacryl S400HR in which the transposase-containing fraction was equilibrated with 6 M guanidium hydrochloride and 50 mM Tris (pH 8.0)
Loaded on a gel filtration column. The transposase fraction was dialyzed against 8 M urea, 50 mM Tris (pH 8.0), 1 mM DTT. The protein is separated into S Sepharose F
Loaded on an F column and eluted with 500 mM NaCl in the same buffer. All steps were performed at room temperature. The protein was added to an ice-cold buffer (50 mM Tris (pH 8.0), 100 mM NaCl, 5 mM
DTT was regenerated by 100-fold dilution into 5 mM MgCl ₂ ). After 30 minutes, insoluble proteins were removed by centrifugation in an Eppendorf centrifuge for 15 minutes. The transposase concentration was 200 mg / ml and the purity was estimated to be over 90%.

In vitro rearrangement reaction Standard reaction conditions: 25 mM Tris (pH 8.0), 25 mM NaCl, 1 mM DTT, 10% ethylene glycol, 5 mM MgCl ₂ (or 2.5 mM EDTA), 4 mM spermidine, 0.05 mg / ml BSA. After pre-incubating 200 ng of the donor plasmid with 2.5 ml of nematode extract or 0.25 ml of purified protein for 5 minutes on ice, 2.5 mg of target DNA in a total volume of 50 ml was added. Incubation was performed at 30 ° C. for 1 hour. 5.5 ml of 250 mM Tris (pH 8
.0), the reaction was stopped by adding 50 mM EDTA, 5% SDS, 2 mg / ml proteinase K. After 1 hour at 37 ° C., the DNA was precipitated and resuspended in 50 ml of water.

Mapping of In Vitro Cleavage Sites Basically as described (Craxton, 1991), 5 ml of template and 0.5 pmol
Using the above primers, linear PCR amplification was performed in 20 ml over 20 cycles of 94 ° C. for 1 minute, 60 ° C. for 1 minute, and 72 ° C. for 1 minute. Sequence primer: BIGR = 5'AGATTTCCACTTAT
ATCATGTTTTATGTTTTGC (SEQ ID NO: 8), R2 (Van Luenen and Plasterk, 1994).

Genetic Assay Electrocompetent DS941 lambda lysogen (Flinn et al., 1989) bacteria were prepared and used as described (Zabarovsky and Winberg, 1990). The donor plasmid contains a lambda origin of replication and cannot replicate in DS941 lambda lysogen. The target plasmid has a Col E1 origin of replication. Use 1-5 ml of DNA per electroporation,
After dilution of 5% of the bacteria, they were plated on ampicillin. The remaining bacteria were plated in double selection. This resulted in up to 200 transformants depending on efficiency.

Table 1: Transposition frequency. An in vitro Tc1 transposition reaction was performed with the supercoiled (SC) or linear donor plasmid and the indicated protein source, and the ratio of amp ^R -kan ^R colonies to amp ^R colonies ( ^* 10 ⁶ ) was determined in two independent rounds. The experiment will be described. No integrated product was recovered when the reaction was performed in the presence of EDTA.

Table 2: Formation of G418 resistant colonies after co-transfection of pRP466.SV-neo1.PGK-tk2 with pcDNA1 / Tc1A or pcDNA1 / amp. PRP466.SV.-neo1.P to cell line 911
GK-tk2 was co-transfected with pcDNA1 / Tc1A or pcDNA1 / amp. Transfected cells were placed in G418 selection medium for 18 days. Next, G418 resistant colonies were stained with methylene blue and counted. pcDN of pRP466.SV-neo1.PGK-tk2
Co-transfection with A1 / Tc1A yielded about 40% more G418-resistant colonies than co-transfection with pcDNA1 / amp. This indicates that in addition to random integration, jumping of the Tc1-neo transposon into the cell genome resulted in the formation of G418 resistant colonies.

Table 3: HSV-tk expression in G418 resistant cell lines co-transfected with pRP466.sv-neo1.PGK-tk2 and pcDNA1 / Tc1A or pcDNA1 / amp. pRP466.sv-neo1.PGK-t
Co-transfection of k2 with pcDNA1 / Tc1A gave more HSV-tk negative (ganciclovir-resistant) colonies than co-transfection with pcDNA1 / amp.

[0061]

[Table 1]

[0062]

[Table 2]

[0063]

[Table 3]

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gene transfer in C. elegans: extrachromosomal maintenance and integratio
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ransposable elements in Caenorhabditis elegans ". Ph. D. thesis. Washingt
on University, St. Louis, MO.Muller, H.-P. and HE Varmus, 1994.DNA bending create favored sites fo
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lex nucleoprotein structures.Genes & Dev. 7: 1521-1534.Plasterk, RHA 1991.The origin of footprints of the Tc1 transposon of
Caenorhabditis elegans, EMBO J. 10: 1919-1925. Plasterk, RHA, 1995. The Tc1 / mariner transposon family. In Transposab
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[Sequence list]

[Brief description of the drawings]

FIG. 1. Southern blot analysis of in vitro Tc1 transposition reaction products. The products of the in vitro rearrangement reaction were separated on a 1% agarose gel, transferred to nitrocellulose, and probed with radiolabeled Tc1. Standard reactions contained MgCl ₂ (lanes 1-3 and 5-11) or EDTA (lanes 4). Prior to electrophoresis, the product was converted to ScaI in vector DNA (lanes 2, 7, 10)
Alternatively, digestion was performed with ApaI (lane 3) in Tc1 DNA. Lanes 5, 8, and 11 show the reaction products when the substrate was linearized with ScaI prior to in vitro cleavage. Lanes 1-5
Shows a reaction product using pRP466 having a complete Tc1 element as a substrate (see FIG. 4). Lanes 6 to 8 show the case where pRP467 in which the left end of Tc1 was deleted was used as a substrate, while lanes 9 to 11 show the case where pRP4678 in which the right end of Tc1 was deleted was used as a substrate. REC
And LEC refer to right and left cuts, respectively. RH and LH indicate the positions of the right and left halves of Tc1. A schematic diagram of pRP466 linearized with ScaI is shown at the bottom of the figure.

FIG. 2. Mapping of in vitro cleavage sites at the nucleotide level. The reaction product obtained in the presence of MgCl ₂ (+) or EDTRA (−) using pRP466 as a donor was subjected to primer extension by PCR. To demonstrate the addition of one additional nucleotide at the end of the PCR product by Taq polymerase, a control reaction was performed using pRP466 digested with EcoRV (RV lane) (see Clark, 1988). The products were analyzed on a sequencing gel. The sequencing reaction (GATC) was filled in as a marker. PCR was performed with primer R2 (right panel) or primer BIGR (left panel). Relevant sequences are indicated by a boxed EcoRV site and an underlined TA target site. The cleavage site is indicated by an arrow. The same result was obtained when the cleavage position at the other transposon end was determined.

FIG. 3 is a schematic diagram of a gene transfer assay. Donor plasmid, pACB10 with lambda origin of replication
Four derivatives (Boyd and Sherratt, 1995) contain a Tc1 element that carries an antibiotic resistance gene. The target plasmid pRP475 has a 1.4 kb HindII gpa-2 fragment and a Col E1 origin of replication (pSP72, Promega). The reaction products were electroporated into lambda lysogenic E. coli strains to counterselect against the donor. Integration events were selected with dual antibiotics.

FIG. 4. Target site selection. Comparison of the distribution of in vitro (black bars) and in vivo (hollow bars) Tc1 inserts. In a standard in vitro transposition reaction using nematode extract, pR
P472 was used and pRP475 as target. Each point on the X axis represents a TA dinucleotide in the gpa-2 fragment, as detailed elsewhere (Van Luenen and Plasterk, 1994).

FIG. 5. Purification of Tc1 transposase from E. coli. Analysis of transposase purified from inclusion bodies on a 12% SDS-polyacrylamide gel. Lane M: molecular weight marker (
Lane 1: bacterial lysate before induction; lane 2: bacterial lysate after induction: lane 3: purified inclusion bodies; lane 4: purified transposase after refolding.

FIG. 6. Mutations at the extreme end of Tc1 affect excision. As shown at the top, pRP480 (wt), pRP481 (TA), pRP482 (CA), pRP483 (GT) or pRP484 (
An in vitro reaction product was obtained using a nematode extract in the presence of MgCl ₂ (+) or EDTE (−) using BS). The products were separated on a 1% agarose gel, transferred to nitrocellulose and probed with a radiolabeled Kan ^R gene fragment. The donor plasmid contains a 28-mer cloned into the SmaI site (wt sequence) and HindII site (wt or mutant sequence) of pUC19, with the interposed pUC4K Kan ^R cassette in between. TA was mutated to CG, CA to TG, and GT to AC. In transposase binding site mutations,
The BalI and EcoRV sites are mutated to TCCCA and GGGCCC, respectively (Vos and Plaster
k, 1994).

FIG. 7: Model of Tc1 transposition. 2bp 3 'staggered overhang
g) Model of non-replicating Tc1 transposition representing a excised linear element with g). Built-in is TA
This results in duplication of target sites. Repair of double-strand breaks leads to the generation of a characteristic footprint (see also Van Luenen et al., 1994).

FIG. 8. Tc1 transposase is not toxic to human cells. 2μg pRc / CM per 5cm
V. Tc1A or 2 μg of pRc / CMV was transfected into cell line 911 and a monoclonal cell line was established from G418 resistant colonies. 50 μg of whole cell extract from each cell line
After separation on a 0% PAA gel, it was analyzed for Tc1 transposase. pRc / CMV.Tc1A
Tc1 transposase was detected in the cell line transfected (lanes 1 to 5), but Tc1 transposase was not detected in the negative control cell line (lanes 6 to 8) transfected with pRc / CMV. Was.

FIG. 9. pRP466.SV-neo1. Catalyzed by Tc1 transposase purified from E. coli.
Southern blot of the in vitro excision reaction product of PGK-tk2. Donor plasmid pRP466.
Incubation of SV-neo1.PGK-tk2 with purified Tc1 transposase (lane 1) results in excision of the Tc1.Sv-neo transposon (lane 1), but not with NEB (lane 2). Thus, plasmid p
RP466.SV-neo1.PGK-tk2 can serve as a Tc1.SV-neo transposon donor plasmid.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // C12N 9/22 C12N 5/00 B (81) Designated country EP (AT, BE, CH, CY, DE) , DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML) , MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN , MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventors Shouten, Hofeld Johann The Netherlands, Ener-2321 Aer Rieden, Da Costastrat 82 (72) Inventors Plasterk, Ronald Hans Anton The Netherlands, Ener-1405 Aer Bussum, Zwaldweg 5 (72) Inventors Juan Ruenen, Hendricks Hellhard Adrians Maria Enel-1108 Beem Amsterdam, The Netherlands 143F Over-time (reference) 4B024 AA20 BA07 CA01 DA02 EA02 FA11 FA15 GA11 GA18 HA01 HA08 4B050 CC03 DD01 LL01 4B065 AA90X AA95Y AA97Y AB01 BA02 CA29 CA44 4C084 AA13 NA10 4C087 AA01 AA02 BC83 CA12 NA10

Claims (15)

[Claims] 1. A vector for integrating an additional nucleic acid substance into cells of a genome belonging to the first genus, said vector comprising two transposase binding sites and a cleavage site corresponding to each of said transposase binding sites. A vector, wherein the transposase binding site is derived from a transposon found in the second genus, wherein the transposase binding sites are each in close proximity to a corresponding cleavage site, and additional nucleic acid material comprises the two transposases in the vector. A vector that integrates between the binding sites. 2. The vector according to claim 1, wherein the transposase binding site and the cleavage site are based on corresponding sites in a transposon from the Tc1 / mariner superfamily of transposons. 3. The transposase binding site and the cleavage site
The vector according to claim 1 or 2, which is based on a Tc1-like transposon. 4. The vector according to claim 3, comprising at least 26 base pairs of the terminal Tc1 as a transposase binding site and a cleavage site for Tc1 transposase. 5. The method according to claim 1, further comprising a means for transducing the target cell.
The vector according to 2, 3, or 4. 6. The vector according to claim 1, further comprising a nucleic acid sequence encoding a transposase activity functional for a transposase binding site and a cleavage site present in the vector. 7. The vector according to claim 1, 2, 3, 4, 5, or 6, whereby the additional nucleic acid material encodes a protein. 8. The method of claim 1, 2, 3, 4, 5, 6, or 7 wherein the additional nucleic acid material provides a blocking nucleic acid such as an antisense RNA.
The described vector. 9. The vector according to claim 1, 2, 3, 4, 5, 6, 7, or 8 wherein said vector is derived from a viral vector, preferably an adenovirus, retrovirus or adeno-associated virus vector. 10. A method for providing a target cell having an additional nucleic acid substance integrated into a genome, the vector according to claim 1, 2, 3, 4, 5, 6, 7, 8, or 9. Transfecting said cells with and providing said target cells with a transposase activity functional for transposase binding and cleavage sites present on said vector. 11. The method according to claim 10, whereby the transduction is carried out via infectious particles consisting of a vector. 12. A recombinant target cell obtained by the method according to claim 10. 13. Use of a functional transposon found in a genus in the integration of a nucleic acid of interest into the genome of cells of a different genus. 14. Use according to claim 13 in a gene therapy regime. 15. Use according to claim 13 for producing transgenic animals.