DE102011118018B4 - Minicircles with transposition cassettes and their use for transforming cells - Google Patents

Abstract

A minicircle comprising: a) at least one nucleic acid sequence of interest, and b) transposase recognition sequences on either side of the nucleic acid of interest, characterized in that the transposase recognition sequences can be recognized by a specific transposase and used for transposition, the specific transposase preferably a Sleeping Beauty transposase or a PiggyBac transposase, wherein the minicircle comprises no resistance gene and no selection marker for amplification in prokaryotic cells.

Description

Technical area

The present invention is in the field of biotechnology. More particularly, the invention relates to the provision of minicircle-based vectors for the transformation of cells by means of transposons.

background

Conventional methods for gene therapy usually use plasmid DNA as vectors for introducing desired DNA segments into the target cells. However, plasmids have several disadvantages in such applications. On the one hand, there is the possibility of uncontrolled plasmid replication after transformation, which can lead to the spread of existing on the plasmid resistance genes to antibiotics. In addition, plasmids contain other prokaryotic DNA sequences, the z. B. in eukaryotes can lead to immunogenic reactions. Nonviral vectors with as few prokaryotic sequences as possible are therefore very useful for gene therapy.

For this purpose, minicircles have been developed, which are understood to mean small, circular DNA molecules containing a desired expression cassette and as few unwanted prokaryotic sequences as possible. Methods for the preparation of minicircles have been described in WO 96/26270 A1 . US 2003/0104444 A1 and Schleef & Schmeer (Pharm. Unserer Zeit 2011, 40 (3): 220-224).

Bigger et al. (J. Biol. Chem. 2001, 276: 23018-23027) describe the preparation of minicircles by introducing plasmids with loxP sites into bacteria capable of expressing cre recombinase. The plasmid further comprises a eukaryotic expression cassette and a marker sequence. Upon induction of Cre, the plasmid is cleaved into minicircle and miniplasmid, with the minicircle containing only the expression cassette. In addition, the loxP sites are mutated so that the reversibility of recombination is reduced.

Other publications also describe the preparation of minicircles using other recombination systems, e.g. In Kreiss et al. (Appl. Microbiol Biotechnol 1998, 49: 560-567) using λ integrase and Chen et al. (Mol. Ther. 2003, 8: 495-500) and Conley el al. (Curr. Opin. Mol. Ther. 2008, 10 (5): 456-463) using ΦC31 integrase. Therefore, minicircles are now established as alternative vectors for the transfection of eukaryotic cells.

For the permanent genetic transformation of target cells, the introduction of transposition cassettes on a vector can be used, which can be converted by means of a transposase from the vector into a cellular nucleic acid. WO 2008/106985 A1 and Conley et al. (Curr. Opin. Mol. Ther. 2008, 10 (5): 456-463) disclose such systems. Izsvák et al. (Bioessays 2010, 32: 756-767) describe the particularly efficient transposon system "SB100X".

However, it is known that in many transposon systems, transposition efficiency decreases with increasing length of the transposition cassette (see, for example, BDJ Lampe et al., Genetics 1998, 149 (1): 179-187; JC Way et al., Genetics 1985, 111 (1984). 4): 705-713). The length of DNA segments that can be efficiently transposed into the DNA of a target cell is therefore limited. There is therefore a need for methods that allow efficient transformation of cellular DNA by transposing even longer DNA segments.

Brief description of the invention

The present invention is based on the idea to combine the minicircle technology for the transfection of cells with the transformation by means of transposon systems. In addition to the aforementioned advantages, which offers known transfection with minicircles, the introduction of a transposition cassette, z. A Sleeping Beauty-based transposition cassette on a minicircle surprisingly at higher transposition rates than the introduction on conventional vectors such as a plasmid.

Accordingly, the invention relates to a minicircle comprising at least one nucleic acid sequence of interest that can be transposed into another genetic environment, and transposase recognition sequences on both sides of the nucleic acid of interest, characterized in that the transposase recognition sequences of a specific transposase, or the derivative of a transposase, and can be used for recombination, wherein the specific transposase is preferably a Sleeping Beauty Transposase or a PiggyBac Transposase.

In one embodiment, the at least one nucleic acid sequence of interest may comprise an expression cassette comprising at least one gene, at least one isolator sequence or a combination thereof, or else several genes, a plurality of isolator sequences or a combination thereof.

The minicircle may contain at least one identification sequence, inside or outside the region between the transposase Recognition sequences. If there are multiple identification sequences, they can be identical or different.

Further, the sequence of the minicircle may comprise a pair of recombinase recognition sequences located on either side of the nucleic acid of interest within the transposase recognition sequences.

The minicircle contains no resistance gene and no selection marker for selection in amplification in prokaryotic cells. The minicircle may be an annular, preferably an annular and supercoiled, DNA molecule.

In another embodiment, the invention also includes parent plasmids from which any minicircle according to the invention can be recovered by recombination. A parental plasmid is a plasmid containing the complete sequence of the desired minicircle and additionally a plasmid backbone (miniplasmid region), the minicircle region and miniplasmid region being separated by recombinase recognition sequences arranged that they can be specifically recombined by a recombinase.

The invention also relates to cells comprising a minicircle according to one of the mentioned embodiments.

In another aspect, the invention relates to a method of insertional mutagenesis or genetic transformation of a eukaryotic target cell, the method comprising providing a minicircle of the invention comprising a nucleic acid of interest within the target cell, and transfecting the target cell with a vector comprising a nucleic acid sequence encoding encoding a transposase specific for the minicircle transposase recognition sequences, wherein expression of the transposase from the vector results in recombination of the nucleic acid sequence of interest with a nucleic acid of the target cell. The target cell is not a human embryonic stem cell derived from embryo destruction.

The provision of the minicircle can be achieved by transfecting the target cell with the minicircle or else by creating the minicircle within the target cell. Furthermore, the provision of the minicircle and the transfection with the vector can be carried out simultaneously in a cotransfection or sequentially in any order. The target cell may be a vertebrate cell, e.g. A human cell.

In another embodiment, the invention also includes cells obtained by a method according to the invention.

Finally, the invention also encompasses a kit for insertion mutagenesis or for genetic transformation of a target eukaryotic cell comprising a minicircle according to one of the mentioned embodiments and a vector which codes for a transposase which is specific for the transposase recognition sequences of the minicircle. For the transposase recognition sequences in the minicircle, a Sleeping Beauty transposase may be specific and the transposase encoded by the vector may be this Sleeping Beauty transposase or a derivative thereof, e.g. For example, the hyperactive Sleeping Beauty Transposase SB100X (also called SB100).

Description of the pictures

1A shows the nucleic acid sequence of the DR element at the outer end of an IR element (SEQ ID NO: 1).

1B shows the nucleic acid sequence of the DR element at the inner end of an IR element (SEQ ID NO: 2).

1C shows the amino acid sequence of Sleeping Beauty Transposase SB10 (SEQ ID NO: 3).

1D shows the amino acid sequence of Sleeping Beauty Transposase SB100X (SEQ ID NO: 4).

1E shows the nucleic acid sequence of a forward primer which can be used to amplify a GFP coding sequence from the plasmid pCMV-GFP (SEQ ID NO: 5).

1F shows the nucleic acid sequence of a reverse primer which can be used to amplify a GFP coding sequence from the plasmid pCMV-GFP (SEQ ID NO: 6).

2 shows a sketch of the minicircle MC-IR-GFP with a GFP expression cassette as a nucleic acid sequence of interest; GFP: GFP coding sequence; Prom: promoter sequence; pA: polyadenylation sequence; IR: IR element (transposase recognition sequence); rec-tag: remainder of the recombinase recognition sequences and identification sequence formed during recombination.

3a shows a sketch of the parent plasmid pP-IR-GFP, from which the minicircles MC-IR-GFP can be formed; GFP: GFP coding sequence; Prom: promoter sequence; IR: IR element (transposase recognition sequence); rec: recombinase recognition sequence for parA resolvase; 2 day: two identification sequences; Backbone: A plasmid backbone containing the sequences necessary for the amplification of the plasmid and an expression cassette for the parA resolvase. The recombinase recognition sequences for cleavage into miniplasmid and minicircle are shown in dark gray.

3b shows a sketch of the parent plasmid pP11-IR-GFP, from which the minicircles MC-IR-GFP can be formed; GFP: GFP coding sequence; Prom: promoter sequence; IR: IR element (transposase recognition sequence); rec: recombinase recognition sequence for parA resolvase; 1 day: an identification sequence; Backbone: Plasmid backbone containing the sequences necessary for the amplification of the plasmid and an expression cassette for the parA resolvase. The recombinase recognition sequences for cleavage into miniplasmid and minicircle are shown in dark gray.

4 Figure 4 shows a vector map of the plasmid pT2 / BH (Addgene Accession No. 26556) which contains appropriate IR elements and can be used as starting material for the cloning of original plasmids for Sleeping Beauty minicircles.

5 shows a schematic representation of a suitable method from the Kloplierung of starting plasmids (upper part), on their Klovierung for the generation of Parentalplasmiden (middle part, eg, pP-SB10 and pP-IR-GFP) and their recombination (respective formation and purification from minicircles) to transfection or cotransfection of a eukaryotic target cell (bottom). The two minicircles shown below contain the transposase expression cassette (here SB10, left) and a GFP expression cassette bounded by IR elements. The names of the constructs are given in their middle. GFP: GFP coding sequence; Prom: promoter sequence; pA: polyadenylation sequence; IR: IR element (transposase recognition sequence); rec-tag: a residue of the recombinase recognition sequences formed during recombination and at the same time 2 specific identification sequences; MCS: multiple cloning site; SB10: SB10 coding sequence; AmpR: ampicillin resistance cassette; ori: origin of replication. The recombinase recognition sequences for cleavage into miniplasmid and minicircle are shown in dark gray.

6A shows the nucleic acid sequence of the left IR / DR element Lmut44 (SEQ ID NO: 7).

6B shows the nucleic acid sequence of the right IR / DR element Rmut13delta13 (SEQ ID NO: 8).

7 schematically shows a preferred construction of Parentalplasmiden for the production of minicycles according to the invention. NOI: nucleic acid of interest; FRT: recombinase recognition sequences for Flp recombinase; IR / DR: transposase recognition sequences; rec: recombinase recognition sequence for parA resolvase.

Detailed description of the invention

MINI CIRCLES

The present invention relates in one aspect to a minicircle comprising at least one nucleic acid sequence of interest that can be transposed into another genetic environment, and transposase recognition sequences on either side of the expression cassette, characterized in that the transposase recognition sequences of a transposase, or the derivative of a transposase, can be recognized and used for transposition. Preferred transposases are sleeping beauty transposases and PiggyBac transposases.

"Minicircle" in the sense of this invention refers to a circular double-stranded DNA which contains at least one nucleic acid sequence of interest and is substantially free of prokaryotic nucleic acid sequences as typically found in plasmids, such as origins of replication, marker genes or resistance genes. In particular of promoters, minicircles contain, if at all, only those which serve for the expression of genes of the expression cassette. As a result, minicircles are particularly suitable for introducing desired nucleic acid sequences in target cells, since effects of undesired DNA elements, such as the expression or recombination of resistance genes, can be excluded and z. B. superfluous and functionless sequences can be avoided.

A "marker gene" within the meaning of the invention is a DNA sequence which gives a cell or an organism a survival advantage under certain conditions (eg resistance genes to certain antibiotics) or is used to find successfully transformed or transfected cells. For example, a marker gene may be a gene for expression of a fluorescent protein, such as GFP.

A "nucleic acid sequence of interest" may be any double-stranded DNA sequence that is to be introduced into a eukaryotic target cell and transposed into a nucleic acid of the target cell. Preferably, a nucleic acid sequence of interest is an expression cassette for one or more genes, e.g. B. 1, 2, 3, 4, 5 or more genes. In another embodiment, it may be a nucleic acid sequence that does not encode a protein. For example, the nucleic acid sequence of interest serve to generate a gap between two DNA sections or DNA-binding factors (eg.

Nucleic acids or proteins) under certain conditions to bind or dismiss and thus interfere with the gene regulation of the target cell. Furthermore, the nucleic acid sequence of interest may be an RNA coding sequence (eg, for siRNA or shRNA) or other genetic elements to be effective in the target cell, e.g. B. isolator sequences (that is, sequence motifs which, when integrated into the genome, affect the regulatory effect of one chromosomal segment on its neighboring regions). Also, a nucleic acid sequence of interest may be a marker sequence which is introduced into the cellular DNA in the course of an insertion mutagenesis - and optionally also removed again. By means of DNA sequencing or PCR starting from the marker sequence, the insertion site of the sequence can then be determined, for example. In addition, a nucleic acid sequence of interest may have an intended effect in the form of stimulation of cellular or immunological responses (eg, by CpG motifs, see A. Krieg et al., Nature 1995, 374: 546-9).

An "expression cassette" is a DNA sequence that contains one or more genes and sequences that control the expression of the genes. In particular, an expression cassette comprises promoter sequences, open reading frames that encode the polypeptides to be expressed, and 3 'untranslated regions that usually contain a polyadenylation sequence when expressed in eukaryotes. An expression cassette may also contain more than one gene (eg, 2, 3, 4, 5 or more genes), particularly a cluster of genes commonly used for stem cell programming. Such a cluster may contain, for example, 3, 4 or 5 genes.

"Transposase recognition sequences" are those DNA sequences which can be recognized, bound and reacted by a natural transposase or a derivative of a natural transposase such that, under appropriate conditions, the recombination of the DNA located between two recognition sequences with another ( trans) or the same (cis) DNA molecule. In turn, transposases are enzymes from transposon systems responsible for the transfer of transposons to other areas of the genome. A transposase is considered to be "specific" to given recognition sequences if it recognizes, binds and reacts them so that, under appropriate conditions, the transposition of the DNA located between two recognition sequences into another (trans) or the same (cis) DNA Molecule is enabled.

For the purposes of this invention, "derivatives" of a given transposase are understood as meaning those transposases which are specific for the same recognition sequences as the given transposase. The amino acid sequence of a derivative is generally related to the amino acid sequence of the original transposase and, for example, at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 99%. identical with her.

The minicircles of the invention contain several transposase recognition sequences. Preferably, minicircles of the invention contain two transposase recognition sequences, one on either side of the nucleic acid sequence of interest, such that the nucleic acid of interest is within the two transposase recognition sequences. The transposase recognition sequences may be directly attached to the nucleic acid of interest, or the sequences may be separated by a spacer. Spacers may be attached to either side of the nucleic acid of interest, or only on one side. The spacers preferably have a length of 1-1000 bp, z. 1, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 bp. If spacers are present on both sides of the nucleic acid of interest, they may have the same or different lengths. By "spacer" is meant herein any DNA sequence that is between the nucleic acid of interest and the transposase recognition sequences. In particular, a spacer may also comprise one or more identification sequences as described below.

Depending on the type of transposase used for transposition, the recognition sequences may be of different nature. In some embodiments, the recognition sequences contain repeats. Examples of different types of repetitions are long terminal repeats (LTRs), inverse repeats (IRs), or direct repeats (DRs). Repetitions of these types can also be combined with one another, for example as DRs within IRs (IR / DR). "Repetitions" here are not only completely identical sequences, but also sequences that are sufficiently similar to each other that a homology can be detected. For example, the in 1A and B represented DR sequences from a recognition sequence of Sleeping Beauty transposases about 80% identical to each other.

It will also be apparent to those skilled in the art that mutations can be introduced into recognition sequences, as long as their function is not thereby adversely affected. In any case, the recognition sequences must be of such a nature and be placed in such an orientation and location in the minicircle that they can be recognized, bound and reacted by a specific transposase such that, under appropriate conditions, the transposition within the DNA is Transposase recognition sequences DNA is enabled.

In principle, recognition sequences of any transposase can be used in minicircles of the invention. Examples of transposases include PiggyBac transposases, Sleeping Beauty transposases and their derivatives. The amino acid sequence of a derivative is generally related to the amino acid sequence of the original transposase and, for example, at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 99%. identical with her. The derivative of a transposase also possesses transposase activity and may be specific for the same or different recognition sequences as the original transposase.

In a preferred embodiment, the minicircle contains two IR / DR type recognition sequences, for which Sleeping Beauty transposases (also called Sleeping Beauty or SB transposases) are specific. The recognition sequences recognized by SB transposases are IR sequences of about 210-250 base pairs in length and each contain two DR sequences, one at each end, which are over 80% identical to one another. The DR sequence at the outer end, farther from the center of the DNA segment to be transposed, in this case has the sequence of SEQ ID NO: 1 (see also FIG 1A ) and the DR sequence at the inner end closer to the midpoint of the DNA segment to be transposed has the sequence of SEQ ID NO: 2 (see also US Pat 1B ). An SB transposase can also use two IR sequences for transposition, even if the IR sequences do not represent exact repetitions, but contain sequence differences in the regions between the respective DRs, as long as the DRs themselves contain the sequences shown in SEQ ID NO: 1 and 2 (see Z. Ivics et al., Cell 1997, 91: 501-510). Accordingly, in further preferred embodiments, at least one or both of the transposase recognition sequences have a length of 210-250 base pairs (e.g., 225-240 base pairs), at the end farther from the nucleic acid of interest, the sequence of SEQ ID NO: 1 and at the end closer to the nucleic acid of interest is the sequence of SEQ ID NO: 2.

Sleeping beauty transposases (SB transposases) are based on the artificial transposase SB10 (SEQ ID NO: 3; see also 1C ), which in turn was designed based on the inactive transposases of several Tc1-like elements (IcEs) from several species of true bony fish (see Z. Ivics et al., Cell 1997, 91: 501-510). While the naturally occurring TcEs are all inactive, the SB10 transposase has been mutated to catalyze transcriptional functions at the IR / DR sequences of TcEs (for example, the T element from Tanichthys albonubes) and is fully functional. Another SB transposase is SB100X (also called SB100), a derivative of SB10 that has an approximately 100-fold increased transposition rate to SB10 (SEQ ID NO: 4, see L. Mátés et al., Nat. Genet. 753-761, see also 1C ).

2 shows a sketch of an exemplary minicircles according to the invention with a GFP expression cassette as a nucleic acid sequence of interest.

The SB transposon system has the particular advantage that it is active in a large number of different vertebrate cells from various species (see Z. Ivics et al., Cell 1997, 91: 501-510) and therefore a versatile use of minicircles in recombinations - and integration experiments. In particular, SB minicircles can be used to prepare induced pluripotent stem cells.

Minicircles according to the present invention can have very different sizes, for example between 500 and 15000 bp, such as 500 bp, 1000 bp, 1500 bp, 2000 bp, 2500 bp, 3000 bp, 4000 bp, 5000 bp, 6000 bp, 8000 bp , 10000 bp, 12000 bp or 15000 bp. Preferably, sizes between 1000 bp and 6000 bp, more preferably sizes between 2000 bp and 4000 bp.

Preferably, the minicircles of the invention are supercoiled.

Preferred minicircles of the invention may be divided into two regions, an "inner" and an "outer" region. The inner region comprises the region comprising the nucleic acid of interest up to and including the two transposase recognition sequences and is thus also a transposition cassette. The outer region comprises the region between the outer ends of the two transposase recognition sequences.

According to the invention, minicircles with outer ranges ≦ 500 bp are preferred. Particularly preferred are minicircles with outer ranges ≤ 300 bp, z. B. with outer areas of 30, 50, 75, 100, 150, 200, 250 or 300 bp.

It has been observed that DNA sequences flanked by transposase recognition sequences are surprisingly transposed more efficiently by the corresponding transposase if the DNA sequences are present on minicircles instead of on plasmids. The resulting higher transposition rate results in that the frequency and efficiency of integration of desired DNA sequences into the DNA of a target cell can be significantly increased if these sequences are provided in the form of transposition cassettes on minicircles. This is especially true when the minicircles are supercoiled.

Provision of transposition cassettes in minicircles, for example, for the purpose of insertional mutagenesis or transformation in a target cell thus allows, on the one hand, to take advantage of the general advantages of minicircle technology in avoiding the introduction of unnecessary and possibly immunogenic prokaryotic sequences into the target cells. In addition, however, the transposition efficiency is also increased, which represents a significant further advantage of the technology described herein. As already described in the Background section, it is known that in many transposon systems, the transposition efficiency decreases as the length of the transposition cassette increases. The length of DNA segments that can be efficiently transposed into the DNA of a target cell is therefore limited. The observed increase in transposition efficiency in providing the transposition cassette on a minicircle can therefore be used to advantage in transposing longer DNA segments into the DNA of a target cell with efficiency that could only be achieved at significantly shorter intervals with previous methods. Especially for the transposition of sections containing multiple genes, minicircle transposon constructs are very interesting.

According to one embodiment of the invention, the nucleic acid sequence of interest in the minicircle is an expression cassette having at least one gene. The gene preferably encodes a therapeutically useful protein. However, any other genes can also be used in the minicircles of the invention. The gene may encode a wild-type protein, a mutant protein or even a fusion protein, e.g. For a GFP fusion protein. The expression cassette may also contain multiple genes or other genetic elements. As mentioned above, this may be, for example, a sequence that does not code for a protein, but merely serves to generate a distance between two DNA segments or DNA-binding factors (eg nucleic acids or proteins) bind or release certain conditions and thus intervene in the gene regulation of the target cell. Furthermore, the sequence in the expression cassette may be an RNA coding sequence (eg for siRNA or shRNA) or other genetic elements to be effective in the target cell, e.g. B. isolator sequences (that is, sequence motifs which, when integrated into the genome, affect the regulatory effect of one chromosomal segment on its neighboring regions). Also, a nucleic acid sequence of interest may be a marker sequence which is introduced into the cellular DNA in the course of an insertion mutagenesis - and optionally also removed again. By means of DNA sequencing or PCR starting from the marker sequence, the insertion site of the sequence can then be determined, for example. In addition, a nucleic acid sequence of interest may have an intended effect in the form of stimulation of cellular or immunological responses (eg, by CpG motifs, see A. Krieg et al., Nature 1995, 374: 546-9).

In another embodiment, the minicircles in the inner region, flanking the nucleic acid of interest, contain a pair of recombinase recognition sequences for which a corresponding recombinase is specific. A recombinase specific for given recombinase recognition sequences is any enzyme capable of catalyzing directional recombination at the recombinase recognition sequences. In the present case, the two recombinase recognition sequences must be oriented such that upon recombination, the intervening DNA segment is excised as a cyclic molecule.

With the aid of these recombinase recognition sequences, after insertion mutagenesis, the nucleic acid of interest which has been integrated into the genome of the target cell by means of the transposase can be removed from the genome or exchanged again by providing the corresponding recombinase specific for the recognition sequences within the target cell. The provision is preferably carried out by inducible expression of the recombinase in the target cell. Preferred recombinase recognition sequences for such embodiments of the invention are FRT sequences for which the Flp recombinase from the plasmid is 2μ specific (see, e.g., B.J. Andrews et al., Cell 1985, 40 (4): 795-803). However, other recombinases and recombinase recognition sequences may also be used.

MANUFACTURE OF MINICIRCLES

The minicircles of the invention may be prepared by recombination from appropriate plasmids as described in U.S. Pat WO 96/26270 A1 or EP 1620559 B1 described. This is based on a plasmid ("parent plasmid"), which contains the complete sequence of the desired minicircle (minicircle region) and additionally a plasmid backbone (miniplasmid region) (see 3 ). Between the minicircle region and the miniplasmid region there are recombinase recognition sequences on both sides for which a corresponding recombinase is specific. A recombinase specific for given recombinase recognition sequences is any enzyme capable of catalyzing directional recombination at the recombinase recognition sequences.

The parent plasmid is recombined by the specific recombinase into a minicircle and a remainder, the miniplasmid. Here, the two recombinase recognition sequences must be oriented so that the recombination into minicircle and miniplasmid occurs and not a single modified plasmid is formed.

The recombinase recognition sequences may be immediately adjacent to the transposition cassette, or the sequences may be separated by a spacer. Spacers may be attached to either side of the transposition cassette, or only on one side. The spacers preferably have a length of 1-1000 bp, z. 1, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 bp. If there are spacers on both sides of the Transposition Cassette, they may have the same or different lengths. By "spacer" is meant herein any DNA sequence located between the transposition cassette and the recombinase recognition sequences. In particular, a spacer may also comprise one or more identification sequences as described below.

Examples of possible recombinases are the integrases from bacteriophages λ (see A. Landy et al., Science 1977, 197 (4309): 1147-1160), P22 and 080 (see JM Leong et al., J. Biol. Chem. 1985, 260 (7): 4468-4470), the HP1 integrase from Haemophilus influenzae (see MA Hauser et al., J. Biol. Chem. 1992, 267 (10): 6859-6864), the Cre integrase the phage P1, the integrase from the plasmid pSAM2 (see EP350341 ), the Flp recombinase from the plasmid 2μ (see BJ Andrews et al., Cell 1985, 40 (4): 795-803) or the ΦC31 integrase (see JR Bateman et al., Genetics 2006, 173 (2) : 769-777). Alternatively, recombinases from transposons of the Tn3 family can be used, such as the RPA parA resolvase (see L. Eberl et al., Mol. Microbiol., 1994, 12 (1): 131-141), the resolvases of the transposons Tn3 , Tn21 or Tn522 (see WM Stark et al., Trends Genet., 1992, 8 (12): 432-439), or the gin invertase from the bacteriophage, and the like.

As will be apparent to those skilled in the art, care must be taken that the recombinase recognition sequences used are different from, and not different from, any recombinase recognition sequences (as described above) present within the minicircle region (eg, in the transposition cassette) Enzyme can be recombined. An exemplary arrangement of recombinase and transposase recognition sequences on a parental plasmid according to the invention is described in U.S. Pat 7 displayed. The "rec" sequences are used to make the minicircle. The transposition cassette is restricted by the IR / DR sequences and contains the nucleic acid of interest (NOI), which is additionally flanked by two FRT sequences. After transposition, the nucleic acid of interest can be excised from the genome of the target cell by expression of Flp recombinase and recombination of the FRT sequences.

Parental plasmids according to the invention can be prepared by microbiological strategies and methods known to the person skilled in the art. As a starting point, for example, the plasmid pT2 / BH (Addgene accession number 26556, see 4 ) containing two IR / DR elements. In between is a multiple cloning site (MCS) into which one or more expression cassettes can be cloned. The resulting transposition cassette is flanked by restriction sites for BamHI and can be excised with this enzyme if there is no BamHI site within the transposition cassette. Alternatively, such a fragment can also be obtained by amplification by PCR from primers that are outside, but close to, the desired sequence fragment.

The resulting restriction fragment comprising the transposition cassette can now be cloned into a precursor plasmid containing the miniplasmid region as well as the recombinase recognition sequences of the desired parental plasmid. If necessary, the fragment may also be first cloned into another plasmid in order to use a precursor plasmid-compatible restriction enzyme. The cloning of a PCR fragment can be carried out either in the same way or via a shuttle vector for the cloning of PCR fragments. In all cases, the insertion site on the precursor plasmid must be in the area between the recombinase Detection sequences that does not contain the miniplasmid area. This creates the necessary for the formation of minicircle and miniplasmid Parentalplasmid.

Alternatively, the transposase recognition sequences with the intermediate MCS (an "empty" transposition cassette) can first be cloned from pT2 / BH into the precursor plasmid and then the desired expression cassette inserted between the IR / DR elements on the resulting empty parental plasmid.

This "insertion" may be by classical ligation of appropriate DNA ends (blunt or "sticky") or by recombination (as in Hartley, JL, Temple, GF, and Brasch, MA (2000) DNA Cloning Using in vitro Site-Specific Recombination, Genome Research 10, 1788-1795), whereby care must be taken that the recombination mechanisms used in the latter case do not interfere with those of cleavage into minicircle and miniplasmid.

Of course, a suitable parental plasmid can also be constructed on the basis of plasmids other than those mentioned. The restriction enzymes used may also be different. Crucial is the structure of the resulting parental plasmid, which must contain a transposition cassette between two suitably oriented recombinase recognition sequences.

Recombination of the parental plasmid can be in vitro or in vivo. An advantage of in vivo recombination in host cells is that the parental plasmid need not be purified if the expression of the corresponding recombinase can be induced in the host cells. The antigenic gene coding for the recombinase may be present in the host cell on another nucleic acid molecule. It is particularly advantageous if the recombinase gene is present on the parent plasmid within the miniplasmid region. Preferably, the expression of the recombinase is inducible, for example via a change in temperature or addition of a metabolite. After induction of recombinase expression in the host cell, the parental plasmid is cleaved into two supercoiled circular molecules, the minicircle and the miniplasmide, which can be separated from each other after cell lysis or already in the cell to encode the information encoded on the nucleic acid sequence.

The host cell can thus also be the target cell, whereby the expression control of the recombinase must be ensured in eukaryotic cells. Conversely, the transposase may also be inducible in prokaryotic cells, e.g. B. to modify them in the genome.

Alternatively, a minicircle may also be prepared by conventional restriction and ligation techniques from a parent plasmid. Here, the region of the plasmid which is to form the minicircle, for example, cut out of the plasmid by means of a restriction enzyme and ligated to a minicircle. The mini-circle can also be composed of several separate sections. It should be noted, however, that a minicircle produced in this way is generally not superhelical unless it is subsequently supercoiled in vitro.

In one embodiment of the invention, the minicircle and / or its parent plasmid may furthermore contain at least one identification sequence for purifying the minicircle, but also for other purposes. The identification sequence can be any sequence that allows the minicircles to be separated from the miniplasmids. In particular, these can be sequences which bind to a specific ligand and thus can complex the minicircle with the ligand. The ligand may be a DNA-binding protein or a nucleic acid. The purification can then z. B. on chromatographic methods, in particular directed against the identification sequence affinity chromatography using the immobilized ligand made. Suitable purification systems include, for. For example, triple helix affinity chromatography (THAC, see P. Wils et al., Gene Therapy 1997, 4 (4): 323-330), the lacO / lacI system (see J. Lundeberg et al., Genet. Techn. Appl. 1990, 7: 47-52), the repU / dso system (see A. Muller et al., Nucleic Acids Research 1995, 23 (11): 1894-1900) or systems from the bacteriophage λ repressor or bacteriophage 434 and the corresponding promoter for which the particular repressor is specific.

The identification sequence may be located in the minicircle or miniplasmid region of the parental plasmid prior to recombination. Preferred embodiments are those in which the identification sequence is in the minicircle region of the parental plasmid, or in particularly preferred embodiments in the transposition cassette, ie, together with the nucleic acid of interest between the transposase recognition sequences. If spontaneous and unintentional recombination between the transposase recognition sequences or the recombinase recognition sequences takes place in such a minicircle region of a parent plasmid (or its parent or precursor plasmids) during the production process, a defective product is formed, in which, in addition to the Nucleic acid sequence of interest also the identification sequence is deleted. By directed against the identification sequence affinity purification thus only intact minicircles are isolated. This avoids the problem of contamination of minicircle preparations by defective deletion products without a transposition cassette.

A minicircle may also contain more than one identification sequence. In this case, the identification sequences can be identical and can be present as direct repeats or repeats separated by a spacer. However, it is also possible to use two or more different identification sequences for different purposes. For example, a sequence capable of forming a triple helix with an oligonucleotide ligand may be present outside the transposition cassette to purify the minicircles prior to transfection, and at the same time contain a lacOs sequence within the transposition cassette to detect transposition into the genome after transfection or to measure their efficiency.

Illustrated at the Parental Plasmid 7 For example, the region within the rec sequences in which the nucleic acid of interest (NOI) is located is capable of accepting one or more identifying sequences for identifying a minicircle and separating it from the miniplasmid resulting from its production. The region between the IR / DR sequences in which the NOI is located is suitable for receiving one or more identification sequences with which a transposed genetic element is to be identified and optionally reisolated from the genome. The miniplasmic region outside the rec sequences is suitable for containing one or more identification sequences for identifying a miniplasmide and separating it from the minicircle resulting from the preparation.

CELLS

In another aspect, the invention also relates to a cell containing a minicircle according to the invention. This can be a cell from any species. In particular, prokaryotic and eukaryotic cells containing minicircles according to the invention are encompassed by the invention. In some embodiments, the cell is a vertebrate cell, preferably a human cell. In some embodiments, the cell is a stem cell, preferably an embryonic stem cell or inducible pluripotent cell.

Cells containing a minicircle according to the invention may be prepared by conventional transfection methods known to those skilled in the art. For example, chemical transfection using calcium phosphate (see FL Graham et al., Virohgy 1973, 52 (2): 456-467) can be used with dendrimers (see HL Fu et al., Journal of Control Release 2007, 124 (3)). : 181-188) or by means of cationic polymers (see EP 1505089 A1 ). Further methods are lipofection (see PL Felgner et al., PNAS 1987, 84 (21): 7413-7417), electroporation (see E. Neumann et al., EMBO J. 1982, 1 (7): 841-845), optical transfection (see M. Tsukakoshi et al., Applied Phyrics B-Photophysics and Laser Chemistry 1984, 35 (3): 135-140), magnetofection (see F. Scherer et al., Gene Ther. 2009, 9 (2) : 102-109) or impalfection (see TE McKnight et al., Nano Letters 2004, 4 (7): 1213-1219). Also, particle-based methods such as the gene gun can be used (see US 5,219,746 A ). Preferred methods are calcium phosphate transfection, lipofection and electroporation.

Furthermore, a minicircle according to the invention can be prepared directly in the target cell, as already mentioned above. In this case, the target cell is first transfected with a parent plasmid of the minicircle and then causes the expression of the corresponding recombinase in the target cell, so that the processing of Parentalplasmids is made possible by recombination to minicircle and miniplasmide. For this, the expression control of the recombinase must be ensured in eukaryotic cells, and a gene coding for the recombinase must be present in the target cell. The recombinase gene can be integrated into the genomic DNA of the target cell or else present on another nucleic acid molecule. It is particularly advantageous if the recombinase gene is present on the parent plasmid within the miniplasmid region.

METHOD FOR INSERTION MUTAGENIC OR TRANSFORMATION

In a further aspect, the invention includes a method for insertion mutagenesis or for genetic transformation of a eukaryotic target cell. The target cell may be any eukaryotic cell, but is preferably a vertebrate cell. Particularly preferred are human cells including, for example, embryonic stem cells or inducible pluripotent cells.

In the process, a minicircle according to the invention is provided in the target cell and the target cell is transfected with a vector containing a gene for a corresponding transposase specific for the minicircle transposase recognition sequences. In particular, the transposase may be an SB transposase or a transposase Piggy-Bac transposase. Especially preferred are vectors which code for the transposases SB10 or SB100X.

As described above, providing the minicircle in the target cell preferably involves transfecting the target cell with the minicircle. For this purpose suitable trans fektionsverfahren are known in the art, as also described above. Particularly preferred methods are calcium phosphate transfection, lipofection and electroporation. Alternatively to transfection with the minicircle, however, the minicircle can also be made directly in the target cell.

The vector containing the corresponding transposase gene (transposase vector) may be any type of vector of the art suitable for transfecting eukaryotic target cells. In particular, the vector may be a plasmid, a miniplasmid, a cosmid, a minicircle, a single, double or multiple stranded linear DNA molecule, an RNA, a protein, a viral vector or a cellular viral propagule intermediate, a virus-like particle (VLP) or a microorganism. Particularly preferred in the context of the invention is a minicircle, since this preserves the important advantage of not introducing any unnecessary foreign material into the target cell, which could, for example, lead to undesired immunogenic side effects. It is crucial that the expression control of the transposase is ensured in eukaryotic cells. Preferably, the expression of the transposase in the target cell is inducible, for example via a temperature change or the addition of a metabolite.

The transfection of the target cell with the transposase vector can occur before, simultaneously with or after the provision of the minicircle in the target cell. For example, the transposase vector and the minicircle can be introduced simultaneously into the target cell in a cotransfection. If the minicircle is generated within the target cell from the parental plasmid, the transfection with the transposase vector can take place before, at the same time as or after the transfection with the parent plasmid. An embodiment of the method is shown schematically in FIG 5 shown.

After both the minicircle and the transposase vector have been provided in the target cell, expression of the transposase gene is initiated in the target cell. This results in recombination of the transposase recognition sequences and integration of the nucleic acid sequence of interest in the genome of the target cell.

KITS

Furthermore, in a further aspect, the invention also includes kits for insertion mutagenesis or for genetic transformation of a eukaryotic target cell. A kit hereby comprises a minicircle according to the invention and a corresponding transposase vector which codes for a transposase specific for the transposase recognition sequences on the minicircle. In a particular embodiment, the transposase recognition sequences in the minicircle are from the SB transposon, while the transposase vector is derived from an SB transposase, e.g. B. SB10 or SB100X, encoded. In a further embodiment, the transposase can be coded on a minicircle or both - the transposase and the transposition cassette - each lie on different minicirclem molecules or the same minicirclemolecule.

The minicircle and / or the transposase vector can be stored dry in the kit, e.g. B. lyophilized, are present. Alternatively, they may be dissolved in a buffer and the solution may be liquid or frozen. Furthermore, they may be mixed or separate from one another in different containers. In addition to the minicircle and the transposase vector, the kit may also contain other ingredients useful for carrying out a method according to the invention, such as chemicals, reagents, buffers, solvents or media for carrying out the methods of the invention. In particular, the kit may contain reagents necessary for chemical transfection or lipofection. Some or all of the reagents may be in measured unit amounts, e.g. B. to minimize the pipetting effort of the user to a minimum.

In addition, the kit may also contain descriptions of the minicircle and / or transposase vector, such as vector maps or sequences. Likewise, instructions for carrying out the relevant embodiment of the invention may be part of the kit.

Examples

EXAMPLE 1:

Construction of a parental plasmid (GFP-parentalplamide) for minicircles comprising a transposition cassette for GFP

The plasmid pCMV-GFP (3487 bp, Item No. PF463, Plasmid Factory, Bielefeld, DE) containing a Green Fluorescent Protein (GFP) expression cassette is used as a starting material for targeted amplification of the GFP expression cassette contained thereon. The two Primer (Forward: 5'-CGCCGAATTCGCCGGTTAATTAλATCAATTACGGGGTCATTAG TTCATAGCCCATATA G-3 ', see also 1E ; and reverse: 5'-GCGCGAATTCCGGCGTT AATTAACCCCTGAACCTGAAACAAAATGAATGCAATTGTTG-3's. also 1F ) generate by PCR an EcoRI fragment comprising said expression cassette. The fragment is purified by agarose gel electrophoresis and a NucleoSpin Extract II ^® Gel Extraction Kit (Macherey-Nagel, Düren, DE).

The plasmid pT2 / BH (Addgene accession number 26556, see 4 ) contains the left IR / DR element Lmut44 (see also 6A ; SEQ ID NO: 7) and the right IR / DR element Rmut13delta13 (see also 6B ; SEQ ID NO: 8). In between is a multiple cloning site (MCS) into which the amplified expression cassette can be cloned. This plasmid is cut with the enzyme EcoRI (Article No. R0101L, NEB, Frankfurt, DE) in the MCS and dephosphorylated with alkaline phosphatase (Article No. M0290L, NEB, Frankfurt, DE). Subsequently, the EcoRI fragment is ligated with the GFP expression cassette with T4 ligase (Item No. M0202L, NEB, Frankfurt, DE) into the linearized vector to obtain the plasmid pT2-IR-GFP.

After amplification and preparation of the plasmid, the transposition cassette comprising the IR / DR elements and between them the expression cassette is excised from the plasmid with BamHI (Article No. R0136L, NEB, Frankfurt, DE). The overhanging single stranded ends are filled in with Klenow polymerase (DNA polymerase I, article no. M0210L, NEB, Frankfurt, DE) and the fragment is purified as described above.

The precursor plasmid used to accommodate the transposition cassette (see P. Mayrhofer et al., J. Gene Med. 2008, 10 (11): 1253-1269) contains an MCS flanked by parA resolvase recombinase recognition sequences , two identification sequences and an expression cassette of the parA resolvase outside the recombinase recognition sequences. This plasmid is cut with the enzyme EcoRV (Article No. R0195L, NEB, Frankfurt, DE) in the MCS and dephosphorylated with alkaline phosphatase (Article No. M0290L, NEB, Frankfurt, DE). Subsequently, the transposition cassette with T4 ligase is ligated into the linearized vector to give the GFP parental plasmid pP-IR-GFP ( 3a ; Cloning steps in 5 , right).

EXAMPLE 2

Construction of another parental plasmid (GFP parental plasmid) for minicircles comprising a transposition cassette for GFP

The plasmid pCMV-GFP (3487 bp, Item No. PF463, Plasmid Factory, Bielefeld, DE) containing a Green Fluorescent Protein (GFP) expression cassette is used as a starting material for targeted amplification of the GFP expression cassette contained thereon. The two primers (Forward: 5'-CGCCGAATTCGCCGGTTAATTAAATCAATTACGGGGTCATTAG TTCATAGCCCATATATG-3 ', see also 1E ; and reverse: 5'-GCGCGAATTCCGGCGTT AATTAACCCCTGAACCTGAAACATAAAATGAATGCAATTGTTG-3's. also 1F ) generate by PCR an EcoRI fragment comprising said expression cassette. The fragment is purified by agarose gel electrophoresis and a NucleoSpin Extract II ^® Gel Extraction Kit (Macherey-Nagel, Düren, DE).

The plasmid pT2 / BH (Addgene accession number 26556, see 4 ) contains the left IR / DR element Lmut44 (see also 6A ; SEQ ID NO: 7) and the right IR / DR element Rmut13delta13 (see also 6B ; SEQ ID NO: 8). In between is a multiple cloning site (MCS) into which the amplified expression cassette can be cloned. This plasmid is cut with the enzyme EcoRI (Article No. R0101L, NEB, Frankfurt, DE) in the MCS and dephosphorylated with alkaline phosphatase (Article No. M0290L, NEB, Frankfurt, DE). Subsequently, the EcoRI fragment is ligated with the GFP expression cassette with T4 ligase (Item No. M0202L, NEB, Frankfurt, DE) into the linearized vector to obtain the plasmid pT2-IR-GFP.

After amplification and preparation of the plasmid, the transposition cassette comprising the IR / DR elements and between them the expression cassette is excised from the plasmid with BamHI (Article No. R0136L, NEB, Frankfurt, DE). The overhanging single stranded ends are filled in with Klenow polymerase (DNA polymerase I, article no. M0210L, NEB, Frankfurt, DE) and the fragment is purified as described above.

The precursor plasmid pP11, which serves to accommodate the transposition cassette (see P. Mayrhofer et al., J. Gene Med. 2008, 10 (11): 1253-1269, but now without MCS, without spacer and with one instead of two identification sequences) an MCS flanked by parA resolvase recombinase recognition sequences and a parA resolvase expression cassette outside of the recombinase recognition sequences. This plasmid is cut with the enzyme PmeI (Article No. R0560L, NEB, Frankfurt, DE) in the MCS and dephosphorylated with alkaline phosphatase (Article No. M0290L, NEB, Frankfurt, DE). Subsequently, the transposition cassette with T4 ligase is ligated into the linearized vector to give the GFP parental plasmid pP11-IR-GFP ( 3b ) to obtain.

EXAMPLE 3

Construction of a parental plasmid (SB10 parental plasmid) for minicircles comprising an SB10 expression cassette

The plasmid pSB10 (4732 bp, Addgene accession number 24551), which codes for the Sleeping Beauty Transposase SB10, is labeled with the restriction enzymes SalI (Article No. R0138L, NEB, Frankfurt, DE) and EcoRI (Article No. R0101L, NEB, Frankfurt, DE) to produce a fragment comprising the expression cassette. The overhanging single stranded ends are filled in with Klenow polymerase (DNA polymerase I, article no. M0210L, NEB, Frankfurt, DE) and the fragment is purified as described above.

The precursor plasmid of Example 1 (see P. Mayrhofer et al., J. Gene Med. 2008, 10 (11): 1253-1269) contains an MCS flanked by parA resolvase recombinase recognition sequences, two identification sequences, and one Expression cassette of the parA resolvase outside of the recognition sequences. This plasmid is cut with the enzyme EcoRV (Article No. R0195L, NEB, Frankfurt, DE) in the MCS and dephosphorylated with alkaline phosphatase (Article No. M0290L, NEB, Frankfurt, DE). Subsequently, the transposition cassette is ligated with T4 ligase into the linearized vector to generate the SB10 parental plasmid pP-SB10 (cloning steps in FIG 5 , left).

Instead of the precursor plasmid of Example 1 (P.Mayrhofer et al., J. Gene Med. 2008, 10 (11): 1253-1269), pP11 of Example 2 can also be used to accommodate the SB10 transposition cassette. For this purpose, this plasmid is cut with the enzyme PmeI (Article No. R0560L, NEB, Frankfurt, DE) in the MCS and dephosphorylated with alkaline phosphatase (Article No. M0290L, NEB, Frankfurt, DE). Subsequently, the transposition cassette is ligated with T4 ligase into the linearized vector to obtain the SB10 parental plasmid pP11-SB10.

Instead of the plasmid pSB10, the plasmid pSB100X (L. Mátés et al., Nat. Genet. 2009, 41: 753-761), which codes for the Sleeping Beauty Transposase SB100X, can likewise be used in this process. The SB100X parental plasmid is obtained as pP-SB100X (2 days).

EXAMPLE 4

Production of minicircles from GFP or SB10 parental plasmids

The cultivation is carried out at 37 ° C. in an MBR bioreactor (MBR BIO REACTOR, Switzerland) with a total volume of 7 l with a filling quantity of 5 l. The adjustment of the pH to pH 7 is carried out with 2 M sodium hydroxide solution and 2 M phosphoric acid. The flow rate of the air is set to 5 l / min. The oxygen concentration (60%) is controlled by varying the stirrer speed in the range of 500 to 2000 per minute. LB medium is used without the addition of antibiotics. The procedure is carried out with both pP-IR-GFP and pP-SB10 parental plasmids.

The bioreactor is inoculated with 50 ml of a preculture of E. coli K12 transformed with parental plasmid and cultured for approximately 15 hours at 28 ° C. The precultures are cultured under selective conditions with the addition of 75 mg / ml kanamycin. The LB medium of the precultures is enriched with glucose to prevent premature expression of the parA resolvase, which is under the control of the P _BAD promoter.

The expression of the parA resolvase is induced at an OD ₆₀₀ between 3.5 and 5.0 by the addition of L-arabinose to the medium. After one hour of further growth, the cells are harvested by centrifugation for 6 min at 9039 g, transferred to storage bags, frozen and stored at -20 ° C before the recombination products are purified.

During cultivation in the bioreactor, samples are taken with an automatic sampler and stored at 4 ° C for further analysis. The OD ₆₀₀ is measured and the plasmids are to determine purified (NucleoBond ^® PC 100, Macherey-Nagel, Duren) plasmid yield and the recombination efficiency.

The recombination minicircle and miniplasmid are using commercially available Plasmidisolierungskits (NucleoBond ^® PC 10 000 Macherey & Nagel, Düren) purified from the resulting biomass.

For the isolation of the minicircle from the mixture of minicircle and miniplasmid we used a specific affinity chromatography. For this purpose, a biotinylated repressor of the lactose operon is coupled to Streptavidin Sepharose High Performance (GE Healthcare) (see Mayrhofer et al., 2008).

5 ml of this chromatographic matrix is filled into a ZK 16 chromatography column and equilibrated with the five-fold column volume of 50 mM Tris pH 8, 400 mM NaCl. The mixture of recombination products (1 mg / ml in 50 mM Tris pH 8, 400 mM NaCl) is then applied to the column material in an AKTA system (GE Healthcare) at a flow rate of 0.5 ml / min. The column is washed with 50 mM Tris pH 8, 400 mM NaCl at a flow rate of 1 ml / min until the UV _{260 nm} signal detected on the instrument drops to a stable baseline. The minicircle DNA is then eluted with 50 mM Tris pH8, 500 mM NaCl, 5 mM IPTG, then the column is washed with 50 mM Tris pH 8, 1 M NaCl and 50 mM Tris pH 8 and re-equilibrated for further use.

Precipitation removes the DNA from the high salt mixture and finally resuspends in water.

The DNA of the MC-IR-GFP and MC-SB10 are each adjusted to a concentration of 1 mg / ml.

Instead of the parent plasmid pP-SB10, alternatively, the parent plasmid pP-SB100X can be used as the starting material. This will get the minicircle MC-SB100X.

In addition, instead of the parent plasmid pP-IR-GFP, alternatively the parent plasmid pP11-IR-GFP can be used as starting material. The minicircle MC-IR-GFP is obtained with one instead of two tag sequences.

EXAMPLE 5

Cotransfection of target cells with GFP and SB10 minicircles and transposition of the expression cassette

Jurkat cells (human T cell leukemia line, DSMZ) are cultured in RPMI 1640 medium in the presence of FCS (fetal calf serum, PAA, Linz) at 37 ° C and 5% CO ₂ .

For transfection, the cells are resuspended in serum-free medium to give a cell density of approximately 7.5 × 10 ⁵ cells / ml. 400 μl of this cell suspension are mixed with 20 μg of MC-IR-GFP and 10 μg of MC-SB10 and mixed thoroughly by gently inverting. The electroporative gene transfer is then carried out with a Bio-Rad Gene-Pulser II (Bio-Rad, Foster City, CA, USA). For this purpose, the cells are placed in corresponding cuvettes with an electrode gap of 4 mm and electroporated at a set voltage of 200 V and a capacity of 1600 μF. The cuvette with the cell suspension is immediately stored on ice for 5 min, then the cells are pipetted into culture dishes with prewarmed culture medium (37 ° C., with FCS) and cultivated further as described above.

The cotransfection of the two DNAs (20 μg MC-IR-GFP and 10 μg MC-SB10) leads to the constitutive expression of the SB10 transposase in the target cells. After reaching a sufficient amount of SB10 protein in the cell, this interacts with the transposition-relevant DNA structures and the transposition takes place. Transfection omitting MC-SB10 (ie without transposase expression) leads to no transposition, but to the expression of GFP from the episomal vector.

The detection and quantification by the GFP expression of fluorescent cells occurs at defined times, eg. B. 24 h, 48 h, etc. after transfection with a commercially available fluorescence microscope with a corresponding filter set. The determination of the efficiency of the transposition is based on the assumption that, in addition to a time-deficient transient GFP expression of non-transposed sequences, GFP expression of the expression units integrated into the chromosome of the target cell remains constant over time. As a measure of the transposition efficiency, therefore, the constant basal level of GFP expression is used, which is measured after the transient expression has subsided.

Instead of the minicircle MC-SB10, alternatively 10 μg of the minicircle MC-SB100X can be mixed with 20 μg MC-IR-GFP in this method and the procedure can be analogous.

In addition, only one of the two cotransfected DNA can be a minicircle, while the other is a standard plasmid (see also Example 6).

The processes of Examples 1-5 are also schematically shown in FIG 5 shown.

Transposition after cotransfection can be controlled by the use of inducible promoters which are functional in eukaryotic cells. In the example shown here it was unregulated.

EXAMPLE 6

Comparison of transposition efficiency between transposon minicircle and transposon plasmid

The experiment of Example 5 is repeated, but instead of the minicircle MC-IR-GFP, an equimolar amount of the comparison plasmid pT2-IR-GFP (see Example 2) is transfected. The transposition efficiency is compared with that of Example 5.

The efficiency of transposition compared between plasmid-based transposition (transfection with pP-IR-GFP) and minicircle-based transposition (transfection with MC-IR-GFP, see Example 5) shows that the number of cells transformed by minicircle transposons is higher than that whose transposon is encoded on a plasmid. Efficiency is not enhanced by the fact that SB10 transposase is minicircle-based rather than plasmid-based, provided that equimolar amounts of MC-SB10 and pSB10 DNA per cell are compared.

This is followed by a sequence protocol according to WIPO St. 25. This can be downloaded from the official publication platform of the DPMA.

Claims (12)

A minicircle comprising: a) at least one nucleic acid sequence of interest, and b) transposase recognition sequences on both sides of the nucleic acid of interest, characterized in that the transposase recognition sequences can be recognized by a specific transposase and used for transposition, wherein the specific transposase is preferably a Sleeping Beauty Transposase or a PiggyBac Transposase, wherein the minicircle comprises no resistance gene and no selection marker for amplification in prokaryotic cells , The minicircle of claim 1, wherein the at least one nucleic acid sequence of interest comprises an expression cassette having at least one gene, at least one isolator sequence or a combination thereof. The minicircle of any one of claims 1 or 2, wherein the minicircle comprises at least one identification sequence within the region between the transposase recognition sequences, or wherein the minicircle comprises at least one identification sequence out of the region between the transposase recognition sequences. The minicircle of any one of claims 1-3, wherein the minicircle comprises a pair of recombinase recognition sequences located on either side of the nucleic acid of interest within the transposase recognition sequences. The minicircle of any of claims 1-4, wherein the minicircle is an annular, supercoiled DNA molecule. A cell comprising the minicircle of any one of claims 1-5, wherein the cell is not a human embryonic stem cell. A method of insertional mutagenesis or genetic transformation of a eukaryotic target cell that is not a human embryonic stem cell derived from destruction of embryos comprising the method: a) providing a minicircle comprising a nucleic acid sequence of interest from any one of claims 1-5 within the target cell; b) transfecting the target cell with a vector comprising a nucleic acid sequence encoding a transposase specific for the minicircle transposase recognition sequences; c) expression of the transposase from the vector; and d) recombination of the nucleic acid sequence of interest with a nucleic acid of the target cell, in which i) steps a) and b) can be carried out simultaneously in a Kotransfekon; or ii) wherein step a) can be carried out prior to step b); or iii) wherein step a) after step b) can be performed. The method of claim 7, wherein the providing from step a) is achieved by transfecting the target cell with the minicircle or by generating the minicircle within the target cell. The method of any of claims 7 or 8, wherein the target cell is a vertebrate cell, and is preferably a human cell. A cell obtained by a method according to any one of claims 7-9. A kit for insertion mutagenesis or genetic transformation of a eukaryotic target cell comprising: a) the minicircle of any one of claims 1-5, and b) a vector encoding a transposase specific for the minicircle transposase recognition sequences. The kit of claim 11, wherein for the transposase recognition sequences in minicircle a) a Sleeping Beauty transposase is specific and the transposase encoded by vector b) is the Sleeping Beauty Transposase, the Sleeping Beauty Transposase preferably being the Hyperactive Sleeping Beauty Transposase SB100X.

Images