DE19956513A1 - Resolvase-catalyzed, sequence-specific DNA recombination in eukaryotic cells - Google Patents

Abstract

The invention relates to a method for the sequence-specific recombination of DNA in eukaryotic cells, comprising the following steps: introducing a first DNA sequence which contains a res sequence into a cell, introducing a copy of the first DNA sequence and performing the sequence-specific recombination using the influence of a resolvase or its derivative. The invention also relates to a method for testing topoisomerase inhibitors in a eukaryotic cell, comprising the following steps: introducing a first DNA sequence according to SEQ ID NO:1 into a cell, introducing a copy of the first DNA sequence according to SEQ ID NO:1 in direct orientation, introducing a wild-type resolvase and adding the topoisomerase inhibitors which are to be tested.

Description

The present invention relates to a method for sequence-specific recombination of DNA in eukaryotic cells, comprising introducing a first DNA into a cell Sequence, inserting a copy of the first DNA sequence and performing the sequence specific recombination by the action of a resolvase or its derivative. The The present invention further relates to a method for testing topoisomerase inhibitors in a eukaryotic cell comprising introducing a first DNA sequence into a cell according to SEQ ID NO: 1, the insertion into a cell according to a copy of the first DNA sequence SEQ ID NO: 1 in direct orientation, the introduction of a wild-type resolvase, and the addition of topoisomerase inhibitors to be tested.

Controlled manipulation of eukaryotic genomes is an important method for Research into the function (s) of certain genes in the living organism. Furthermore, plays they play a role in gene therapy procedures in the medical field. The production of transgenic animal strains, the modification of genes or gene segments (so-called "genes targeting ") and the targeted integration of foreign DNA into the genome of higher eukaryotes are of particular importance in this context. By characterizing and These technologies could be used in application of sequence-specific recombination systems have been significantly improved recently.

Conservative, sequence-specific DNA recombinases are divided into two families. Members of the first, the so-called "integrase" family catalyze the separation and Relinking of DNA strands between two defined nucleotide sequences that are in the hereinafter referred to as recombination sequences. These can either be on two different or on a DNA molecule. Then there is an inter- or intramolecular recombination. In the latter case, the result of the reaction depends on the  the respective arrangement of the recombination sequences from one another. Are they Recombination sequences in inverted, i.e. H. opposite, orientation to each other, there is an inversion of the DNA segment in between. Are they Recombination sequences as direct, i.e. H. rectified, repetitions on a DNA Substrate, deletion occurs. In intermolecular recombination, i.e. H. if the two Recombination sequences are placed on two different DNA molecules, it can fusion of the two DNA molecules. While members of the Integrase family usually catalyze both intra- and intermolecular recombination Recombinases of the second family, the so-called "invertases / resolvases", only for the intramolecular Capable of recombination. It should be noted that the "invertases / resolvases" can only perform intramolecular recombination if the Recombination sequences either as inverted repeats (invertases) or as direct ones Repetitions (resolvases) are present. The action of the invertases always leads to Inversion of a DNA segment lying between the recombination sequences, whereas Resolvases always cause a deletion.

The recombinases currently used mainly to manipulate eukaryotic genomes belong to the integrase family. These are the bacteriophage Cre recombinase P1 and the yeast Flp recombinase; see. Müller, U. (1999) Mech. Develop., 82, pp. 3. The Recombination sequences to which Cre recombinase binds are called loxP. loxP is a 34 bp nucleotide sequence, each inverted from two 13 bp long Nucleotide sequences and an intermediate 8 bp long spacer; see. Hoess, R. et al. (1985) J. Mol. Biol., 181, pp. 351. The binding sequences for Flp, referred to as FRT, have a similar structure, but differ from loxP; see. Kilby, J. et al. (1993) Trends Genet., 9, pp. 413. The recombination sequences can therefore not against each other be exchanged, d. H. Cre cannot recombine FRT and FLP loxP sequences. Both recombination systems are active over long distances, i. H. the one to invert or deleting DNA segment flanked by two loxP or FRT sequences can be several 10,000 base pairs (kb) long.

With these two systems z. B. tissue-specific recombination in the mouse model, chromosomal translocation in plants and animals, and a controlled induction of the Gene expression achieved; see. Review article by Müller, U. (1999) Mech. Develop., 82, pp. 3rd  

For example, the DNA polymerase β gene was deleted in certain mouse tissues; see. Gu, H. et al. (1994) Science, 265, pp. 103. Another example is the specific activation of Oncogene from the SV40 DNA tumor virus in the mouse eye lens, causing tumor formation in led exclusively to these tissues. The Cre-loxP strategy was also implemented in the Connection used with inducible promoters. As a result, z. B. Expression the recombinase with an interferon-inducible promoter, resulting in the deletion of a certain genes in the liver and not - or only to a small extent - in other tissues led; see. Kühn, R. et al. (1995) Science, 269, pp. 1427.

So far, two members of the invertase / resolvase family have been manipulating eukaryotic cells Genomes have been used. A mutant of invertin gin from bacteriophage Mu can be without Cofactors catalyze the inversion of a DNA fragment in plant protoplasts. Indeed this mutant was found to be hyper recombinant, i. H. also on others than theirs natural recombination sequences catalyzed DNA strand separations; see. Maeser, S. & Kahmann, R. (1991) Mol. Gen. Genet., 230, pp. 170. This leads to unwanted, partial lethal recombination events in the genome of plant protoplasts. The plasmid-encoded β-recombinase from Streptococcus pyogenes catalyzes recombination between two directly repeated recombination sequences in mouse cell cultures, leading to excision of the Segment leads. In addition to the deletion, however, inversion was also detected; see. Diaz, V. et al. (1999) J. Biol. Chem., 274, pp. 6634. This is the controlled use of this Strong members of the invertase / resolvase family for manipulating eukaryotic genomes restricted or unsuitable.

The γδ resolvase is the prototype of the resolvase subfamily and is characterized by the bacterial Transposon γδ (also referred to as Transposon 1000) encoded. The γδ resolvase is phylogenetically closely related to the Tn3 resolvase. In nature the γδ transposon comes as Part of the F factor episomal in the Escherichia coli bacterium. The biological The function of a transposon-encoded resolvase is usually to resolve the to catalyze so-called co-integrates by sequence-specific DNA recombination. The KO- Integrat consists of two directly repeated copies of the transposon and is transient through the process of transposition ("jumping" of the mobile genetic element into one other genomic locus) within a bacterial cell. The resolvase catalyzes the Recombination between two identical recombination sequences, commonly known as res  be designated. Res comprises 145 nucleotides and, like the resolvase gene, is more natural Part of the transposon. In general, class II transposons consist of one res, a tnpA gene coding for the transposase and a tnpR gene coding for the Resolvase encoded. When the co-integrate is formed, the transposon is doubled, so that two copies of the transposon with the res sequence, the tnpA gene and the tnpR gene as there is a direct repetition; see. Review by Grindley, N.D.F. (1994) Nucl. Acids and Mol. Biol., 8, pp. 236. The resolvase has a total of six binding sites in one res. H. each binding site is bound by a single resolvase molecule. Since the wild type Resolvase in solution (and thus also intracellularly) as a homodimer bind a total of 3 Dimers to a res. Res therefore consists of three dimer binding sites, designated I, II and III. Two of the three binding sites (II and III) are called auxiliary sequences. The occupation these sequences by two resolvase dimers is a necessary prerequisite for Activation of the actual recombination reaction, which ultimately leads to DNA Strand exchange leads. This takes place in the center of the imperfect palindromic Binding site I in res, the so-called "crossover" region, and is bound by the attached one Resolvase dimer catalyzed.

Members of the resolvase family require negative torsional stress (so-called (-) DNA Supercoiling) of the DNA substrate to catalyze recombination between two res can. An additional protein cofactor is not required for the reaction. In addition to (-) DNA Supercoiling is another prerequisite for recombination that both res as direct Repeats occur on a DNA molecule. Resolvases can therefore only intramolecular deletion of a DNA segment located between two res sequences catalyze. The reason for this restriction regarding localization and orientation of the res lies in the formation of a specific synaptic complex, the so-called synaptosome, between at least six resolvase dimers and the two, now paired, res copies. The Formation of the synaptosome is a necessary prerequisite for activation in any case the recombination reaction by the resolvase. A comprehensive overview of the Resolvase-catalyzed recombination reaction is e.g. B. in Grindley (1994).

There are resolvase mutants, e.g. B. the Tn3 resolvase and the γδ resolvase, regardless of the presence (-) supercoilings in the DNA substrate molecule, recombination can catalyze between two complete res. The molecular basis for this phenotype is  not yet clearly clarified. Apparently the changed dimerization properties play the mutant resolvase monomers play an important role here; see. Arnold et al. (1999) EMBO J., 18, pp. 1407. In this case, as with the reaction with wild-type resolvase, it occurs observed, for the deletion of the DNA segment lying between the res sequences.

Another special feature of the mutant resolvase is the presence of a (-) supercoiling in that these mutant resolvases can be recombined without the two auxiliary sequences II and III and can only be carried out with the palindromic binding site I in res. It follows that the recombination can also take place between two DNA segments that contain only the binding sites I and are therefore much shorter than that complete res. The recombination is independent of the location and Orientation of the recombination sequences, i.e. H. the two partner sequences can be on one or lie on two different DNA molecules and as direct or inverted Repetitions occur. As a result, intermolecular recombination can occur which results in a fusion of the two different DNA molecules, or, in the case of the intramolecular reaction depending on the respective orientation of the binding sites I to each other, d. H. as direct or inverted repetitions, for deletion or inversion of a DNA segment lying between the binding sites I.

The starting point, which is important in this context, results from the known properties of the resolvase mutants described and the wild-type enzyme:

• 1. Unmodified resolvase is strictly dependent on the non-relaxed state of the DNA Substrate that must also carry two complete res sequences as direct repeats. There is only intramolecular recombination and the resulting deletion of one DNA segments.
• 2. The mutated variants, e.g. B. γδ-resolvase ^E124Q or γδ-resolvase ^{E102Y, E124Q,} however, the recombination ^reaction to relaxed, z. B. linear, catalyze substrate molecules, but the complete res sequences (I, II and III) must be on the same DNA molecule in direct repetition. Even with the mutant resolvases, the complete res sequences only result in intramolecular recombination and the resulting deletion of a DNA segment.
• 3. However, the mutant resolvase can also perform the recombination if only the res- Sequences with the binding site I are present, regardless of the torsional tension of the DNA,  d. H. with or without (-) supercoiling. The localization of the res sequences on a DNA The molecule and the orientation are not important. Therefore, the resolvase mutants perform both intra- and intermolecular recombinations.

Intracellular genomic and episomal DNA is found in higher eukaryotes except for a few areas in a topologically relaxed state, i.e. H. there are hardly any vacancies present (-) superhelical turns in the DNA, z. B. the wild-type resolvase for the Could use catalysis of recombination. Therefore, the resolvase has not been used for Recombination used in eukaryotic cells.

The topological state of genomic DNA in the cell is determined by a special one Regulates the enzyme class called topoisomerases. If the enzymatic activity of these proteins, for example through mutations or the action of inhibitors, topological tension accumulates in genomic DNA as a result of other DNA Transactions such as transcription; see. Review article by Wang, J.C. (1996), Annu. Rev. Biochem., 65, pp. 635. There are a number of substances that make up the intracellular Can selectively inhibit topoisomerase activities. A special class of these substances is z. B. represented by camptothecin and its derivatives, which are already in use as anti-cancer agents are of therapeutic use and selectively inhibit type I topoisomerases; Review article by Chen and Liu (1994), Annu. Rev. Pharmacol. Toxicol., 34, pp. 191. A major problem in the development of novel and more effective topoisomerase inhibitors However, at the moment there is still no simple test system, either in cell culture or in living organism, such as. B. the mouse, to determine the level of inhibition and / or Tissue specificity of such a substance.

Thus, it is an object of the present invention to be simple and controllable To provide the recombination system and the necessary tools. Further It is an object of the present invention to be simple and reliable To provide a test system for eukaryotic topoisomerase inhibitors.

The tasks are solved by the subject defined in the patent claims.

The method according to the invention is illustrated by the following figures.  

FIG. 1A shows a schematic representation of the γδ-resolvase-catalyzed recombination reaction and FIG. 1B shows a schematic representation of the γδ-res sequence. (A) A circular, topologically relaxed DNA is shown, which carries two res as direct repetitions (black arrows). Binding the resolvase dimers to each res leads to the formation of the synaptic complex, the synaptosome, in which the DNA strand breaks are catalyzed (indicated by open arrowheads). Both synapse formation and DNA strand exchange require negative superhelical tension on the DNA substrate. After the strand exchange, religation and release of the recombination product, a dimeric, simply linked DNA catenane, occurs. (B) The structure of a res. This consists of three separate binding sites for each resolvase dimer (represented by inverted black arrows as the respective half-sequence and marked by I to III). The distance between the centers of the three binding sites is given in base pairs (bp) and marked by the thin arrows under the res. The actual DNA strand exchange takes place during the recombination at the center of the binding site I (marked by open arrowheads).

FIG. 2A shows a schematic illustration of the γδ-resolvase expression vectors and FIG. 2B shows a schematic illustration of the substrate vector and the product vector resulting from the recombination. (A) A schematic representation of the eukaryotic expression vectors for the wild-type γδ resolvase (pPGKγδ) and their mutants γδ ^{E102Y, E124Q} (pPGKγδ102) and γδ ^E124Q (pPGKγδ124) is shown. The position and nature of the respective mutation in the resolvase gene is marked. There is a version with and one without a nuclear localization ^sequence (NLS) at the C-terminal end of the gene of the wild type and the two mutants, γδ ^{E102Y, E124Q} and γδ ^E124Q . The genes are expressed by the phosphoglycerate kinase promoter (P-PGK). This promoter is active in all eukaryotic cells examined to date. (B) A schematic representation of the substrate vector is shown, with which recombination is tested in eukaryotic cells. pCH-RNRZ denotes the non-recombined vector. In addition to the ampicillin resistance gene for amplification in E. coli, it contains the early SV40 promoter, which before the recombination cassette consists of res, the gene for neomycin (neo) resistance, res and the lacZ gene, which codes for the β-galactosidase. The location and respective orientation of the two PCR primers P-SV40prä and P-Seqanti is indicated by small arrows above and below the vector. The recombination between the two res leads to the deletion of the intermediate neo gene, the simultaneous loss of a res copy and the expression of the lacZ gene. The vector resulting from the recombination is called pCH-RZ. The relevant interfaces in both vectors for the restriction enzyme BamHI are additionally marked.

Fig. 3 shows a schematic representation of a Western analysis.

After the introduction of the respective vector, as indicated, into CHO cells, cell lysates were prepares and proteins in an SDS polyacrylamide gel electrophoresis according to their Molecular weight separated. The presence of the γδ resolvase and its derivatives was confirmed visible through polyclonal mouse antibodies directed against wild-type γδ resolvase made. The position of γδ resolvase in the gel is marked by an arrow. Lane 1, control with Cre vector; Lane 2, control with cleaned resolvase.

Fig. 4 shows the quantitative analysis of transient transfection experiments shows co-in CHO cells.

The respective γδ expression vectors were separated using the substrate pCH-RNRZ Lipofection introduced into CHO cells. The enzymatic activity of the β-galactosidase was increased after 72 hours in cell lysates determined photometrically on the protein content of the lysates standardized and statistically evaluated. A bar chart is shown with the respective Standard deviations from the mean.

Fig. 5 shows a schematic representation of the detection of the deletion in reporter H5 by PCR and subsequent Southern hybridization after separation of the DNA molecules in agarose gels (1.2% w / v). A Southern analysis of the PCR products achieved by the respective expression vectors is shown. The term unrec. or rec. indicate the position of the PCR products in the gel that result from the non-recombined (pCH-RNRZ) or recombined vector (pCH-RZ).

Fig. 6A shows a representation of the quantitative evaluation of the influence of the topoisomerase inhibitor camptothecin at recombination with wild-type γδ resolvase. The term "transposon" used here denotes a mobile genetic element which comprises at least one recombination sequence, a transposase and a resolvase and which is derived from bacteria.

The term "resolvase" as used herein means an enzyme derived from a transposon is encoded, and that the resolution of the co-integrate structure of the transposon by sequence catalyzes specific recombination like its derivatives, e.g. B. mutant resolvases.

The term “derivatives” used here denotes nucleic acid or amino acid sequences, the one or more deletions, substitutions, additions, insertions and / or inversions exhibit.

The term "direct orientation" or "direct repetition" used here means that sequences are each in the 5'-3 'direction.

The term "transformation" or "transform" as used herein means anything Introducing a nucleic acid sequence into a cell. The introduction can e.g. B. a transfection or be lipofection, by the calcium phosphate method, electroshock method or an oocyte injection can be carried out.

The term "vector" used here means naturally occurring or artificial created constructs for the uptake, multiplication, expression or transfer of Nucleic acids, e.g. B. plasmids, phagemids, cosmids, artificial chromosomes, bacteriophages, Viruses or retroviruses.

One aspect of the present invention relates to a method for sequence-specific Recombination of DNA in eukaryotic cells comprising simultaneous or sequential introduction into a cell of two copies of a first DNA sequence and that Carrying out the sequence-specific recombination by the action of a resolvase. A method is preferred in which a copy of the first DNA sequence in direct Orientation on the same DNA molecule as the other copy is introduced. In particular a method is preferred, the first DNA sequence being a res sequence according to SEQ ID NO: 1 or SEQ ID NO: 2.

To carry out the method according to the invention, the first DNA sequence in addition to the Recombination sequence include other DNA sequences that integrate into one allow desired target location in the genome of the eukaryotic cell. This integration is ongoing  about homologous recombination, which is caused by internal recombination mechanisms is conveyed. To do this, the other DNA sequences must be homologous to the target DNA and both 3 'and 5' of the two direct repeats res sequences lie. What is the degree of homology and how long are the respective 3 'and 5' sequences must have a sufficient probability for the homologous recombination expires is known to the person skilled in the art; see. Review article by Capecchi, M. (1989) Science, 244, pp. 1288.

The inventive method can, for. B. be used to delete the DNA segment lying between the directly oriented recombination sequences in eukaryotic cells in an intramolecular recombination. The recombination sequences are directly oriented if the sequences are inserted in the 5'-3 'direction. Are the recombination sequences, e.g. B. res with the binding sites I, II and III (SEQ ID NO: 1) or res with the binding site I, via homologous recombination each in an intron sequence 5 'and 3' of an exon on the same DNA molecule and the recombination by the action of the resolvase, e.g. B. the γδ-resolvase ^E124Q or γδ-resolvase ^{E102Y , E124Q} , the exon is deleted. This can, for. B. the polypeptide encoded by the corresponding gene lose its activity or function or the transcription is stopped by the deletion so that no (complete) transcript is produced. In this way, e.g. B. the biological function of the encoded polypeptide can be examined. The resolvase or its derivative can carry a nuclear localization sequence at the C-terminal end, which ensures better intracellular transport of the recombinase into the cell nucleus.

Furthermore, the method according to the invention can be used to in eukaryotic cells in the case of an intramolecular recombination, that between the not directly oriented Recombination sequences to invert lying DNA segment. To do this, the Recombination sequences res with the binding site I in reverse orientation on the same DNA molecule.

The method according to the invention can also be used to in eukaryotic cells to perform an intermolecular recombination. To do this, the Recombination sequences res with the binding site I regardless of the orientation  two different DNA molecules. The recombination then leads to a fusion of the two DNA molecules at the binding sites I.

However, the first DNA sequence can also comprise further nucleic acid sequences which include a encode one or more polypeptide (s) of interest. So z. B. with the Recombination sequences a structural protein, an enzyme or a regulatory protein in the Genome are introduced, which eliminates again after intramolecular recombination whereby the expression and activation of the polypeptide is abolished. Will through the intramolecular recombination does not delete the polypeptide of interest but inverted, the expression and activation of the polypeptide can follow. The introduced polypeptide can be endogenous or exogenous. Furthermore, a marker protein be introduced. The person skilled in the art is aware that this list of applications of the The inventive method is only exemplary and not exhaustive. examples for Applications according to the invention with the previously used Cre and Flp recombinases were carried out, for. B. in the review by Kilby, N. et al., (1993), Trends Genet., 9, pp. 413.

To carry out the process according to the invention, a resolvase must be placed on the Recombination sequences act. The resolvase or the resolvase gene can already do so before introducing the first DNA sequence and / or the copy of the first DNA sequence in the eukaryotic cell or after the introduction of the first DNA sequence and / or Copy of the first DNA sequence are introduced. The one for the sequence specific Resolvase used in recombination is preferably expressed in the cell in which it is Reaction. For this purpose, a second DNA sequence, comprising a resolvase gene, is inserted into introduced the cells. Preferred resolvase genes are listed in SEQ ID NO: 3, 5 and 7 and also include their derivatives. A method in which the second DNA sequence into the eukaryotic genome of the cell via homologous recombination or is integrated indiscriminately. Also preferred is a method in which the second DNA sequence regulatory DNA sequences comprising a spatial and / or temporal expression of the Resolvase gene effect.

Spatial expression in this case means e.g. B. that the recombinase only in one certain cell type, e.g. B. liver cells, kidney cells, nerve cells or cells of the  Immune system, are expressed through the use of cell type-specific promoters can and only catalyzes recombination in these cells. A temporal expression at the Regulation of resolvase expression can be achieved by promoters that start from or in at a certain stage of development or at a certain point in time in the adult Organism are active. Furthermore, the spatial and / or temporal expression by the Use of inducible promoters, e.g. B. by interferon- or tetracycline-dependent Promoters can be achieved; see. Review article by Müller, U. (1999) Mech. Develop., 82, pp. 3. A spatial and / or temporal activation of the recombination can not only can be achieved by the regulation of resolvase expression, but is also by that Effect of topoisomerase inhibitors on constitutive expression of the wild-type resolvase reachable.

The resolvase used in the method according to the invention can be both the wild-type resolvase and a modified resolvase. The use of a modified resolvase is preferred. The resolvases preferably used in the method according to the invention comprise the amino acid sequences listed in SEQ ID NO: 4, 6 and 8. The modified resolvase is modified in such a way that it can carry out the recombination reaction without (-) DNA supercoiling. The preparation of modified polypeptides and the screening for the desired activity are state of the art and easy to carry out; see. Erlich, H. (1989) PCR Technology. Stockton Press. The use of the γδ-resolvase according to SEQ ID NO: 4 is preferred. Two γδ-resolvase mutants which are used as γδ-resolvase ^E124Q (SEQ ID NO: 7) and γδ-resolvase ^{E102Y, E124Q} (SEQ ID NO: 5) be designated; see. Arnold et al. (1999), see above. γδ-resolvase ^E124Q contains a glutamine residue instead of a glutamate residue at position 124. γδ-resolvase ^{E124Y, E124Q} contains, in addition to the exchange of mutant 124, a tyrosine residue instead of the glutamate residue at position 102 compared to wild-type γδ- Resolvase. Both γδ resolvase mutants were produced via PCR mutagenesis of the γδ resolvase gene. These mutants can perform recombination between two directly repeated, complete res sequences without the cofactor (-) supercoiling.

The method according to the invention can be carried out in all eukaryotic cells. The Cells can e.g. B. present in a cell culture and all types of plant or animal Cells include. For example, cells can be oocytes, embryonic stem cells, hematopoietic  Stem cells or any type of differentiated cells. A method is preferred in which the eukaryotic cell is a mammalian cell. A method is particularly preferred in which the Mammalian cell a human cell, monkey, mouse, rat, rabbit, hamster, goat, Is a cow, sheep or pig cell. The method according to the invention thus also relates eukaryotic cells comprising res according to SEQ ID NO: 1 or 2 in at least one and preferably two copies and / or the resolva gene according to SEQ ID NO: 3, 5 or 7 or the polypeptides encoded by the genes, or their respective derivatives.

The invention relates to the use of a res sequence according to SEQ ID NO: 1 or SEQ ID NO: 2 in a sequence-specific recombination of DNA in eukaryotic cells. The eukaryotic cells can also be found in the cell structure of an organism, e.g. B. a mammal, that does not contain a resolvase. This organism can be used to cross with other organisms can be used, which in turn contain the resolvase in their cells, so that progeny arise in the cells then the sequence-specific recombination by the action of Resolvase is performed. The invention thus also relates to the use of a Resolvase or a resolvase gene in a sequence-specific recombination in eukaryotic cells. In particular, the invention relates to the use of the resolvase gene according to SEQ ID NO: 3, the resolvase 102 according to SEQ ID NO: 5 and the resolvase 102/124 according to SEQ ID NO: 7.

The invention further relates to transgenic organisms, e.g. B. mammals, especially transgenic monkeys, Mice, rats, rabbits, hamsters, goats, cows, sheep or pigs, the res according to SEQ ID NO: 1 or SEQ ID NO: 2 in one or two copies and / or the resolvase gene according to SEQ ID NO: have 3, 5 or 7 integrated in their cells.

The method according to the invention can also be used to test topoisomerase inhibitors in a eukaryotic cell can be used, comprising a) introducing into a cell first DNA sequence according to SEQ ID NO: 1, b) the introduction into a cell of a copy of the first DNA sequence according to SEQ ID NO: 1 in direct orientation, and c) the introduction of a Wild-type resolvase, and d) addition of the topoisomerase inhibitors to be tested.

The eukaryotic cells can be in culture (ex vivo) or in the organism (in vivo) for the inventive methods are used. The method is preferred here  Organism, since here the cell and tissue-specific physiological influences on the possible degradation or modification of the topoisomerase inhibitors and the resulting resulting effects can be tested in terms of their effectiveness. The Topoisomerase inhibitors can be on the cells before or after the expression of the resolvase act, which leads to the activation of the recombination and z. B. via enzymes or Marker proteins are easy to detect. The efficiency of each substance in terms of its inhibitory effect on topoisomerases correlates with the activation of recombination.

A spatial and / or temporal activation of the recombination can be regulated the resolvase expression can be achieved. A spatial and / or temporal activation is also by regulating the action of topoisomerase inhibitors in constitutive Expression of wild-type resolvase achievable.

A method is preferred, a second DNA sequence additionally comprising a Wild-type resolvase gene is introduced into the cell. A method is particularly preferred, in which the wild-type resolvase is a wild-type γδ resolvase. The invention further relates to a eukaryotic cell, comprising two DNA sequences according to SEQ ID NO: 1. Preferred is one eukaryotic cell, further comprising a wild-type resolvase gene, especially a wild-type γδ resolvase contains. The invention further relates to a transgenic organism, e.g. B. one Mammals, especially transgenic monkeys, mice, rats, rabbits, hamsters, goats, cows, sheep or pig, the two copies of res according to SEQ ID NO: 1 in direct orientation and one Resolve gene, in particular a γδ-resolve gene according to SEQ ID NO: 3 in their cells have integrated.

Examples 1. Preparation of the expression and substrate vectors 1.1 Expression vectors

The eukaryotic expression vectors for γδ-resolvase (pPGKγδ / pPGKγδ ^NLS ) as well as for γ δ-resolvase mutants γδ ^E124Q (pPGKγδ124 / pPGKγδ124NLS) and γδ ^{E102Y, E124K} (pGGPGLS) from pGGPDL Cologne (pPGGD) Cologne ). The vector contains the phosphoglycerate kinase promoter (P-PGK) and the polyadenylation signal sequences from the small Simian Virus 40 (SV40) tumor antigen. The γδ resolvase genes cloned by PCR were ligated between these elements in the vector.

The following primers were used for cloning the resolvase genes:

The amplification of the wild-type resolvase gene with or without a nuclear localization sequence (NLS) was carried out with the primers P-γδA and P-γδB, or P2972 after a first Denaturation step at 94 ° C (5 min.) With 28 cycles of denaturation (95 ° C, 30 sec.), Primer binding (56 ° C, 30 sec.) And DNA synthesis (72 ° C, 45 sec.), Followed by one last Synthesis step (72 ° C, 4 min.). The resulting PCR fragment, restricted with PstI and XbaI, replaces the Cre gene excised with the same enzymes in pPGKcrebpa.

The resolvase mutants were produced by assembly PCR. Here were in the first Assembly PCR step, in two separate PCR approaches with the primers P-γδA and P-γδ 124Qanti and P-γδ124Q and P-γδB, respectively, amplified the two γδ resolvase gene fragments, which in the overlapping area contain the desired mutation, which is then the second step of the Assembly PCR in equimolar amounts as a template for the following third PCR with the primers P-γδA and P-γδB were used. The amplifications took place after a first Denaturation step at 94 ° C (5 min.) With 30 cycles of denaturation (94 ° C; 45 sec.), Primer binding (60 ° C; 45 sec.) And DNA synthesis (72 ° C; 45 sec.), As well as a last one Synthesis step (72 ° C; 6 min). Analogously, the oligonucleotides P-γδA, P-γδ102Y, P-γδ102Yanti and P-γδB introduced the second mutation E102Y into the gene. The mutants were then by PCR with the primers P-γδA and P2972 among them Provide reaction conditions with an NLS at the C-terminus of the protein.

1.2 substrate vectors

The substrate vectors are derivatives of pCH110 (Pharmacia). Here are the Recombination cassettes under the control of the SV40 promoter, which is a strong constitutive  Expression guaranteed. The plasmid also has the polyadenylation signal sequences of the SV40 tumor antigen. The one in the output vector together with the prokaryotic promoter contained β-galactosidase gene (lacZ) was first cut out with HindIII and BamHI, to do it after PCR amplification with the primers P-lacZ1 and P-lacZanti1 without prokaryotic Insert promoter again. A first denaturation step (94 ° C; 5 min.) carried out, followed by 30 cycles of denaturation (94 ° C; 1 min.), primer binding (56 ° C; 1 min.) And DNA synthesis (72 ° C; 4 min.) And a final synthesis step (72 ° C; 10 min.).

The vector pSV2Neo (Southern, P.J., Berg, P. (1982) J. Mol. Appl. Genet. 1, pp. 327) with the restriction enzymes Sma1 and BglII the neomycin resistance gene cut out and flanked with γδ-resolvase recognition sequences prepared via PCR. These recombination sequences (res) are arranged in the construct in the same orientation.

The res was amplified using the primers P-RES1 and P-RESanti1 and P-RESanti2. It started with a first denaturation step (94 ° C; 5 min.) And 30 cycles of Denaturation (94 ° C; 30 sec.), Primer binding (56 ° C; 30 sec.) And DNA synthesis (72 ° C; 30 sec.). A final synthesis step followed (72 ° C; 4 min.). The at the 5 'terminus of Res residing neomycingens was ligated to the neomycin gene via BglII, the second res linked to the 3 'end of the gene by a blunt-end ligation. The resulting one Recombination cassette res-Neo-res was inserted over the HindIII interface between SV40- Promoter and lacZ gene cloned into the substrate plasmid. This was called pCH-RNRZ designated.

The above oligonucleotides are listed below:

The plasmid pCH-RNRZ was generated in vivo in E. coli strain DH5α cells (Hanahan, D. (1983) J. Mol. Biol., 166, pp. 557), recombined. This bacterial strain is characterized in that it carries an F 'plasmid encoding wild-type γδ resolvase. The resulting recombined Plasmid pCH-RZ was used as a positive control in the further experiments.  

Expression plasmid DNAs and pCH-RZ were obtained from E. coli strain DH5α Affinity chromatography (Qiagen, Germany) isolated. Substrate plasmid pCH-RNRZ was from E. coli strain JC5547 (Willets, N.S., Clark, A.J. (1969) J. Bacteriol., 100, pp. 231). The sequence control of all expression and substrate vectors, as well as all PCR generated Products were analyzed using the fluorescence-based 373A DNA sequencing system (Applied Biosystems). PCR reactions were carried out using the "Master Mix Q - Kit" (Quiagen, Germany). All DNAs were analyzed by agarose gel electrophoresis (0.8% - 1.5% w / v) analyzed in TBE buffer.

2. Cell culture and construction of the reporter cell lines

The transient expression and recombination analyzes were carried out with an ovary Hamster cell line CHO (Puck, T. T. (1958) J. Exp. Med., 108, pp. 945) and a human epithelial cervical carcinoma cell line HeLa (Geye, G. O., et al. (192) Cancer Res., 12, pp. 264) carried out. HeLa cells were cultivated in α-MEM (Life Technologies, Inc.), which with 10% fetal calf serum was enriched, and streptomycin (0.1 mg / ml), penicillin (100 units / ml), as well as sodium pyruvate (1 mM). CHO cells were grown in DMEM with the same Additives, without sodium pyruvate, cultivated.

HeLa reporter cell lines that have stably integrated pCH-RNRZ into the genome were constructed as follows: approx. 20 μg of the plasmid DNA were linearized with BamHI, purified by phenol / chloroform extractions, precipitated with ethanol and in approx. 5 × 10 ⁶ cells were transfected using a lipofect (30 μl Fugene ^TM 6, Boehringer Mannheim). Stable cell lines were selected with 500 µg / ml G418 / Geneticin (Life Technologies) and then characterized by PCR, DNA sequencing and Southern analysis.

3. In-vivo recombination analysis

In order to carry out intramolecular recombination in vivo, approximately 1 × 10 ⁵ cells were sown in 3.5 cm plates. After 24 hours, the circular expression vector was cotransfected, together with the circular superhelical wound substrate plasmid, in a ratio of 3: 1, with the Lipofect Fugene ^TM 6 (Boehringer Mannheim) according to the manufacturer (2 µg DNA; 3 µl Fugene). To demonstrate the recombination that occurred, either a β-Gal staining or a β-Gal assay was carried out after 72 h, since in the recombined state the lacZ gene can be transcribed directly by the SV40 promoter and thus expressed, which is due to the starting substrate the recombination cassette in between does not work. After fixation of the cells with 0.5% glutaraldehyde, the β-gal staining was carried out by adding the staining solution (5 mM K ₄ Fe (Cn ₆ ) × 3H ₂ O, 5 mM K ₃ Fe (Cn ₆ ), 2 mM MgCl ₂ , 4 mg / ml X-Gal in PBS) and subsequent incubation for 7-8 h at 37 ° C. The number of blue colored cells was determined microscopically. The β-Gal assay was carried out with the aid of the "Galacto Light Kit" (Tropix, Perkin Elmer) according to the manufacturer, with amounts reduced to 33%. Relative light units (RLUs) were measured in the Lumat LB9501 luminometer (Berthold) and standardized to RLU / µg protein in the cell lysate.

In order to analyze recombination of wild-type γδ resolvase after topoisomerase I inhibition, 1 × 10 ⁵ cells were first sown in 3.5 cm plates and transfected with 2 μg expression vector (pPGKγδNLS). In order to ensure sufficient expression of the recombinase, the cells were cultivated for 72 hours before the addition of the topoisomerase inhibitor camptothecin (50 μg / ml). After 3 hours of incubation and a change of medium, a co-transfection of pCH-RNRZ and expression vector in a ratio of 1: 1 was carried out. Another 72 h later, β-gal staining was performed as described above.

The recombination analysis in the stable HeLa cell lines (H5) was carried out directly at the DNA level. For this purpose, 5 × 10 ⁶ cells were seeded in 10 cm plates and, after 24 h, were transfected with 14 μg circular expression vector and 21 μl Fugene ^TM 6. After 72 hours, the cells were harvested in order to isolate the genomic DNA via affinity chromatography (QIAamp Blood Kit; Qiagen, Germany), according to the manufacturer.

About 100-400 ng of the genomic DNA were amplified by PCR in order to detect the deletion of the neomycin resistance gene and a complete res caused by resolution. 50 µmol of the following oligonucleotides were used for this:

The amplification was carried out after a first denaturation at 94 ° C. (5 min.) With 34 cycles of Denaturation (94 ° C, 1 min.), Primer binding (63 ° C, 1 min.) And DNA synthesis (72 ° C, 2 min.), followed by a final synthesis step for 4 min at 72 ° C.  

The amplified PCR products were separated by agarose gel electrophoresis, the recombination product was eluted (QIAquick Gel Extraction Kit, Qiagen, Germany) and then sequenced. In order to demonstrate the purity of the negative controls, the PCR products were transferred to a nylon membrane by means of Southern hybridization, and hybridized with the recombined PCR product as ³² P-dATP and ³² P-dCTP (Amersham) radiolabelled probe. Another PCR with the primers P-SVNeo1 and P-SVNeo2 served as evidence of a possible inversion product.

4. Western analysis

Cell lysates from transiently transfected cells were removed by boiling (15 min.) The cells in Sample buffer (New England Biolabs) made. The proteins were in a 12.5% SDS polyacrylamide gel separated by molecular weight and overnight on a Nitrocellulose membrane (Immobilon P, Millipore) transferred. The membrane was 1%. Blocking solution (BM Chemiluminescence Western Blotting Kit; Boehringer Mannheim, Germany) treated and with polyclonal mouse antibodies against γδ-resolvase in one Dilution of 1: 3000 incubated (antibody from N. Grindley, USA). With the peroxidase coupled secondary antibodies was the position of the resolvase on the membrane made visible (BM Chemiluminescence Western Blotting Kit; Boehringer Mannnheim, Germany). Purified γδ resolvase acted as a control. For size comparison, the kD marker (Broad range marker, New England Biolabs) used.

5. Results 5.1 Synthesis of wild-type and mutant γδ resolvase in CHO cells

To test whether γδ resolvase or one of the mutant recombination in eukaryotic cells catalyze, it was first necessary to demonstrate that the recombinase from the Cells can be synthesized. For this, the eukaryotic expression vectors, pPGKγδ and their derivatives that the resolvase gene or the genes for the derivatives under the Control of the PGK promoter contains used. After inserting the Expression vectors in CHO cells by lipofection were cell lysates 72 hours later prepared and examined by Western analysis. The recombinase was detected using mouse polyclonal antibodies directed against wild-type γδ resolvase. As Control, pPGKCre, which expresses the Cre recombinase of phage P1, was introduced into the cells brought in.  

The results show that proteins with the expected molecular weights in the Cells were present that were treated with the resolvase expression vectors in lipofection were. This protein was undetectable when the control vector pPGKCre was used has been.

5.2 Resolvase-catalyzed intramolecular recombination in CHO and HeLa cells

Western analysis shows that, based on the respective expression vectors, the Resolvase protein or its respective derivatives is synthesized in CHO cells. To test, which of the different resolvase proteins is the recombination between two res sequences can catalyze in CHO cells, the expression vectors were each with the Recombination substrate pCH-RNRZ transiently introduced together. The recombination leads to the deletion of the DNA segment from the β-galactosidase gene, which is then by the SV40 Promoter can be expressed. Successful recombination events can therefore can be detected by an enzyme activity determination. It turned out that in such Co-transfection experiments all mutant resolvase proteins, both with and without the C-terminal NLS that can perform recombination with high efficiency. The wild-type In contrast, resolvase, whether with or without NLS, is inactive; H. there can be no recombination this way.

A human reporter cell line (H5) was constructed in order to test whether the recombination substrate can also be recombined from the resolvase or its derivatives as a stable component of the eukaryotic genome. This cell line contains a copy of the substrate vector (pCH-RNRZ) randomly integrated into the genome by non-homologous recombination. This was demonstrated in advance using a Southern analysis. Approximately 72 hours after the introduction of the respective expression vectors into the cell line H5, genomic DNA was isolated and an attempt was made to detect the recombination via PCR with the primers P-SV40prä and P-CHSeqanti and subsequent Southern analysis. The PCR product, which is obtained with unrecombined substrate, is longer than that created after the deletion of the neo gene by recombination, and thus both products can be separated from one another in an agarose gel electrophoresis. The results show that recombination took place in the presence of the two resolvase mutants γδ ^E124Q and γδ ^{E102Y, E124Q} and that one - presumably very low - activity can also be measured with wild-type γδ resolvase (without NLS).

The results so far show that both γδ resolvase mutants are both episomal and can recombine genomic substrates. A significant effect in terms of efficiency the recombination by the NLS attached to the C-terminal end was not possible be determined. A very low activity of the wild-type γδ resolvase is only possible with the Cell line H5, but not in transient co-transfection experiments. Out of it it follows that the topological state of the genomic or episomal intracellular DNA is predominantly relaxed, or activation of the wild-type γδ resolvase is not permitted.

5.3 Activation of the wild-type γδ resolvase by the topoisomerase inhibitor Camptothecin

To test whether the wild-type γδ resolvase was exposed to the topoisomerase inhibitor Camptothecin (CPT), which indirectly changes the topological state of intracellular DNA, can be activated, the expression vector pPGKγδ was initially alone in HeLa tents introduced by lipofection. Approx. 72 hours later, the cells were treated with CPT and after a further 3 hours the expression vector together with the substrate pCH-RNRZ in the cells pretreated in this way, again by lipofection. After another 72 The cells were stained for hours by treatment with X-Gal. A blue color of the Cells shows that the enzyme β-galactosidase is expressed in the cells, which in turn successful recombination events. The results show that without CPT no recombination by the wild-type γδ resolvase takes place in HeLa cells, whereas by the action of the CPT, blue cells can be detected in all experiments. In Control experiments also ensured that this effect did not affect CPT Treatment of the cells per se is attributed (not shown). Thus, as a result of Action of a topoisomerase inhibitor on the topological state of intracellular DNA the recombination can be demonstrably activated by the wild-type γδ resolvase.  

SEQUENCE LOG

Claims (27)

1. A method for sequence-specific recombination of DNA in a eukaryotic cell, comprising

• a) introduction into a cell of a first DNA sequence,
• b) inserting a copy of the first DNA sequence into a cell, and
• c) performing the sequence-specific recombination by the action of a resolvase.

2. The method of claim 1, wherein the copy of the first DNA sequence in direct Orientation on the same DNA molecule is introduced. 3. The method according to claim 1 or 2, wherein the first DNA sequence is a res sequence according to SEQ ID NO: 1 or its derivative or a res sequence according to SEQ ID NO: 2 or its Derivative includes. 4. The method according to any one of claims 1 to 3, wherein in addition a second DNA sequence comprising a resolvase gene is introduced into the cell. 5. The method of claim 4, wherein the second DNA sequence further a regulatory DNA sequence comprising a spatial and / or temporal expression of the resolvase gene causes. 6. The method according to any one of claims 1 to 5, wherein the resolvase is a modified Resolvase is. 7. The method according to any one of claims 1 to 5, wherein the resolvase is a γδ resolvase. 8. The method of claim 7, wherein the γδ resolvase is a modified γδ resolvase. 9. The method according to claim 8, wherein the modified γδ-resolvase is γδ-resolvase ^E124Q or γδ-resolvase ^{E102Y, E124Q} . 10. The method according to any one of claims 1 to 9, wherein the first and / or the copy of the first DNA sequence further comprises DNA sequences that integrate the first and / or the copy of the first DNA sequence into the genome of the eukaryotic cells by means of causes homologous recombination. 11. The method according to any one of claims 1 to 10, wherein the first and / or the copy of the first DNA sequence further comprises a nucleic acid sequence comprising a polypeptide of Interest encoded. 12. The method of claim 10, wherein the polypeptide of interest is a structural protein endogenous or exogenous enzyme, a regulatory protein or a marker protein. 13. The method according to any one of claims 1 to 12, wherein the eukaryotic cell Is mammalian cell. 14. The method according to claim 13, wherein the mammalian cell is a human cell, a monkey, Mouse, rat, rabbit, hamster, goat, cow, sheep or pig cell. 15. Use of a res sequence according to SEQ ID NO: 1 or its derivative or a res- Sequence according to SEQ ID NO: 2 or its derivative in a sequence-specific recombination of DNA in eukaryotic cells. 16. Nucleic acid sequence according to SEQ ID NO: 5 or its derivative. 17. Amino acid sequence according to SEQ ID NO: 6 or its derivative. 18. Use of the nucleic acid sequence according to claim 16 and / or Amino acid sequence according to claim 17 for use in a sequence specific Recombination of DNA in eukaryotic cells. 19. A method for testing topoisomerase inhibitors in a eukaryotic cell, comprising

• a) introduction into a cell of a first DNA sequence according to SEQ ID NO: 1,
• b) the introduction into a cell of a copy of the first DNA sequence according to SEQ ID NO: 1 in direct orientation,
• c) the introduction of a wild-type resol vase, and
• d) addition of the topoisomerase inhibitors to be tested.

20. The method according to claim 19, wherein additionally comprising a second DNA sequence Will-type resolvase gene is introduced into the cell. 21. The method of claim 20, wherein the second DNA sequence further comprises a regulatory DNA sequence comprising a spatial and / or temporal expression of the resolvase gene causes. 22. The method according to any one of claims 19 to 21, wherein the first and / or the copy of the first DNA sequence further comprises DNA sequences that integrate the first and / or the copy of the first DNA sequence into the genome of the eukaryotic cells by means of Recombination causes. 23. The method according to any one of claims 19 to 22, wherein the first and / or the copy of the first DNA sequence further comprises a nucleic acid sequence comprising a polypeptide of Interest encoded. 24. The method of claim 23, wherein the polypeptide of interest is a structural protein endogenous or exogenous enzyme, a regulatory protein or a marker protein. 25. Eukaryotic cell, comprising two DNA sequences according to SEQ ID NO: 1 or SEQ ID NO: 2 and optionally a resolvase gene. 26. The eukaryotic cell according to claim 25, wherein the resolvase gene encodes a wild-type resolvase, a γδ-resolvase, a γδ-resolvase ^E124Q or γδ-resolvase ^{E102Y, E124Q} . 27. Transgenic organism, comprising res sequences according to SEQ ID NO: 1 or SEQ ID NO: 2 and / or a resolvase gene according to SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 or their derivatives.