DE10207313A1 - In vitro or in vivo site-specific DNA recombination, useful e.g. for gene inactivation, using Cre recombinase that lacks heterologous protein-transduction domain - Google Patents

Abstract

Site-specific DNA recombination in eukaryotic cells in vitro comprising using Cre recombinase that lacks heterologous protein-transduction domains, is new. Site-specific DNA recombination in eukaryotic cells in vitro comprises: (a) introducing a polypeptide (I) with Cre recombinase activity but not containing any heterologous protein-transduction domains, into a eukaryotic cell culture where the cells contain DNA region(s) with recognition site(s) for (I); and (b) culturing the cells under suitable conditions for DNA recombination. Independent claims are also included for: (1) a polypeptide (Ia) with Cre recombinase activity comprising fully defined sequences of 343 amino acids (S2) or 371 amino acids (S4), as given in the specification, or their derivatives, homologues and active fragments, excluding the native enzyme from phage P1; (2) nucleic acid sequences (II) comprising fully defined sequences of 1032 base pairs (S1) or 1116 base pairs (S3), as given in the specification, encoding (I); (3) isolated cells that contain (Ia) or (II); (4) a reporter system for detecting site-specific DNA recombination in eukaryotic cells; (5) isolated cells containing the reporter system of (4); (6) preparing (Ia) by recombinant expression; (7) a kit for the new process or the method of (6); and (8) use of glycerol for performing or improving protein transduction.

Description

Technical area

The present invention relates to novel methods and kits for introducing a polypeptide having Cre recombinase activity in eukaryotic cells. The invention further relates Polypeptides with Cre recombinase activity and a reporter system, which after introduction into a corresponding cell digestion about successful DNA recombination. In the end The invention relates to kits for the detection of site-specific DNA Recombinations.

State of the art

Sequence-specific recombinations leading to a directed Insert, cut or inversion of Nucleic acid sections can be used for a variety of applications be of great use. In bacteria, bacteriophages and yeasts were already found some site-specific recombinases and characterized. They belong to the family of λ integrases (also referred to as "tyrosine recombinases"), for which over 100 now Members were identified based on sequence homologies. Recombinases recognize specific DNA sequences and catalyze the mutual (reciprocal) exchange of the DNA sections between these recognition sequences. The best studied Examples are the integrase from the bacteriophage λ, the bacterial recombinases XerC and XerD, yeast Flp recombinase as well as belonging to the Cre / lox system Cre recombinase from the Bacteriophage P1.

The function of the Cre / lox system in the propagation cycle of the Bacteriophage P1 is among other things in the cyclization of the linear Genome and the dissolution of dimeric phage plasmids to order an efficient division of the phage genome during the bacterial cell division (Austin et al. (1981) Cell 25: 729-36; Abremski et al. (1983) Cell 32: 1301-11). The Target sequence of Cre recombinase, the so-called loxP sequence exists from 2 recombinase binding elements of 13 bp (base pairs), the as "inverted repeats" flank a central sequence of 8 bp. The central 8 bp sequence determines the orientation of the 34 bp loxP sequence. For a recombination event, four cre- Molecules and two loxP sequences are cross-shaped Intermediate structure that is resolved by the reciprocal strand exchange (Gou et al. (1997) Nature 389: 40-6).

The Cre / lox system is useful because of its specificity Tool for many applications in genetic engineering (Sauer (1998), Methods 14: 381-392; Kilby et al. (1993) Trends Genet. 9: 413-21). The loxP sequences can be used, for example, in vectors or in the Genome can be integrated by other cells and there as Target sequence for targeted integrations, inversions or excisions serve. A DNA section consisting of two loxP sequences Flanked (i.e., "flopped") is flanked by the Cre activity cut out. The addition of an excess "flooded" DNA to a target sequence with one or two loxP- Can provide an integration of this DNA at the appropriate Position result. Lox sequences in opposite Orientation, on the other hand, enables the inversion of the flanked ones Sequence. For the recombination process, in addition to the Recombinase and the loxP sequences no further factors required (Abremski and Hoess (1984) JBC 259: 1509-14). This facilitates the application of the Cre / lox system in the various Cell types and in cell-free systems.

By a Cre-induced inactivation of genes after completed development of an organism becomes for example the functional study of genes responsible for the Embryonic development of the corresponding organism are essential, allows. Also, the removal of exogenous sequences allows if their effect is no longer desirable. So could for example, primary hepatocytes and myogenic cells Expression of the flooded SV40 large T antigen reversible immortalized and thus expanded indefinitely in vitro (Kobayashi et al. (2000) Science 287: 1258-62; Berghella et al. (1999) Hum Gene Therapy 10: 1607-17).

By cutting out a DNA segment, genes can also be used be activated by z. B. in the vicinity of an active Promoters (Lakso et al. (1992) PNAS 89: 6232-6). Cre mediated activation of genes can also be achieved by inversion be reached by DNA sections.

Another application that is becoming increasingly important is the targeted integration of transgenes (Gorman and Bullock (2000) Current opinion in Biotechnology 11: 455-60). Furthermore, were already using the Cre / lox system synthetic antibody Libraries (Tsurushita et al. (1996) Gene 172: 59-63), directed chromosomal translocations (Qin et al. (1994) PNAS 91: 1706-10) and mosaic genomes (Betz et al. (1996) Curr Biol 6: 1307-16) constructed or humanized animal models by directional Gene Exchange (Zou et al. (1994) Curr Biol 4: 1099-103).

The use of the Cre / lox system in the site-directed Recombination of DNA in eukaryotic cells was also reported in the U.S. Patent 4,959,317.

A noteworthy feature of Cre recombinase is the Ability to enter the cell nucleus and thus the Recombination process independent of the dissolution of the nuclear membrane during the Initiate mitosis. This is especially for one Use in stationary, postmitotic eukaryotic cells useful. Initially, Cre recombinase was thought to be responsible for its small size of 38 kilodaltons passively through the nuclear pores diffused. Some groups have classic Nuclear localization sequences fused with the Cre recombinase to increase the efficiency of the Core transport (Gu, et al. (1993) Cell 73: 1155-1164; Bergemann et al. (1995) NAR 23: 4451-56). In 1999, Le et al. (NAR 27: 4703-9), however, demonstrated that the Cre protein is endogenous Nuclear localization signals, which is unusual for a Protein that performs its function in seedless bacterial cells.

The Cre / lox-specific recombination in intact cells was in the previously established applications only by the introduction allows a Cre-encoding nucleic acid in the target cells. The Cre gene was z. B. by transfection of a Expression vector or by infection with retro or adenoviruses (Bergemann et al. (1995) NAR 23: 4451-56; Kanegae et al. (1995) NAR 23: 3816-3821), or it was crossed by crossing with a transgenic organism merged with the lox sequences. A Control of Cre expression or Cre activity was included achieved these approaches in various ways. The cre gene can z. B. be placed under the control of a promoter, the time or tissue-specific regulation (Gu et al. (1994) Science 265: 103-6).

Inducibility of Cre activity may be due to expression of Cre fusion proteins with so-called hormone-binding domains (HBDs) of the progesterone or estrogen receptor (Metzger et al. (1995) PNAS 92: 6991-5; Kellendonk et al. (1996) NAR 24: 1404-11). The Cre-HBD fusion protein is in the cells constitutively expressed, but it does not arrive until after the addition of the appropriate steroid hormone in the nucleus and can then initiate the recombination. For induction, hormone Analogues used only with the HBD of the fusion protein, but do not interact with the endogenous steriod receptors. Nevertheless the application of these steroids is not safe. On Another disadvantage of the system is that on the one hand, the induced Recombination also in the application of high "inducer" amounts is not complete and that on the other hand, even in the absence of Inducers a residual activity of Cre recombinase are observed (Moltendonk et al. (1999) J Mol Biol. 285: 175-82; et al. (1998) NAR 26: 1427-32). This background activity makes especially in vitro (Zhang et al., 1996 NAR 24: 543-8 Guide Benzakein et al. 2000 NAR 28: e99; Cheng et al. 2000 NAR 28: e108).

With the help of the Tet system (Gossen and Bujard (1992) PNAS 89: 5547-51), Cre expression could be due to the presence of Tetracyclines suppressed (St-Onge et al. (1996) NAR 24: 3875-77) or induced (Saam and Gordon (1999) JBC 274: 38071-82). This method avoids the application of the questionable hormone Analogues and instead uses a well-studied, non- toxic drug for induction. But the Tet system has the disadvantage that in addition to the "flooded" target sequence and the Cre Gene under the tTA-responsive promoter another transgene necessary for the tet repressor transactivator fusion protein (tTA) or its reverse form rtTA encoded.

The introduction of a Cre-encoding nucleic acid generally harbors considerable risks: On the one hand, there is a risk of one Insertional mutagenesis due to non-specific integration of the exogenous DNA into the genome of the target cell (Stocking et al., (1988) Cell 53: 869-79). Another risk factor is the one with the most Systems permanent or for a longer period of time sustained expression of Cre recombinase. So was recently in two independent works demonstrated that a sustained Expression of Cre recombinase slows growth and causes chromosomal aberrations in the examined cells (Silver and Livingston (2001) Mol Cell 8: 233-43; Loonstra et al. (2001) PNAS 98: 9209-14). The dependence of Cre-mediated cytotoxicity from their endonuclease activity suggests that cryptic loxP sequences found in the genomes of mammalian, yeast and Bacterial cells were found at high expression of the Recombinase be recognized and recombined.

The object of the present invention is therefore the provision of site-specific DNA recombination methods in eukaryotic cells which inhibit the introduction of a polypeptide Recombinase activity in cells (preferably those that themselves do not have such an enzyme) and the described disadvantages of the methods established in the prior art, Transfer of Cre recombinase-encoding nucleic acids, not respectively.

It has now surprisingly been found that one (e.g. recombinantly produced) Cre recombinase, which does not require additional, heterologous protein transduction domain has, when added to the Culture medium of intact eukaryotic cells that are neither electrically were still chemically pretreated, is absorbed by these and in the nucleus of the cells a specific DNA Recombination process of DNA regions that have corresponding Have recognition sequences, catalyze. The Recombination rate can already after a single application over 50%.

According to the invention, the object is thus achieved by an in-vitro Method for site-specific DNA recombination in eukaryotic Cells in which a polypeptide with cre recombinase Activity that is not a heterologous protein transduction domain which adds to a eukaryotic cell culture whose cells (or its DNA) at least one DNA region (DNA segment) containing at least one recognition sequence of the polypeptide with Cre recombinase activity is recognized, and the cells are cultured under conditions that produce a DNA Allow recombination.

In general, even lipophilic proteins with a molecular mass greater than 0.5 kD do not pass the cell membrane.

Some larger proteins, such as B. the homeobox protein Antennapedia (Antp) from the fruit fly Drosophila, the HIV proteins Tat and Vpr or the glycoprotein PreS2 from the hepatitis B virus (Ford et al., (2001) Gene Therapy 8: 1-4; Schwarze et al. (2000). Trends in Cell Biol 10: 290-5; Oess and Hildt (2000) Gene Ther 7: 750-8), on the other hand, are able to pass through the plasma membrane in to get intact cells. The mechanism of the so-called Protein transduction is largely unexplained. The Amino acid sequences that mediate presumptive passive uptake have been reported for Antp, Tat and PreS2 on relatively short sections (11 to 16 Amino acids) are limited. The transduction domains of Tat and PreS2 can produce relatively large reporter proteins such as β- Galactosidase (β-gal, 120 kD) or the green fluorescent Protein from the jellyfish Aequorea victoria (GFP, 27 kD) intact Introducing cells (Schwarze et al. (1999) Science 285: 1569-72; Oess and Hildt (2000) Gene Ther 7: 750-8).

According to a recently published paper (Jo et al. (2001) Nature Biotechnology 19: 929-33) was a Cre fusion protein with the membrane translocation sequence of the Kaposi fibroblast Growth factor ("MTS" from FGF-4) used to produce a specific To achieve recombination in vitro and in vivo.

All the more surprising is the invention of the underlying Recognizing that a Cre recombinase without heterologous Transduction domain is also able to access the plasma membrane of the cell overcome and get into the cell interior (see Example 3).

The inventive use of a Cre protein without heterologous protein transduction domain has over the use of the Cre-MTS fusion protein from Jo et al. (loc.cit.) the advantage that less heterologous peptide sequences, which may be the Could influence the target cell, be "transduced" Efficiency of recombination on application of the Cre protein without heterologous protein transduction domain in serum-containing medium just as high as in the application of the method described by Jo et al. shown Cre-MTS fusion protein in serum-free medium. Thus, too Cell types that are sensitive to serum deprivation (such as Example murine bone marrow cells), in the inventive Methods are efficiently transduced in serum-containing medium.

The use of such a membrane permeable Cre recombinase opens up the possibility of directed recombination events to perform, with the introduction of a Cre recombinase encoding nucleic acid can be dispensed into the host cells, and the above-mentioned disadvantages can thus be avoided. The polypeptide also becomes relatively fast in the host cells degraded and not - as when introducing one for the polypeptide encoding nucleic acid - permanently over a longer period of time reproduced. This will increase the risk of cytotoxic Minimizes effects. There is also no possibility of nonspecific integration of DNA into the genome of the used Host cells. Another advantage of the invention is the faster procedure of polypeptide transfer (less than 4 hours), whereas after a nucleic acid transfer the Cre Recombinase must first be synthesized ribosomally. The Specific DNA recombination can be carried out by the method according to the invention thus within a short and defined period of time be performed.

In the context of the present invention is meant by the Term "DNA recombination" generally means a physical process, which leads to an exchange of DNA areas between two different DNA molecules or two different areas of a single DNA molecule and thus to a rearrangement of the genetic Starting material leads. In a "site-specific DNA Recombination "is in this context a Recombinant DNA derived from a polypeptide having enzymatic Activity is catalyzed, which is the DNA to be recombined Range based on defined DNA sequence sections (so-called. Recognition sequences) recognizes and a DNA strand exchange between two such recognition sequences (but on different DNA molecules / chromosomes can be localized).

In the enzymatically active polypeptides containing such Catalyst process, it can be according to the invention to polypeptides with a Cre recombinase activity associated with the interacting with specific recognition sequences involving the DNA cut and religate. As "polypeptides with cre recombinase Activity "are within the scope of the present invention Polypeptides with enzymatic activity called the same Catalyze reaction such as those from the λ integrase family recombinase-derived Cre recombinase of bacteriophage P1. The polypeptides may be the Cre recombinase activity as have major or minor enzymatic activity. The In addition, polypeptides may also contain other enzymatic and non-enzymatic fulfill enzymatic functions. Of course it can be a polypeptide having Cre recombinase activity, which is described in U.S. Pat method according to the invention can also be applied to the Recombinase of bacteriophage P1 act on its own. The one of the Polypeptide catalyzed with Cre recombinase activity Recombination process may lead to a directed insertion, cut, Exchange or to an inversion of the subject, too Recombinant DNA area lead. The polypeptides with cre recombinase There are no heterologous activities Protein transduction domains on.

As protein transduction domains are within the scope of the invention short amino acid sequences understood that the transition of a mediate larger protein into an intact eukaryotic cell can. For these amino acid sequences, only 11-16 Amino acids may be, for example, to the corresponding transduction domains from the Tat protein, the Homeobox protein Antennapedia or the PreS2 protein act. Of the The term "heterologous" shows that the Protein transduction domain not representing the natural amino acid region Primary structure of the polypeptide with Cre recombinase activity counts.

According to a preferred embodiment of the present invention The invention features the polypeptide having Cre recombinase activity as described in U.S. Pat SEQ ID NO: 2 or SEQ ID NO: 4 indicated amino acid sequences or is a derivative, a homolog (for example, with at least about 70% identity, preferably at least about 85% identity, with a degree of homology of at least about 95% being particular preferred) or a fragment (which is preferably a length of at least 304 amino acids and in particular at least the Amino acids 37 to 340 of SEQ ID NO: 2) thereof. The Designation "SEQ ID NO:" corresponds to the sequence index "<400>" after WIPO Standard St.25.

In the present case, a "derivative" is a polypeptide understood the same primary structure, which in comparison with the respective polypeptide of the invention additional modifications having the peptide side chains. Such modifications are The skilled worker well known. It can be for example, the glycosylation of asparagine, serine or threonine Residues to the formation of disulfide bridges between Cysteine residues to facilitate covalent attachment of fluorescent labels (e.g. Alexa 488) or coenzymes like biotin, lipoic acid or Pyridoxal phosphate, or the phosphorylation of tyrosine, serine or threonine residues. A "homolog" denotes a Polypeptide whose primary structure is about 30% maximum (Preferably at most about 15%, with a maximum of about 5% especially are preferred) of the primary structure of the respective different polypeptide of the invention. For a homologous protein can one or more amino acids of the original sequence against others Be exchanged amino acids. Furthermore, one or more Amino acids of the original sequence deleted or additional Amino acids are inserted. As an "active fragment" is present denotes a polypeptide whose primary structure with a Area of the primary structure of the respective invention Polypeptide matches, wherein the active fragment less Amino acids include as the polypeptide of the invention. An active Fragment of a polypeptide of the invention has the same enzymatic activity such as the polypeptide of the invention, d. H. it catalyzes the same chemical reaction as that polypeptide of the invention.

In the polypeptide having the in SEQ ID NO: 2 specified Amino acid sequence is a recombinant Cre recombinase (hereinafter referred to as "wtCre") other than the corresponding enzyme from the bacteriophage P1 by a Amino acid exchange in position 1 (glycine instead of methionine) different. It has been found that the inventive Polypeptide cross the plasma membrane of intact cells and in penetrate the nucleus, where it is present in appropriate Recognition sequences of the localized genomic DNA of the Cell catalyzes a DNA recombination. Surprisingly it was the polypeptide is able to function without any heterologous domains, after external addition to a culture of mouse fibroblasts Catalyze loxP-specific DNA recombination (Example 6). The one given in SEQ ID NO: 4 and designated "nlsCre" Polypeptide for purposes of better detectability of the Polypeptides in the cells in addition to that shown in SEQ ID NO: 2 represented amino acid sequence the antibody epitope 9E10 (c-myc; Amino acids 360-371) as well as an SV40 localized at the N-terminus. Nuclear localization sequence (amino acids 11-17). The Nuclear localization sequence is intended to increase the efficiency of the transport of the Increase polypeptide into the nucleus.

With regard to the eukaryotic cells in which the DNA Recombination can be performed is subject to inventive method no restrictions. So can For example, cell cultures of isolated mammalian cells, such as Cells of the mouse or pig, as well as isolated cells human Origin serve as target cells. The target cells can do this originate in various areas of the body. The cells are intact according to the invention and require neither a chemical still physical pretreatment. According to a preferred Embodiment of the present invention are in the eukaryotic cells around human or murine cells. According to one Particularly preferred embodiment is in the human or murine cells around bone marrow cells or Fibroblasts.

The cultivation conditions, the successful introduction a polypeptide having Cre recombinase activity in the eukaryotic cells (into the cell interior) as well as a subsequent DNA Allow recombination are dependent on the chosen one Cells in which the DNA recombination is to be performed. They generally correspond to the cultivation conditions that the Specialist in a cell culture of the corresponding cells apply Accordingly, they are the most detailed in the art described. They include u. a. the cultivation and propagation of the isolated cells in different conventional media. As media come u. a. Dulbecco's Modified Bagle's Medium (DMEM) or Eagle's Minimum Essential Medium (MEM). For the It is understood by those skilled in the art that other media and cytokines Can be used to cre transfer to the target cells too allow. The media can use antibiotics such as Penicillin, streptomycin or kanamycin in the common Concentrations, cytokines (eg Il-3, Il-6, SCF) and others for the Cultivation of cells useful additives, such as fetal calf serum (FCS) or bovine serum albumin (BSA), in contain different concentrations.

Cultivation and cultivation of the cells may each be in the for the respective cells recommended temperature range in conventional Culture dishes are carried out. The introduction of the polypeptide with Cre recombinase activity in the cells can according to the invention by simply adding the polypeptide to the supernatant of the Cell culture. In this case, the polypeptide in a buffered aqueous solution, such as. In a PBS solution. The Cell breeding as well as the choice of alternative methods of addition, the are suitable in the context of the method according to the invention, are in Range of the average skill of the specialist. The Efficiency of transduction is primarily used by the Concentration of the polypeptide with Cre recombinase activity dependent (see Example 4). According to a preferred Embodiment of the present invention, the addition of the Polypeptide with Cre recombinase activity in a concentration of 100 nM up to 30 μM. After addition of the polypeptide, the cells preferably for a period of 2-12 h continue at the chosen Conditions are incubated. Subsequently, the culture medium be replaced with fresh nutrient medium.

In the recognition sequences derived from the polypeptide with Recombinase activity can be detected in the present case Nucleotide sequence sections are similar to those in the field Those skilled in the art are known as "loxP" sequences. It acts these are sequence sections of 34 bp, which consist of two inverted ones Repeated repeats of 13 bp each consist (see SEQ ID NO: 5, bp 1-13 and 22-34), which a flank an asymmetric spacer of 8 bp (see SEQ ID NO: 5; bp 14-21). The intermediate element gives the entire loxP- Sequence section its orientation. Depending on the localization and Orientation of the loxP sequence sections results in the Recombination into different DNA arrangements; so can the flanked nucleotide sequence region in the course of recombination cut out, exchanged, inserted or inverted. For example, flanking two loxP recognition sequences identical orientation results in a specific DNA region by the polypeptide having Cre recombinase activity catalyzed recombination process in a cut (or a Exchange) between the loxP recognition sequences isolated ("flogged") DNA region. The term "identical Orientation "refers to an identical 5 '→ 3' base sequence of the of the inverted repeats enclosed intermediate elements of two loxP recognition sequences. All bases of the intermediate element with Exception of a position (position 18 in SEQ ID NO: 5) vary, with only recombinations between two loxP sequences are efficient with homologous intermediates (Hoess et al. (1986) NAR 14: 2287-2300). The DNA region to be recombined can in the context of the present invention both on one or several chromosomes of a eukaryotic cell, as well one or more mobile genetic elements, such as a plasmid, for example. According to a preferred Embodiment of the present invention are the Recognition sequences loxP sequences of identical orientation with in SEQ ID NO: 5 indicated nucleotide sequence. Included in the invention are nucleotide sequences that differ in terms of Base exchanges, deletions and insertions of one or more Differentiate nucleotides from these sequences. In each of the "inverted Repeats "8-10 bases of the 13 bases must match loxP, to support a Cre-mediated recombination (Abremski et al. (1988), J. Mol. Biol. 202: 59-66; Abremski and Hoess (1985) J. Mol. Biol. 184: 211-220).

The invention further relates to a polypeptide with Cre recombinase Activity which is one of SEQ ID NO: 2 or SEQ ID NO: 4 has indicated amino acid sequence or a derivative, a homolog or a fragment thereof, whereby the natural cre- Recombinase is excluded from the bacteriophage P1.

The invention further relates to a method for Preparation of a Polypeptide Having Cre Recombinase Activity. object is therefore also a process for the preparation of a polypeptide having the sequence given in SEQ ID NO: 2 or SEQ ID NO: 4, in which one coding for said amino acid sequence Nucleic acid sequence, preferably those shown in SEQ ID NO: 1 or SEQ ID NO: 3 given sequence, ligated into an expression vector, man introduce this in suitable for the vector host cells and man the cells are suitable for expression of the polypeptide Cultivated conditions. If necessary, an insulation and purification step. The SEQ ID NO: 2 and SEQ ID NO: 4 were shown polypeptides with Cre recombinase activity z. B. recombinantly produced and as glutathione-S-transferase (GST) fusion protein heterologously expressed in E. coli and by means of Purified affinity chromatography (see Example 1). The Fusion proteins were each incubated in the presence of thrombin, to the GST portion of the Cre recombinase polypeptides separate. Of course, the expression of the polypeptides can also be using other established procedures or in others Organisms such. B. in yeast or insect cells. Appropriate Method of heterologous expression of polypeptides and suitable purification strategies of the proteins or fusion proteins are well known in the art and include u. a. the baculovirus expression system, which is 6xHis tagged Expression System, Maltose Binding Protein (MBP) - Expression system.

The invention also relates to a nucleic acid which is a polypeptide with any of those given in SEQ ID NO: 2 or SEQ ID NO: 4 Amino acid sequence encoded. The nucleic acids preferably have the in SEQ ID NO: 1 or SEQ ID NO: 3 indicated nucleotide sequence on, being the degeneration of the genetic code is included according to the invention. In addition, the invention relates isolated host cells comprising a polypeptide of the invention or contain a nucleic acid according to the invention or a express polypeptide according to the invention. Regarding the choice of Host cells, the invention is not limited, and it In addition to bacterial cells (such as E. coli), yeast cells (such as z. S. cerevisiae or P. pastoris), insect cells and (higher) Euraryote cells, such as. For example, CHO or BHK cells included.

In addition, the invention relates to the use of a polypeptide with Cre recombinase activity to carry out a Site-specific DNA recombination in eukaryotic cells, in which one Cells containing at least one DNA region, the contains at least one recognition sequence derived from the polypeptide with Cre recombinase activity is detected in the presence of the Polypeptides are cultured under conditions involving a DNA recombination allow.

• a) ein erstes Reportergen von zwei Erkennungssequenzen identischer Orientierung flankiert und ein zweites Reportergen stromabwärts des ersten Reportergens lokalisiert ist, wobei stromaufwärts der beiden Erkennungssequenzen ein Initiationskodon und eine Ribosomen-Bindungsstelle lokalisiert sind, die eine Translation des ersten Reportergens ermöglichen, die durch ein Stopkodon zwischen den beiden Erkennungssequenzen beendet wird, und im Bereich zwischen den beiden Reportergenen kein funktionstüchtiges Initiationskodon lokalisiert ist; und
• b) durch eine ortsspezifische DNA-Rekombination das zweite Reportergen in eine Position zu dem Initiationskodon gebracht wird, die eine Translation des zweiten Reportergens ermöglicht; und
• c) die Genprodukte der beiden Reportergene voneinander zu unterscheiden sind.

In a further aspect, the invention further relates to a reporter system for detecting recombinant DNA in eukaryotic cells (for example, triggered by use of a polypeptide having recombinase activity), in which

• a) flanking a first reporter gene from two recognition sequences of identical orientation and a second reporter gene located downstream of the first reporter gene, upstream of the two recognition sequences being located an initiation codon and a ribosome binding site enabling translation of the first reporter gene through a stop codon between the two recognition sequences is terminated, and in the region between the two reporter genes no functional initiation codon is located; and
• b) by site-specific recombinant DNA, the second reporter gene is brought to a position to the initiation codon which allows translation of the second reporter gene; and
• c) the gene products of the two reporter genes are to be distinguished from one another.

The reporter system allows fast and quantitative Detection of site-specific DNA recombination in one eukaryotic cell. It opens the possibility to check immediately whether under given experimental conditions in the transduction of a Polypeptides with recombinase activity a sufficiently high Transduction efficiency was achieved. The reporter system allows thus an accompanying quality control of the Protein preparation and the transduction conditions (medium Composition, time of incubation, temperature etc.).

In the recognition sequences of a polypeptide with Recombinase activity can be detected, for example loxP (see SEQ ID NO: 5), to frt recognition sequences or to their homologues, polypeptides with Cre or Flp recombinase Activity detected sequences act. The recognition sequences of the reporter system have an identical orientation to each other, what an excision flanked by them ("flooded") DNA region causes. Excision becomes the first Reporter gene removed from the gene construct, with the second Reporter gene in a position to the initiation codon and the Ribosome binding site is brought, the a translation (of the Gene product) of the second reporter gene, d. H. the Initiation codon and the second reporter gene should be used after the DNA Recombination in a reading frame. As site-specific DNA recombinations are based on the base, can be an accurate Predict the expected recombination product. The person skilled in the art is therefore in the construction of the reporter system possible to select the base sequence in this regard and the To ensure proper functioning of the system.

According to a preferred embodiment of the present invention Invention are the recognition sequences loxP recognition sequences with the nucleotide sequence given in SEQ ID NO: 5. According to the invention Included are recognition sequences which differ from the above mentioned. Derive sequences and exchange individually Nucleotides differ from these. The polypeptides with recombinase Activity associated with the invention Reporter system can be used in addition to the polypeptides with Cre recombinase activity also include polypeptides comprising the enzymatic activity of another recombinase, such as for example, those of the lambda integrase Flp, have. nucleotide in eukaryotic cells as an initiation codon, stop codon or Ribosome binding site can be used to count Standard knowledge of the specialist and can be relevant textbooks be removed.

Reporter genes used in the context of the present invention are genes which code for proteins having a qualitative and / or quantitative detection of their expression. Such reporter genes are sufficient in the art described and include u. a. those for the β-galactosidase, the Luciferase or the chloramphenicol acetyltransferase coding Genes. Carrying out the proof of recombination may be the digestion of the cells and / or the addition of additional Substances require their biochemical conversion by the Gene product of a reporter gene is detectable. Expression of β- For example, galactosidase can be both colorimetrically (By staining the cells) by reacting the Substrate o-nitrophenyl-β-D-galactoside (ONPG) or X-Gal, fluorometrically by reacting a 4-methylumbelliferyl-β- galactopyranoside compound or by chemiluminescence the reaction of a 1,2-dioxetane-galactopyranoside derivative prove. Furthermore, genes can be applied to cells Resistance to cytotoxic or other substances that inhibit or inhibit growth. The proof of Expression of these genes takes place by cultivating the Cells under appropriately selective conditions.

According to a preferred embodiment of the present invention Invention encodes at least one of the reporter genes for a fluorescent protein. The measurement of luminescent proteins caused light emissions is particularly suitable for in vivo Measurements, as the cells do not need to be digested and thus be accessible to direct evidence. For this reason is the measurement of fluorescence with less temporal and material effort. Aliquots of the respective cell culture can be during the culture growth with the help of a Fluorescence microscope or by flow cytometry on an emission of Examine the light.

According to a particularly preferred embodiment of the present invention, the fluorescent protein has one of the in SEQ ID NO: 6 or SEQ ID NO: 7 indicated amino acid sequences or is a derivative, a homolog or an active fragment the same. These are the red-fluorescent DsRed1 Protein from Discosoma sp. or the enhanced green fluorescent protein (eGFP), a variant of the GFP from Aequorea victoria.

According to a further, particularly preferred embodiment of the present invention, the reporter system has the nucleotide sequence given in SEQ ID NO: 8. This reporter construct (SFr) was generated by flanking the red fluorescent protein-encoding DsRed1 gene from loxP sequences of the same orientation. Downstream of the second loxP sequence is the open reading frame of an eGFP gene which encodes the enhanced green fluorescent protein and has a deleted ATG start codon. A ribosome binding site and a potential ATG initiation codon in the reading frame of the DsRed gene are located before the first loxP sequence. The transitions are designed so that loxP-specific recombination results in excision of the DsRed gene and at the same time puts the eGFP gene in the correct reading frame to the ATG initiation codon (see Figure 2). The detection of a Cre-specific recombination can be carried out by fluorescence microscopy or by flow cytometry. Flow cytometry allows an accurate determination of the rate of recombination without the need to pre-treat the cells.

The invention thus also relates to a method for detection a site-specific DNA recombination in eukaryotic cells using a polypeptide having recombinase activity the reporter system according to the invention and a polypeptide with recombinase activity in a eukaryotic cell and cultured the cells under conditions that produce a DNA Allow recombination, and in the following the Recombination process using the gene product encoded by the second reporter gene (i.e., by a change or the like) Occurrence of fluorescence when at least one reporter gene for a fluorescent protein encoded).

In this case, the reporter system according to the invention also to be used to detect recombination events in which the Polypeptide with recombinase activity after in the state of Technique described in the cell is introduced. According to a preferred embodiment of the present invention the polypeptide having recombinase activity by transfection or by viral infection with a vector that is one for the Polypeptide containing nucleic acid encoding recombinase activity, placed in the cell, taking the cells under conditions which expresses expression of the polypeptide with recombinase Enable activity. The expression can be under the Control of a constitutive or inducible promoter structure stand. An inducible promoter structure denotes a Promoterstruktur, in which the addition of certain substances a Change in the transcription rate of the to be controlled Nucleotide sequence (such as a gene) results. Furthermore, can a Cre variant (for example, a fusion with a hormone Binding domain), which is first added by adding a substance (for Example, a steroid hormone) becomes "activated", constitutively are expressed. Preferably, this is done only with the addition of this inducing substances are site-specific Recombination. The reporter system facilitates the control of Inducibility of such a system. (Inducible promoter structures, the can be used in the context of the procedure are the Skilled enough known.)

Of course, the reporter system is also suitable also to detect recombination events caused by the inventive method of site-specific DNA recombination and in which the polypeptide has been treated with recombinase Activity for culture supernatant of eukaryotic cell culture was given. According to a preferred embodiment of In the present invention, the polypeptide is a polypeptide with Recombinase activity corresponding to that shown in SEQ ID NO: 2 or SEQ ID NO: 4 has indicated amino acid sequences or a derivative, a Homolog or an active fragment thereof.

According to a further preferred embodiment of the present invention Invention are the recognition sequences loxP sequences with in SEQ ID NO: 5 indicated nucleotide sequence.

The invention further relates to the use of the described Reporter system for the detection of a site-specific DNA Recombination in eukaryotic cells.

As part of an in vivo application, a polypeptide containing Recombinase activity (without heterologous PTD, eg according to SEQ ID No. 2 or 4) injected, for example, into mice that are either transgenic Carry Cre recognition sequences infected with recombinant viruses or a transplant (such as bone marrow) with such Have received sequences. The injection can be directly into the appropriate organ or into the bloodstream can and can, depending by position of the recognition sequences, in a partial Inactivation or activation of genes, or also, for example, in a chromosomal translocation result. The present The invention thus also relates to the use of a polypeptide with Cre recombinase activity (see above) for the preparation of a pharmaceutical preparation for performing a site-specific DNA recombination in eukaryotic cells in vivo. Accordingly Subject of the invention also a polypeptide of the invention with Cre recombinase activity (s. O.) Containing pharmaceutical Preparation which, if appropriate, additionally pharmaceutical contains compatible excipients and carriers.

In addition, the invention includes host cells containing the Reporter system according to the invention, as well as host cells containing the Reporter system according to the invention and the inventive Containing polypeptide with Cre recombinase activity. Regarding the choice of host cells, the invention is not subject Limitations, and besides bacterial cells (like E. coli) also insect cells and (higher) Euraryontenzellen such. B. murine and human cell lines include CHO or BHK cells.

The invention also relates to kits for carrying out a site-specific recombination.

Finally, the present invention relates to kits for Carrying out a method for detecting a site-specific DNA Recombination in eukaryotic cells using a Polypeptides having recombinase activity. The kits can be next to the Reporter system according to the invention u. a. a suitable one Transformation vector and various reagents contained in the Introducing the reporter system into the cells are useful.

The invention will be described in more detail below with reference to examples described.

Description of the figures

Fig. 1 (A) is a schematic representation of the polypeptides nlsCre, PTD-nlsCre, TLM-nlsCre and wtCre after their purification as GST fusion protein and cleavage of the GST portion with thrombin. The only aberration of the recombinant wtCre protein to the native Cre amino acid sequence (Accession No. P06956) is a glycine instead of methionine at position # 1. The modified nlsCre protein has an amino acid substitution (wt threonine instead of alanine) at position 207, the additional SV40 nuclear localization sequence ("NLS", amino acids 11-17 in SEQ ID NO: 4) at the N-terminus and a 9E10 (c-myc) epitope ("myc-tag", amino acids 360-371 in SEQ ID NO : 4) at the C-terminus. In contrast, the proteins TLM-nlsCre and PTD-nlsCre additionally contain the known protein transduction domains PreS2-TLM from HBV and Tat-PTD from HIV-1, respectively. (B) SDS-PAGE for detection of nlsCre, TLM-nlsCre, PTD-nlsCre and wtCre. The Cre proteins were expressed as GST fusion proteins in E. coli, affinity purified, and cleaved from the GST portion with thrombin. The protein preparations were separated by SDS-PAGE in a 12.5% gel, and the proteins were then visualized by Coomassie staining.

Fig. 2 Reporter system for the detection of cre-recombinase activity. In the retroviral reporter construct SFr (SEQ ID NO: 8), the DsRed1 gene (encoded for a red fluorescent protein) is flanked by loxP sequences of the same orientation. Distal to the second loxP sequence is the open reading frame of the eGFP gene (encoded for the "enhanced green fluorescent protein") in which the ATG start codon was deleted. A Kozak consensus sequence and a potential ATG initiation codon in the reading frame of the DsRed gene are located before the first loxP sequence. The transitions are designed so that lox-specific recombination results in excision of the DsRed gene and at the same time puts the eGFP gene in the correct reading frame to the ATG initiation codon. Cells stably transduced with this reporter construct show a red fluorescence. After Cre / lox-specific recombination, eGFP is expressed in place of the DsRed1 gene and the cells show green fluorescence.

Fig. 3 fluorescence micrograph of clonal reporter cells, which were cultured 2 days before in medium with 800 nM recombinant, purified nlsCre protein. Among all treated reporter cell clones, cells with distinct green fluorescence could be detected (shown here for clone 12 and 15) that were not observed in untreated cell populations. The non-homogenous localization of the DsRed1 protein in the cells is probably due to its tendency to aggregate (Clontech product information).

Fig. 4 Detection of lox-specific recombination at the chromosomal DNA level. (A) Positions of primers for a PCR on the reporter construct SFr. Precise, Cre-mediated excision of the DsRed1 gene results in a template that is shortened by 735 bp. (B) Genomic DNA from untreated (-) or recombinant nlsCre protein incubated (+) reporter cells (clones 12 and 15) was used as a template with the PCR primers p1 and p2. In addition to the expected PCR product of 1099 bp for the non-recombined template, the expected product of 364 bp after specific recombination could be detected in the (+) samples. The identity of the visible bands was checked by sequence analysis.

Fig. 5 Dependence of the recombination rate on the concentration of nlsCre protein in the medium. (A) Reporter cells were incubated for 6 hours in medium (MEM with 10% FCS) with different concentrations of nlsCre and analyzed 48 hours later by flow cytometry for eGFP and DsRed1 expression (see also Section B for evaluation). The proportion of eGFP-positive cells is shown as a function of the nlsCre concentration in the medium. Under these conditions, a recombination rate of over 50% was achieved by the addition of 5 μM nlsCre. (B) Reporter cells were incubated for 6 hours with 2.5 μM nlsCre in serum-free medium (MEM without FCS). After 48 hours, the cells were analyzed by flow cytometry and DsRed1 expression against eGFP expression was plotted and evaluated using the "Cellquest" software (Becton Dickinson). While no expression of eGFP could be detected in untreated cells (mock),> 50% of the nlsCre-treated cells were eGFP-positive.

Fig. 6 Cre / lox-specific recombination in murine bone marrow cells after addition of nlsCre protein to the medium. Schematic representation of the endogenously "flooded" C / EBPα locus before and after lox-specific recombination. By the first PCR with the primers p3 and p4 the C / EBPα ^{fl / fl} -Lokus could be proven in the genomic DNA of the bone marrow cells. With the second PCR (primers p5 and p6), it was only in the genomic DNA of nlsCre or PTD-nlsCre-treated bone marrow cells that the specific 400 bp product that results after excision of C / EBPα could be detected. The 400 bp products were verified by sequence analysis.

Fig. 7 specific recombination after addition of unmodified Cre recombinase. Reporter cells were incubated with 1 μM of the wtCre protein for 3 hours. After two days, approximately 1.5% eGFP-expressing cells could be detected by fluorescence microscopy.

Examples

Cloning, annealing of oligonucleotides, phosphorylation of DNA fragments and the preparation of chromosomal DNA were prepared according to standard protocols (Ausubel et al. (1994) Current protocols in molecular biology John Wiley & Sons. New York, USA) carried out. PCR reactions were performed with Taq polymerase (Qiagen) or Platinum PfxI polymerase (Gibco BRL) according to recommendations of corresponding company performed. The mung bean nuclease digestion was carried out according to the recommendation of Promega.

example 1 Preparation of recombinant, purified cerebrum recombinase Example 1a Construction of vectors for the expression of Cre recombinase

As an expression vector for the production of GST-Cre Fusion proteins in E. coli was used pGEX-2T (KT) (SEQ ID NO: 9), a modified pGEX-2T vector with the Multi-cloning region from pEG (KT) (Mitchell et al. (1993) Yeast 9: 715-723). For the cloning of pGEX-nlsCre was pGEX-2T (KT) with XhoI linearized, the overhangs filled with the Klenow fragment and then cut with NcoI. A modified Cre gene (Bergemann et al. (1995) NAR 23: 4451-6) was prepared from R815 (Prassolov et al. (2001) Exp Hematol. 29: 756-65) with the Restriction endonucleases NcoI and XbaI isolated, the XbaI overhang also was smoothed with the help of Klenow fragment. The Cre coding DNA fragment was subsequently mixed with pGEX-2T (KT) ligated.

For the construction of pGEX-TLM-nlsCre the Oligonucleotides TLM + (SEQ ID NO: 10; 5'-GATCCCCGCTGTCTTCCATTTTCTCCCGTATC- GGCGATCCGGGTGGCGGTTC-3 ') and TLM (SEQ ID NO: 11; 5'- CATGGAACCGCCACCCGGATCGCCGATACGGGAGAAAATGGAAGACAGCGGG-3 '), for pGEX-PTD-nlsCre the oligonucleotides PTD + (SEQ ID NO: 12, 5'- GATCCTATGGCCGTAAAAAGCGCCGTCAGCGCCGCCGTCC-3 ') and PTD (SEQ ID NO: 13; 5'-CATGGGACGGCGGCGCTGACGGCGCTTTTTACGGCCATAG-3 ') and phosphorylated. The resulting 52 bp and 40 bp fragments, respectively were each inserted into the BamHI / NcoI-linearized plasmid pGEX- nlsCre inserted.

The introduced DNA fragments encode amino acid sequences from the PreS2 glycoprotein of HBV or from the tat protein of HIV-1. Both sequences were shown to be uptake of recombinant reporter proteins into cells (Oess and Hildt 2000 Gene Therapy 7: 750-8; Schwarze et al., 1999 Science 285: 1569-72). The modified Cre gene from R815 encodes for the Cre recombinase from the bacteriophage P1 with a Amino acid exchange (threonine instead of alanine) at position 207, additionally the SV40 nuclear localization sequence (amino acids 11-17 in SEQ ID NO: 4) at the N-terminus and a 9E10 (c-myc) epitope (Amino acids 360-371 in SEQ ID NO: 4) at the C-terminus. The modified Cre recombinase is hereinafter referred to as "nlsCre" designated.

For the preparation of a Cre recombinase without C and N-terminal Modifications were first performed using PCR with pGEX-nlsCre Template and the oligonucleotides Cre-N (SEQ ID NO: 14, 5'- GAAGAGGAAGGGATCCAATTTACTGACCG-3 ') and Cre-C (SEQ ID NO: 15; 5'- CAGAAATAAGCTTTTGTTCCTAATCGCCATC-3 ') using the PfxI Polymerase (Gibco Life Technologies) performed. The resulting 1060 bp fragment was digested with the restriction endonucleases BamHI and HindIII, and the resulting 683 bp (BamHI) HindIII) and 355 bp (BamHI) fragments were sequentially added to the BamHI-HindIII opened vector pGEX-2T (KT).

Furthermore, the base exchange that became the A207T mutation in nlsCre using oligonucleotides 207+ (SEQ ID NO: 16; 5'-GGTTAGCACCGCAGGTGTAG-3 ') and 207- (SEQ ID NO: 17; 5'-CTACACCTGCGGTGCTAACC-3 ') by means of the overlap extension Method (Ho et al., (1989) Gene 77: 51-59) wtCre gene (SEQ ID NO: 1) in the resulting vector pGEX-wtCre was verified by sequence analysis.

After the expression described in Example 1b, purification and thrombin digestion of the GST-wtCre fusion protein becomes "wtCre" protein (SEQ ID NO: 2), which differs from the described, "natural" Cre recombinase from the bacteriophage P1 (Accession NO: P06956) only at position 1 (glycine instead Methionine).

Example 1b Expression and Affinity Purification of the Cre Fusionsoroteine

Transformants of the E. coli expression strain BL21-CodonPlus (DE3) -RP (Stratagene) with the expression plasmids pGEX-nlsCre, pGEX-TLM-nlsCre, pGEX-PTD-nlsCre and pGEX-wtCre were incubated in 1 l LB (+ 100 mg / 1 ampicillin and 50 mg / l chloramphenicol) to an OD ₆₀₀ of 0.7 and induced for 8 hours at 25 degrees with 0.5 mM IPTG. The digestion of the cells was carried out in 40 ml of PBS, 0.5% Triton-X-100, 0.5 mM Pefabloc (Roche Diagnostics GmbH) by ultrasound. After centrifugation at 4000 × g, the supernatant was incubated with 500 μl glutathione-Sepharose 4B (Amersham Pharmacia) at 4 ° C. for 3 h in the rotary wheel. The Sepharose was washed 3 × 10 min in the rotary wheel with 40 ml PBS, pH 7.0. The identity of the GST-Cre fusion proteins was checked immunologically with the monoclonal anti-myc antibody (9E10). To cleave the Cre proteins from the GST portion, the Sepharose was treated with 10 units thrombin (Amersham Pharmacia) in 1 ml PBS / 100 mM Na ₂ HPO _{4 for} 16 hours at 25 degrees in a spinwheel.

The Cre proteins released by thrombin digestion showed a running behavior in the SDS gel which corresponds to the expected molar masses ( FIGS. 1, A and B). NlsCre, TLM-nlsCre, PTD-nlsCre and wtCre were obtained at a concentration of 100 to 500 ng / μl (2-12 μM).

Example 2 Representation of a reporter system for the detection of Cre activity Example 2a Construction of the Retroviral Reporter Construct SFr ( Figure 2)

• 1. Die loxP-Sequenz vor dem DsRed-Gen (loxP1) wurde durch Hybridisierung der Oligonukleotide loxP1+ (SEQ ID NO: 18; 5'- GGCCGCATGATAACTTCGTATAATGTATGCTATACGAAGTTATC-3') und loxP1- (SEQ ID NO: 19; 5'-CATGGATAACTTCGTATAGCATACATTATACGAAGTTATCATGC-3'), die loxP-Sequenz nach dem DsRed-Gen (loxP2) durch Hybridisierung der Oligonukleotide loxP2+ (SEQ ID NO: 20; 5'- GATCTTAGTCGACGCGTATAACTTCGTATAATGTATGCTATACGAAGTTATCAGGCCTGCAC- 3') und loxP2- (SEQ ID NO: 21; 5'-GTGCAGGCCTGATAACTTCGTATAGCATACATTATACGAAGTTATACGCGTCGACTAA-3') erhalten. Die loxP- Fragmente wurden anschließend mit Polynukleotidkinase (MBI- Fermentas) phosphoryliert.
• 2. Für die Konstruktion von pDsRed1-loxP2 wurde pDsRed1-C1 (Clontech) mit XmaI geschnitten, die Überhänge mit Hilfe des Klenow- Fragmentes geglättet, anschließend mit BglII verdaut und das loxP2-Fragment in den so vorbereiteten Vektor insertiert.
• 3. pKS-loxP1-DsRed1(Cterm)-loxP2 entstand durch eine 3-Fragmente- Ligation des loxP1-Fragmentes und des 326 bp NcoI-BamHI- Fragmentes aus pDsRed1-loxP2 mit dem Not1-BamHI-geöffneten Vektor pBlueskript(KS+) (Stratagene). Durch nachfolgende Insertion des 423 bp-NcoI-Fragmentes aus pDsRed1-C1 in den NcoI-geschnittenen pKS-loxP1-DsRed1(Cterm)-loxP2 in der richtigen Orientierung wurde das Zwischenkonstrukt pKS-loxP1-DsRed1-loxP2 erhalten.
• 4. Die loxP1-DsRed1-loxP2-Sequenz wurde mittels NotI/XmaI-Verdau aus pKS isoliert, und die Überhänge wurden mit Mung Bean-Nuklease (Promega) geglättet. pSFβ91-eGFP(tag) (Wahlers et al. (2001) Gene Ther 8: 477-86, Schambach et al. (2000) Mol. Ther. 2: 435-45) wurde mit NcoI linearisiert, die Überhänge mit Mung Bean-Nuklease geglättet, und das loxP1-DsRed1-loxP2-Fragment wurde insertiert. Nach Überprüfung der Orientierung und der Übergänge mittels Sequenzanalyse konnte pSFβ91-loxP-DsRed1-loxP-eGFP mit einem "in frame"-Übergang zwischen loxP2 und dem eGFP-Gen isoliert werden. Der "in frame"-Übergang gewährleistet die Translation von eGFP nach Cre-vermittelter Excision durch die korrekte Positionierung zum ATG-Startkodon vor loxP1.
• 5. Zur Erhöhung der Expression des retroviralen Reporterkonstruktes in den Zielzellen wurde das HindIII-Fragment mit wPRE (woodchuck HBV posttranscriptional regulatory element, Zufferey et al. (1999) J Virol. 73: 2886-92) aus pKS-γwPRE isoliert (Schambach et al. (2000) Mol Ther. 2: 435-45) und in die HindIII-Stelle von SFβ91-loxP-DsRed1-loxP-eGFP insertiert. Die richtige Orientierung wurde durch Restriktionsverdau überprüft.

The construction of the reporter construct SFr (= SFβ91-loxP-DsRed1-loxP-eGFP-wPRE, SEQ ID NO: 8) proceeded through several intermediate steps. All plasmid amplifications were performed in RecA-deficient E. coli strains (DHSα or DH10B from Gibco BRL) at a growth temperature of 28 ° C.

• 1. The loxP sequence before the DsRed gene (loxP1) was isolated by hybridization of the oligonucleotides loxP1 + (SEQ ID NO: 18, 5'-GGCCGCATGATAACTTCGTATAATGTATGCTATACGAAGTTATC-3 ') and loxP1 (SEQ ID NO: 19; 5'-CATGGATAACTTCGTATAGCATACATTATACGAAGTTATCATGC- 3 '), the loxP sequence after the DsRed gene (loxP2) by hybridization of the oligonucleotides loxP2 + (SEQ ID NO: 20; 5'-GATCTTAGTCGACGCGTATAACTTCGTATAATGTATGCTATACGAAGTTATCAGGCCTGCAC-3') and loxP2- (SEQ ID NO: 21; 5'-GTGCAGGCCTGATAACTTCGTATAGCATACATTATACGAAGTTATACGCGTCGACTAA -3 '). The loxP fragments were subsequently phosphorylated with polynucleotide kinase (MBI-Fermentas).
• 2. For the construction of pDsRed1-loxP2, pDsRed1-C1 (Clontech) was cut with XmaI, the overhangs were smoothed using the Klenow fragment, then digested with BglII, and the loxP2 fragment inserted into the prepared vector.
• 3. pKS-loxP1-DsRed1 (Cterm) -loxP2 was generated by a 3-fragment ligation of the loxP1 fragment and the 326 bp NcoI-BamHI fragment from pDsRed1-loxP2 with the Not1-BamHI-opened vector pBluescript (KS +) ( Stratagene). Subsequent insertion of the 423 bp NcoI fragment from pDsRed1-C1 into the NcoI-cut pKS-loxP1-DsRed1 (Cterm) -loxP2 in the correct orientation yielded the intermediate construct pKS-loxP1-DsRed1-loxP2.
• 4. The loxP1-DsRed1-loxP2 sequence was isolated from pKS by NotI / XmaI digestion and the overhangs were blunted with mung bean nuclease (Promega). pSFβ91-eGFP (tag) (Wahlers et al. (2001) Gene Ther 8: 477-86, Schambach et al. (2000) Mol. Ther. 2: 435-45) was linearized with NcoI, the overhangs were mung bean coated. Nuclease smoothed and the loxP1-DsRed1-loxP2 fragment was inserted. After orientation and transitions were checked by sequence analysis, pSFβ91-loxP-DsRed1-loxP-eGFP could be isolated with an in-frame transition between loxP2 and the eGFP gene. The "in frame" transition ensures the translation of eGFP after Cre-mediated excision by correct positioning to the ATG start codon before loxP1.
• 5. In order to increase the expression of the retroviral reporter construct in the target cells, the HindIII fragment was isolated from pKS-γwPRE with wPRE (woodchuck HBV posttranscriptional regulatory element, Zufferey et al., (1999) J Virol 73: 2886-92) (Schambach et (2000) Mol Ther. 2: 435-45) and inserted into the HindIII site of SFβ91-loxP-DsRed1-loxP-eGFP. The correct orientation was checked by restriction digestion.

Example 2b Cloning of mouse fibroblasts after transduction with SFr

The packaging cell line Phoenix-gp (http: / / www.stanford.edu/group/nolan/) was using the reporter construct SFr as well Expression plasmids for MoMuLV gag / pol (the expression plasmid for gag / pol, pSVgag / pol, contains the gagpol gene from MoMuLV under the Control of the SV40 promoter) and for the ecotrophic coat protein MoMuLV (Morita et al. (2000) Gene Ther. 7: 1063-66) transfected. The mouse fibroblast cell line SC-1 was infected with the virus infected supernatants infected. After three days, SC-1 Cells in which the reporter construct was stably integrated through their red fluorescence microscopically and by flow cytometry be detected. By means of single cell deposits 5 SC-1 Clones (clone 3, clone 8, clone 10, clone 12 and clone 15) isolated which show stable expression of DsRed1. None In the observation period (weeks to Months) a spontaneous recombination can be observed, which in expression of eGFP.

To package the reporter construct SFr in ecotrophic viruses, Phoenix-gp packaging cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 2 mM glutamine and 10% fetal calf serum (FCS, Sigma). Transfection of 10 ⁷ Phoenix-gp with 10 ug expression plasmid for MoMuLV gag, pol, 5 ug SFr and 3 ug expression plasmid for MoMuLV env was determined by the Ca ₃ (PO _{4) 2} - Method (. Ausrubel et al (1994), supra) carried out. For transfection and harvest of supernatants after 44 hours, DMEM / FCS with 20 mM HEPES, pH 7.5 was used. For infection, 50 .mu.l of the virus-containing supernatant in 500 .mu.l of DMEM / FCS / HEPES + 4 .mu.g / ml protamine sulphate were added to 10 ⁵ SC-1 cells (ATCC NO CRL-1404) and centrifuged for 90 min at 20.degree. C. and 800.times.g.

Cultivation of SC-1 reporter cells was done in Eagle's Minimum Essential Medium (MEM, Sigma) with 2mM glutamine and 10% FCS at 37 ° C in the incubator.

Example 3 loxP-specific recombination after addition of purified Cre recombinase without heterologous transduction domain

To investigate whether externally added thereto Cre protein can trigger a lox specific excision into the reporter cells, various SFr-transduced were SC-1 clones (in each case 10 ⁵ cells per cell culture dish with 1.5 cm diameter) with affinity purified nlsCre , PTD-nlsCre or TLM-nlsCre (final concentration 0.8-1 mm in MEM with the supplements mentioned in Example 2 and 50 U / ml penicillin, 50 mg / ml streptomycin) incubated for 3-4 hours in an incubator. After 48 hours, the proportion of eGFP-positive cells was determined by flow cytometry using the FACScaliber (Becton Dickinson). Surprisingly, the proportion of eGFP-positive cells after addition of the nlsCre protein without heterologous protein transduction domain was at least as high as after addition of PTD-nlsCre and TLM-nlsCre (Table 1). (n = number of experiments, range of variation of the proportion of eGFP-positive cells). Table 1

After addition of 0.8 μM nlsCre protein to various clonal reporter cells (clones 3, 8, 10, 12 and 15), microscopic examination revealed that all cells with a weak green fluorescence were detected after 24 h in all batches after 48 h Hours became clearer (see Fig. 3). The eGFP-positive cells showed no or significantly reduced red fluorescence compared to the surrounding cells. The proportion of eGFP-positive cells was by flow cytometric analysis between 2 and 4%. By contrast, no eGFP-positive cells could be detected under the control cells.

To demonstrate Cre-specific recombination at the DNA level, chromosomal DNA was isolated from the nlsCre-treated mixed populations of clones 12 (4% eGFP positive) and 15 (2.5% eGFP positive). The chromosomal DNA was used as template for a polymerase chain reaction in which the (+) primer p1 (SEQ ID NO: 22; 5'-ACGCCGAAACCGCGCCGCGCGTC-3 ') 173 bp before the first loxP sequence and (-) primer p2 (SEQ ID NO: 23; 5'-GCAGATGAACTTCAGGGTCAGCTTGC-3 ') binds 154 bp after the second lox sequence (see Fig. 4A). In the first PCR, the mixed populations show, in addition to the 1099 bp product to be expected from the template of the unrecombined reporter construct, another product of 364 bp, which is the length of the expected product from the recombined template (ie after excision of DsRed1 Gene). The 364 bp product is absent when genomic DNA from the untreated reporter cells was used as a template ( Figure 4B). Sequence analysis of gel-isolated PCR products confirmed the precise, Cre / lox-specific recombination.

This example clearly shows that the nlsCre protein without heterologous protein transduction domain after external addition to the Cell nucleus of reporter cells and there reaches a specific Recombination initiates.

Example 4 Efficient loxP-specific recombination by nlsCre

To increase the efficiency of recombination by nlsCre, were initially higher concentrations of the purified protein used, and the incubation was extended.

For this purpose, reporter cells with 1.0, 1.5, 2.5 and 5.0 mm nlsCre- Concentrations in MEM (with 2 mM glutamine, 10% FCS, 50 U / ml Penicillin and 50 mg / ml streptomycin) for 6 hours at 37 ° C incubated and the medium was changed to fresh MEM. After two days became the proportion of eGFP positive cells determined by flow cytometry with a FACScaliber.

As expected, the recombination efficiency increases with increasing nlsCre concentration. Incubation with 5 μM nlsCre resulted in a recombination rate of over 50% ( FIG. 5A). When incubation was carried out with nlsCre in serum-free medium (MEM with 2 mM glutamine and 50 U / ml penicillin, 50 mg / ml streptomycin), it was possible to measure a recombination rate of more than 50% after the addition of 2.5 μM nlsCre ( FIG. 5B). A reduction in recombination efficiency in the presence of serum (here 40% versus 53% after addition of 2.5 μM nlsCre) may possibly be due to aggregation of the recombinant protein with serum proteins.

Example 5 Externally added nlsCre protein can produce a loxP- initiate specific excision in murine bone marrow cells

Bone marrow cells from a C57Bl / 6 mouse strain in which the endogenous C / EBP gene is flanked with loxP sequences (Zhang et al. (2001) Blood 98: 792a-793a, see Fig. 6) were first removed by rinsing out upper - and lower leg prepared. Aliquots of 2 x 10 ⁶ cells were incubated for 7 hours at 37 ° C in 500 μl of a 1: 1 mixture of 2x DMDM (with doubly concentrated cytokines (see below), 100 U / ml penicillin, 100 mg / ml streptomycin) and PBS (mock) or PBS incubated with purified, recombinant nlsCre protein. Final concentrations were 1.5 μM nlsCre and 1.5 μM PTD-nlsCre, respectively. The cells were spun down and the medium was challenged with IMDM (containing 20% FCS, 0.1% bovine serum albumin, 2 mM glutamine, 5 ng / ml human IL-6, 10 ng / ml murine IL-3 and 50 ng / ml murine SCF; all recombinant growth factors from R & D Systems). After one week of culturing the cells at 37 ° C. in the incubator, genomic DNA of the three batches was prepared and used as template for two different polymerase chain reactions (PCRs).

The first PCR using the oligonucleotides p3 (SEQ ID NO: 24, 5'-TGGCCTGGAGACGCAATGA-3 ') and p4 (SEQ ID NO: 25; 5'-CGCAGAGATTGTGCGTCTTT-3') serves to detect the non-recombined allele C / EBPα ^{fl / fl} , which gives as template a 269 bp fragment as a PCR product. This product could be detected in all batches containing genomic DNA of the C / EBPα ^{fl / fl} mouse strain but not in the negative control without DNA template ( Figure 6). For the second PCR, the oligonucleotides p5 (SEQ ID NO: 26, 5'-GCCTGGTAAGCCTAGCAATCCT-3 ') and p6 (SEQ ID NO: 27; 5'-TGGAAACTTGGGTTGGGTGT-3') were used, containing a 400 bp fragment with the recombined template (ie, after excision of the C / EBPα gene) and theoretically yielded a 6.6 kb fragment with the non-recombined template. While the 6.6 kb fragment did not form under the chosen conditions (standard with Qiagen Taq polymerase, 30 sec denaturation, 45 sec annealing at 60 ° C and 45 sec extension phase per cycle), a 400 bp product was formed detected only in the batches whose template was derived from cells incubated with recombinant nlsCre or PTD-nlsCre ( Figure 6). Sequencing of the 400 bp PCR products confirmed the correct excision of C / EBP.

This example shows that the direct transfer of the recombinant, purified Cre protein not just for a specific Recombination of the reporter construct SFr in the mouse Fibroblast cell line SC-1 is suitable, but in principle also for the recombination of another template (here the "flopped" C / EBPα gene) in primary cells (here mouse bone marrow) used can be.

Example 6

LoxP-specific recombination after transduction of unmodified Cre protein

The investigations set forth in Examples 3 and 4 show a Cre-specific recombination in clonal reporter cells, which recombinant, purified Cre protein over the culture medium was fed. The principal property of Cre recombinase, to get into intact cells, is independent of the known protein transduction domains Tat-PTD or PreS2-TLM. In order to examine whether the "artificially attached" amino acid sequences (the SV40 NLS at the N-terminus or the 9E10 epitope at the C-terminus) of the modified nlsCre protein used here To convey property, a cre protein should be without modifications getting tested.

Reporter cells were incubated with 1 μM of the wtCre protein (see Example 1) for 3 hours. After two days, eGFP-positive cells could be detected by fluorescence microscopy ( FIG. 7). The average recombination rate is about 1.5% (n = 7, 0.6-2.5%).

Since the transduction of the Cre protein is apparently independent of heterologous amino acid sequences, the Cre recombinase from bacteriophage P1 is likely to have an endogenous protein transduction domain. The slightly higher recombination efficiency of nlsCre under the same conditions (see Table 1) can be attributed to the acceleration of the nuclear transport by the SV40 NLS. SEQUENCE LISTING

Claims (37)

1. In vitro method for site-specific DNA recombination in eukaryotic cells, in which a) adding a polypeptide having Cre recombinase activity, which does not comprise a heterologous protein transduction domain, to a eukaryotic cell culture whose cells contain at least one DNA region containing at least one recognition sequence recognized by the polypeptide having Cre recombinase activity , and you b) culturing the cells under conditions allowing recombinant DNA. 2. The method according to claim 1, characterized in that the Polypeptide with Cre recombinase activity one of the in SEQ ID NO: 2 or SEQ ID NO: 4 indicated amino acid sequences or a derivative, a homologue or an active Fragment of the same is. 3. Process according to one of claims 1 to 2, characterized characterized in that the eukaryotic cells are human or murine Cells are. 4. The method according to claim 3, characterized in that the human or murine bone marrow cells or cells Fibroblasts are. 5. The method according to any one of claims 1 to 4, characterized characterized in that the polypeptide with Cre recombinase Activity for culture supernatant of eukaryotic cell culture gives. 6. The method according to claim 5, characterized in that one the polypeptide having Cre recombinase activity in one Concentration of 100 nM to 30 uM admits. 7. The method according to any one of claims 1 to 6, characterized characterized in that the recognition sequences loxP sequences with are the nucleotide sequence given in SEQ ID NO: 5. 8. Use of a polypeptide having Cre recombinase activity, which does not contain a heterologous transduction domain, for Carrying out a site-specific DNA recombination in eukaryotic cells, which contain cells that are at least contain a DNA region containing at least one Contains recognition sequence derived from the polypeptide Recombinase activity is detected in the presence of the Cultured polypeptide under conditions that produce a DNA Allow recombination. 9. Use according to claim 8, characterized in that the Recognition sequences loxP sequences with the in SEQ ID NO: 5 are indicated nucleotide sequence. 10. Use of a polypeptide having Cre recombinase activity, which does not contain a heterologous transduction domain, for Preparation of a pharmaceutical preparation for carrying out a site-specific DNA recombination in eukaryotic Cells in vivo. 11. Polypeptide with Cre recombinase activity, thereby in that the polypeptide has one of the sequences shown in SEQ ID NO: 2 or SEQ ID NO: 4 has indicated amino acid sequences or a derivative, a homologue or an active fragment the same except for the natural Cre recombinase the bacteriophage P1. 12. Nucleic acid containing a polypeptide with cre recombinase Activity encoded, characterized in that the Nucleic acid is one of SEQ ID NO: 1 or SEQ ID NO: 3 having indicated nucleotide sequences. 13. Isolated cell containing a polypeptide with cre recombinase Activity according to claim 11. 14. An isolated cell comprising a nucleic acid according to claim 12 contains. 15. Cell according to claim 13 or 14, characterized in that it is a bacterial cell or a eukaryotic cell is. 16. Cell according to claim 15, characterized in that it has a E. coli cell, a yeast cell or an insect cell. 17. Reporter system for the detection of a site-specific DNA recombination in eukaryotic cells, characterized in that the reporter system is a DNA construct in which a) a first reporter gene flanked by two recombinase recognition sequences in a manner resulting in excision of said first reporter gene in the presence of a polypeptide having recombinase activity, and b) a second reporter gene is located downstream of the first reporter gene, upstream of the two recognition sequences being located an initiation codon and a ribosome binding site enabling translation of the first reporter gene terminated by a stop codon between the two recognition sequences and in the region between the two reporter genes are not localized to a functional initiation codon; and c) by site-specific recombinant DNA, the second reporter gene is brought to a position to the initiation codon enabling translation of the second reporter gene; and d) the gene products of the two reporter genes are to be distinguished from one another. 18. Reporter system according to claim 17, characterized that the recognition sequences loxP sequences with the in SEQ ID NO: 5 nucleotide sequence. 19. Reporter system according to claims 17 or 18, characterized characterized in that at least one of the reporter genes for a encodes fluorescent protein. 20. Reporter system according to claim 19, characterized the fluorescent protein is as described in SEQ ID NO: 6 or having the sequence given in SEQ ID NO: 7. 21. Reporter system according to claims 17 to 20, characterized in that the reporter system has the sequence shown in SEQ ID NO: 8 has indicated sequence. 22. A method for detecting site-specific recombinant DNA in eukaryotic cells using a polypeptide having recombinase activity, wherein a) introducing a reporter system according to claims 17 to 21 and a polypeptide having recombinase activity into a eukaryotic cell and cultivating the cells under conditions allowing DNA recombination; and you b) detecting a successful recombination on the basis of the encoded by the second reporter gene gene product. 23. The method according to claim 22, characterized in that one the polypeptide with recombinase activity by transfection or viral infection with a vector in the cells introducing a coding for the polypeptide Nucleic acid sequence containing the cells under conditions cultured, which allow the expression of the polypeptide. 24. The method according to claim 22, characterized in that one the polypeptide having recombinase activity in the cells introduces the polypeptide with recombinase activity to the culture supernatant of the eukaryotic cell culture. 25. The method according to claim 24, characterized in that the Polypeptide with recombinase activity according to a polypeptide Claim 11 is. 26. The method according to any one of claims 22 to 25, characterized in that the recognition sequences are loxP sequences with the nucleotide sequence given in SEQ ID NO: 5. 27. Use of a reporter system according to claims 17 to 21 for the detection of a site-specific DNA Recombination in eukaryotic cells. 28. Isolated host cell, which is a reporter system according to Claims 17 to 21 contains. 29. Host cell according to claim 28, characterized in that the cell additionally contains a polypeptide with cre recombinase Activity after claim 11 contains. 30. Cell according to claim 28 or 29, characterized in that it is a bacterial cell or a eukaryotic cell is. 31. Cell according to claim 30, characterized in that it has a E. coli cell, a murine or human cell. 32. Method for the production of a polypeptide with Cre Recombinase activity according to claim 11, characterized characterized in that one for the amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 4 encoding nucleic acid sequence ligated into an expression vector, this one in Brings in host cells and the cells underneath to express the Polypeptide suitable conditions. 33. The method according to claim 32, characterized in that the Nucleic acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 3 has indicated sequence. 34. Kit for carrying out a method according to claims 1 to 7. 35. Kit for carrying out a method according to the claims 22 to 26. 36. Pharmaceutical preparation for carrying out a Site-specific DNA recombination in eukaryotic cells. 37. Pharmaceutical preparation according to claim 36, characterized characterized in that the preparation additionally pharmaceutical contains compatible excipients and / or carriers.