WO2002081675A2

WO2002081675A2 - Method for detecting homologous recombination events

Info

Publication number: WO2002081675A2
Application number: PCT/DE2002/001207
Authority: WO
Inventors: Felix Bestvater; Tobias A. Knoch; Eberhard Spiess
Original assignee: Deutsches Krebsforschungszentrum
Priority date: 2001-04-04
Filing date: 2002-04-03
Publication date: 2002-10-17
Also published as: WO2002081675A3; AU2002304884A1; DE10116826A1

Abstract

The invention relates to a method for detecting recombination events which are based on the transfection of the desired cell with one or more vectors which contain different genes for detecting proteins, preferably different fluorescent proteins. According to the invention, recombination event occurring one the basis of homology between different genes leads to a detectable alteration in an expression pattern of said different genes.

Description

Detection method for homologous recombination events

The present invention relates to a method for determining recombination events which is based on the transfection of the desired cell with one or more vectors which contain different genes for detectable proteins, e.g. contain different fluorescence proteins, a recombination event taking place due to homology between the different genes leading to a detectable altered expression pattern of the different genes.

The previous determinations of recombination processes within the cell (e.g. homologous recombination, repair mechanisms) via in vivo or in vitro methods, e.g. for examining the effects of chemical or physical (e.g. radiation, heat etc.) influences etc., are essentially based that the cells are lysed and then the proteins involved in the recombination are quantified with the aid of ELISA tests, Western blots, DNA expression chips or similar methods, from which one can then indirectly deduce the ability, for example, for homologous recombination. Another method is the detection of proteins using immunohistological staining methods. The cells have to be fixed here, then a corresponding dye-carrying antibody is allowed to bind to them and the binding is detected, for example, by microscopic methods. However, the methods used to date have a number of disadvantages. So the previous in vivo investigations of homologous recombination were mostly measured by the amount of a protein / substance involved and not directly by the DNA sequence, which therefore does not allow any statement about the actual activity of the factors involved necessary preparation of the cells is not artifact-free. It is also disadvantageous that the tests cannot be continued on the same cells without cell consumption and that measurements on individual cells are also not possible. Finally, these methods are also expensive due to the need for a number of different substances, kits, etc. These disadvantages also occur with the immunological staining methods, ELISA tests etc. previously used for mass screening. on. In addition, mass screening is only possible to a limited extent and is not possible in vivo, and selection and cultivation of cells with certain recombination properties is hardly possible.

In addition to the detection of proteins, an altered DNA sequence has also been examined directly to investigate recombination events, i.e. obtained from the cells and then sequenced. First, a DNA sequence is introduced into the cells or the organism, which is then changed (e.g. repaired) in the cells themselves or a sequence that is already in the cells is changed. However, this only leads to a positive / negative decision with regard to whether homologous recombination occurs or not, and even with this procedure no in vivo tests are possible. In addition, this process is also associated with high costs and is time-consuming, since the required DNA constructs have to be produced via complex restriction, ligation, transfection, knockout or chemical synthesis processes.

The present invention is therefore based on the technical problem of providing a method for examining homologous recombination which does not have the disadvantages of the previous methods discussed above.

This technical problem is solved by providing the embodiments characterized in the claims. It emerged during the experiments leading to this invention that the disadvantages of the previous methods described above can be avoided by using a method which is based on the transfection of the cell, in the recombination processes (eg homologous recombination, replication, repair mechanisms and similar processes, hereinafter referred to as “RRRP”) are to be investigated, based on one or more vectors which contain different genes for detectable proteins, for example different fluorescent proteins, a recombination event taking place due to a homology between the different genes demonstrably changes in expression patterns of the different genes leads. The following problems can be solved in vitro and in vivo with the method according to the invention:

(1) determining the occurrence and quantification of RRRP in cells under the influence of chemical or physical influences;

(2) Determination of the occurrence and quantification of RRRP in living cells depending on the DNA, chromatin, nucleus, cell structure and cell dynamics, in the cell cycle, in cell differentiation, in different cell types or in dependence on other cell-dependent / physiological factors, eg cell physiological changes such as increased homologous recombination;

(3) determining the occurrence and quantification of RRRP in living cells as a function of the tumor genicity of the cells and, conversely, determining the tumor genicity of the cells;

(4) Determination of the occurrence and quantification of RRRP in tissues / organs in the living organism depending on the above items 1 to 3;

(5) Investigation of repair mechanisms and gene regulation control in cells or in an organism depending on items 1 to 4 above;

(6) Examination of replication processes in cells or in an organism depending on items 1 to 4 above;

(7) Optimization and screening of recombination, replication and repair systems as well as their individual components depending on items 1 to 6 above; and

(8) Selection and cultivation of cells or organisms with certain (desired) recombination properties.

A DNA sequence dependency can also be determined by means of the above investigations, and these can also relate to viruses or the relationship of viruses to cellular organisms.

The advantages which can be achieved by means of the method according to the invention are therefore, among others: (1) The possibility of integral in vivo analysis of the RRRP, ie, for example, the process of homologous recombination is measured directly on the DNA sequence and not, for example, on the amount of a protein or substance involved.

(2) The possibility of in vivo analysis of the issues discussed above.

(3) Extensive freedom from artifacts and cost and time savings due to the elimination of complex preparation methods by in-vivo assessment under a microscope (real-time analysis).

(4) The investigations can be continued on the same cells without cell consumption; Measurements on individual cells are now possible.

(5) Mass screening is easy (and also in vivo).

(6) The possibility of selecting and growing cells with certain recombination properties.

The present invention thus relates to a method for determining recombination events, the method comprising the following steps:

(a) Production of a vector or a plurality of vectors which contain a gene coding for a first detectable protein (NP ^ gene and a gene coding for a second detectable protein (NP ₂ ), both genes having regions which are homologous to one another and wherein before the recombination event to be detected (1 ) no or only one NP is detectable or (2) the two NPs are detectable in different cell compartments;

(b) transfecting the desired cell and culturing the cell; and

(c) Determining the presence and / or concentration of the proteins NP-i and NP ₂ , a changed presence, concentration and / or subcellular localization of the proteins NPi and NP _{2 being} an indication of a recombination event that has taken place.

If a recombination event is detected in step (c), step (d) can follow, which involves the production of a cell lysate, the purification of nucleic acids and the determination of the recombination event Sequencing includes. A cell lysate for the production of the nucleic acids, their purification and sequencing can be produced using routine methods known to the person skilled in the art, for example using the method described in the examples below.

Depending on the design of the method according to the invention, the genes coding for NPi and NP ₂ or other required genetic elements can be distributed on a single vector or on two or more vectors. General methods known in the art can be used to construct vectors containing the NP-encoding genes and appropriate control sequences. These methods include, for example, in vitro recombination techniques, synthetic methods, and in vivo recombination methods, as described, for example, in Sambrook et al., Molecular Cloning: A Laboratoty Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY ( 1989)). are described. The genes coding for NP can also be inserted in connection with a DNA sequence coding for another protein or peptide, so that the NPs can be expressed, for example, in the form of fusion proteins.

The method can be carried out both in vivo and in vitro. The in vivo procedure is carried out on the living organism, while the in vitro procedure works with appropriate cell extracts. Then either the nucleic acid product can be detected by sequencing or by the expression of the corresponding construct. Since protein synthesis is not quite straightforward in vitro, the corresponding nucleic acid can also be brought back into cells in order to be expressed and detected there. Care should then be taken to ensure that recombination etc. can occur again. In order to then quantify the recombination more precisely, an exact calibration of the system is necessary.

The person skilled in the art can also produce the above vector or vectors according to standard methods in such a way that the conditions relating to step (c) are met. For example, in the construction of the vector or vectors that only the gene for NPi is functionally linked to a promoter, but not the gene for NP ₂ . After transfection of the cells, only NP * ι is thus expressed. In an occurred recombination between the gene and NP NPi _2, however, the gene for NP ₂ comes under control of the promoter and thus the expression of NP ₂ is used instead of NP1 is an indication of a recombination event.

For example, a vector according to the invention for the detection of conversion (recombination) events can be constructed from the following four elements, three of these elements being the DNA sequences of genetic marker genes, e.g. encode fluorescent proteins (see also FIG. 6):

(1) The first genetic marker (eg NP ^ is the marker to be analyzed. It is expressed by means of a promoter and can be fused with a specific localization sequence. The marker contains a mutation which inactivates it (knockout), so this construct is only correctly expressed after a conversion The promoter can also be an inducible promoter (eg the TetOn / TetOff system), so the conversion control can be switched on or off.

(2) The second genetic marker (eg NP ₂ ) has the correct sequence ready for the first genetic marker, so after the conversion this correct sequence is introduced into the first genetic marker and as a result the first genetic marker is correctly expressed. The second genetic marker is not expressed and, to prevent expression under foreign promoters, should preferably have no ATG start sequence at its beginning.

(3) The third (optional) genetic marker can be used as an internal standard regarding transfection and expression for the correct quantification of the conversion. It is either expressed via a normal promoter or a bidirectional double promoter which controls both the expression of the third genetic marker and the first genetic marker. The latter procedure has the advantage that the converted first genetic marker is directly connected to the internal standard, thus quantifying the conversion delivers even better results. The third marker must differ from the second genetic marker in such a way that it is not functional as such. This means that the third marker has little homology with the first marker and the second marker and therefore cannot undergo conversion.

(4) The fourth element contains a common resistance gene for selection in bacteria, mammalian cells etc.

The first genetic marker can e.g. a non-mutated XFP (XFP = general name for fluorescent protein), the second genetic marker is another XFP and the third genetic marker is a third XFP (especially DsRed). In this context, reference is also made to the embodiments described in more detail below and the examples below.

The term “recombination event” used here refers not only to homologous recombination events, but also to processes such as replication or repair mechanisms.

The expression "where both genes have regions homologous to one another" used here means that the degree of homology between the two genes is so high that events such as homologous recombination can take place.

The term "nucleic acid" used means DNA or RNA.

The detectable proteins NPi and NP ₂ must differ from each other insofar as they have to be detectable separately using different detection means. In principle, proteins (NP) are suitable for the method according to the invention, which can be detected using the following detection means: different fluorescence, different spectral excitation and / or emission, different lifespan and quantum yield, different diffusion time, etc. Examples of detectable proteins are fluorescent proteins, β-galactosidase, luciferase, resistance genes and other marker genes.

The term "cell" used here refers to any cell, which can be a prokaryotic or eukaryotic cell. Preferred eukaryotic cells are animal cells or tissues / organs, with mammalian cells being particularly preferred. Those skilled in the art are also familiar with methods for transfection and cultivation of the The transfection methods include, for example, transfection with a transfection reagent, for example a reagent described in the examples below, electroporation, Ca precipitation, microinjection, etc.

In a preferred embodiment of the method according to the invention, NPi is a first fluorescent protein (XFP ^ and NP _{2 is} a second fluorescent protein (XFP ₂ ), the two proteins differing in their fluorescence. The fluorescent proteins Blue Fluorescent Protein (BFP) are particularly preferred, Cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP) and green fluorescent protein (GFP) (from Clontech, Heidelberg).

In a further preferred embodiment of the method according to the invention, the NPi to be detected after the recombination event is not originally expressed, wherein in a particularly preferred embodiment of the method according to the invention the gene encoding NP * (for example as a fusion protein) is functionally linked and expressed with a promoter and that NP ₂ coding gene is functionally linked to no promoter or an inactive promoter and is not expressed. After a recombination event, the gene coding for NP ₂ comes under the control of the promoter, is expressed and is only then detectable.

In a further preferred embodiment of the method according to the invention, NP ₂ corresponds to a mutated form of NPi (or vice versa), the mutation leads to a protein that is modified or inactivated in its biological function. The DNA sequence encoding the mutated version of NP1 differs from the sequence encoding the active protein by deletion (s), insertion (s), exchange (s) and / or other modifications known in the art or represents a fragment of the original DNA molecule. Methods for generating the above changes in the DNA sequence are known to the person skilled in the art and are described in standard works in molecular biology, for example in Sambrook et al., supra. The person skilled in the art is also able to determine whether a protein encoded by a DNA sequence modified in this way is changed or inactivated with regard to its biological properties, for example by examining in an XFP whether it still has fluorescence properties.

In a particularly preferred embodiment of the method according to the invention, NPi is an XFP-i and NP _{2 is} a mutated form of XFP-i (or vice versa), the mutation resulting in a protein which is no longer fluorescent. On the other hand, other spectral properties can also be changed, for example a change in excitation / emission, lifetime or quantum yield. Most preferred is a method in which the NP ₂ , preferably XFP ₂ coding gene is functionally linked to a promoter and is expressed, but is inactive due to a mutation, and the NPi, preferably XFP ₂ coding gene with no promoter or an inactive Promoter is functionally linked and is not expressed.

In an alternative embodiment, the method according to the invention is characterized in that

(a) the genes encoding NP and NP _{2 are} each functionally linked to a promoter and are expressed;

(b) NP! and NP ₂ are expressed as fusion proteins, the fusion partners differing in such a way that the fusion proteins are located in different cell compartments; and (c) wherein a change in the localization or the degree of expression in the different cell compartments indicates a homologous recombination event.

The NP, preferably XFP, is replaced by a recombination event in one or both fusion proteins, so that this change is then documented by the new localization in the case of an XFP by a new localization of the different fluorescences within the cell; see also the following examples relating to this embodiment.

The person skilled in the art can produce the fusion proteins required for the process according to the invention or the DNA sequences coding for them according to standard methods, taking care that both partners still have the desired biological properties, i.e. the NP, e.g. XFP, must be detectable, i.e. in the case of an XFP still have fluorescence, the partner required for the localization of the fusion protein must still be able to achieve the desired localization. The localization polypeptide is preferably the N-terminal partner of the fusion protein. Suitable localization signals or polypeptides are known to the person skilled in the art and include e.g. Polypeptides for nuclear localization, for localization in the Golgi apparatus and / or endoplasmic reticulum and / or mitochondria or on / in the cell membrane. The one NP fusion partner is preferably a polypeptide for nuclear localization and the fusion partner for the other NP is a polypeptide for localization in / on the cell membrane. In a further preferred embodiment, the one NP fusion partner is a polypeptide for the nuclear localization and the fusion partner for the other NP is a polypeptide for the localization in the Golgi apparatus and / or endoplasmic reticulum.

In a particularly preferred embodiment of the method according to the invention, the NP fusion partner is a human histone, for example H1, H2A, H2B, H3 or H4, preferably H2A.1, and / or the NP ₂ fusion partner human catepsin B protein.

The method according to the invention also allows the investigation of sequence-dependent recombination effects. Thus, in a further preferred embodiment of the method according to the invention, the vector or the vectors for the investigation of sequence-dependent recombination effects also contain sequences flanking the NP * and / or NP ₂ coding genes. These can be, for example, those which induce a certain curvature in the DNA, which have special symmetries, which bring about splicing or which represent certain binding sequences for proteins.

The vector or the vectors (DNA or RNA vector (s), viruses) used for the method according to the invention can contain further genetic elements, preferably a further genetic marker NP3 as internal standard with regard to the transfection and expression efficiency. This marker can e.g. is another XFP that differs from the XFP1 and XFP2 in terms of its fluorescence properties and homologies. This additional marker is preferably under the control of a constitutive promoter.

It is also possible to carry out a double or multiple simultaneous transfection of cells with the vectors. This should be such that RRRP no longer occurs. RRRP can be prevented by

(a) use of a transporter for the constructs that support RRRP less,

(b) lowering the degree of homology of the constructs used, e.g. by changing the "codon usage". This means that the resulting protein can be the same after transcription, but the degree of homology can be greatly reduced.

It is therefore a method for the separate amplification of several DNA sequences in vivo while avoiding recombination events between individual DNA sequences, the method comprising the following steps:

(a) Production of a vector or several vectors which contain more than one DNA sequence, the DNA sequences having such a low degree of homology to one another that no recombination between the individual DNA sequences can occur;

(b) transfecting the desired cell and culturing the cell; and

(c) Selection of transfected cells and isolation of the amplified DNA sequences.

The present invention also relates to vectors or vector mixtures with the elements or properties defined above, and to a kit containing this vector or a vector mixture and optionally detection means for NP1 and / or NP2. Depending on the design of the kit, the detection means can be freely available or immobilized on a solid support, for example a plastic dish, a test tube, a microtiter plate, a test stick etc. The kit can also contain instructions on how to use the detection means contained in an assay for the determination explain the NP ₁ / NP ₂ activity or localization. The kit can also contain suitable reagents for the detection of labels or for labeling positive and negative controls, washing solutions, dilution buffers etc.

Finally, the present invention also relates to cells (eukaryotic or prokaryotic) or cell lines which are transfected with a vector or vector mixture according to the invention and are suitable for investigating recombination events, for example with regard to the effect of carcinogenic substances or other chemical or physical noxae on the recombination events. or repair rate within the cell. Furthermore, the present invention also relates to organs and whole organisms which are transfected with a vector or vector mixture according to the invention and are therefore suitable for the investigation of recombination events. The present invention further relates to an in vivo and in vitro method for producing nucleic acid sequences. This procedure has the following steps:

(a) producing a vector or a plurality of vectors which contain a first and a second or more partial sequence (s), the partial sequences having regions which are homologous to one another;

(b) transfecting the desired cell with the vector or vectors from step (a) and culturing the cell; and

(c) Selection of transfected cells in which the partial sequences have been recombined.

The aforementioned method allows the production of nucleic acid constructs which normally cannot be completely amplified in vitro or in vivo in a single step and / or can be introduced into a cell. If you want to insert a nucleic acid sequence into a cell whose length is too long or cannot be produced in one piece for various reasons, there is the option of dividing the sequence, taking care that the two sequences overlap, For example, for a 1000 bp sequence, one would make one piece of 1-600 bp and the other of 400-1000 bp, and both would be introduced into cells, tissues, or organisms, where recombination into a single sequence would occur. Entire gene banks can be produced in this way. It is also conceivable, for example, to introduce two non-functional constructs into cells which, after a recombination event has taken place, become a single functional construct. The method can thus be used if a desired nucleic acid construct cannot be introduced into a cell, for example due to a certain folding, etc. This folding can, for example: can be prevented by a certain mutation, but this usually leads to the fact that the construct is no longer functional. This problem can also be avoided by the method according to the invention, namely again by splitting the original construct into two or more partial sequences which have mutually homologous regions, which carry the corresponding mutation and are not functional, but can be introduced into a cell. The desired construct is then obtained by homologous recombination in the cell. Brief description of the figures:

Figure 1:

(A) Physical map for pSV-Hx-XFP

In the examples, pSV-H2A-CFP was used and H2A-YFP or H2A-GFP were expressed.

(B) Physical map of pcDNA3-hCB-eYFP

Figure 2:

Homology comparison of the vectors pSV-H2A-CFP and pcDNA3-HCB-eYFP The enlarged sequences of CFP and YFP reveal a difference of 16 base pairs. Arrows mark the primers used in genomic PCR to demonstrate the sequence conversion from H2A-CFP to H2A-YFP and their directions.

Figure 3:

Possible phenotypes after co-transformation with PSV-H2A-CFP and pcDNA3-hCB-eYFP

Figure 4:

(A. LCLC103H cells express PSV-H2A-CFP in the nucleus and pcDNA3-HCB-eYFP in the endoplasmic reticulum and Goloi apparatus

One nucleus contains a mixture of H2A-CFP and H2A-YFP, which is a positive

Gene conversion indicates.

(B) Overview (false color) of LCLC103H cells. which were transfected with pSV-H2A-CFP (green and pcDNA3-HCB-eYFP (red)

The population showed an enriched fraction of H2A-YFP and H2A-CFP +

H2A-YFP.

Figure 5: The results of the sequencing and a homology comparison of the genomic PCR (see also FIG. 4A) confirm the conversion from the expression of H2A-CFP to H2A-YFP

Figure 6:

Plasmid card for the conversion test

The plasmid can also contain other functional elements such as e.g.

Origins of replication etc. included.

The following examples illustrate the invention. Simultaneous co-transfections with DNA constructs (see FIG. 1), which were equipped with genes encoding different fluorescent proteins (XFPs), were carried out by means of a number of different transfection methods and cell lines (Table 1). In accordance with the constructs and methods used (FIGS. 2 and 3), the fluorescence properties of the DNA constructs were converted (FIG. 4). The fact that the conversion occurred at the DNA level was demonstrated by genomic PCR and sequencing (Figures 2 and 5).

Example 1 Construction of vectors with inserts encoding XFP fusion proteins

(a) PSV-HX-XFP

The region coding for the human histone H2A.1 was amplified (DE 100 13 204.9) and cloned into a eukaryotic expression vector derived from the promoterless plasmid pECFP-1 (Clontech, Heidelberg) via the EcoRI and BamHI restriction sites. This vector contains an SV40 promoter region in reverse orientation (which in this case leads to a higher expression efficiency) and the ECFP (Enhanced cyan fluorescent protein) coding sequence at the 3 'terminus of the fusion gene. A plasmid with the following structure was obtained by this procedure: p- (Hindlll) -P _R y. _l niN- (Hindlin- (EcoRn-hH2A.1- (BamHn-ECFP-1 (Figure 1A)

(b) pcDNA3-hCB-eYFP

The nucleic acid sequence encoding the human cathepsin B protein without the C-terminal propeptide was amplified by PCR from the IMAGE clone ID380482. Restriction sites for Kpnl (5 'end) and Sall (3' end) were added during amplification. The nucleic acid sequence encoding eYFP (enhanced yellow fluorescent protein) was also obtained via PCR from a plasmid (Clontech) derived from pEYFP-1, the restriction sites for Sall (5 'end) and NotI (3' end) being added at the same time. Using these restriction sites, a mammalian expression vector with the following structure was obtained after various cloning steps: pcDNA3- (Kpnn-hCB (flmCpro -) - (Sall) -ECFP- (NotI) (FIG. 1B)

The expression of the fusion protein is under the control of the CMV promoter from the cytomegalovirus. The pcDNA3 is available from Invitrogen.

Example 2 (I) Transfection procedures used

(a Transfection with FuGENE ™ 6

The cells were transfected with the FuGENE ™ 6 transfection reagent (Röche Molecular Biochemicals, Mannheim) according to the standard method. For this purpose, 0.5 × 10 ⁵ cells were sown on Lab-Tek ™ “2-well” chamber supports (Nunc, Wiesbaden) and transfected with the plasmids indicated 12 hours later. The FuGENE ™ 6 reagent was used for each well to be transfected incomplete RPMI-1640 culture medium mixed (final volume: 50 μl) After 5 min. incubation, 1 μg plasmid DNA was added to the transfection mixture and after a further 15 min the mixture was administered to the cells. The following reagents were also tested: Dmrie-C, Cellfectin, Lipofectin and GibcoPlus. (Transfection Reagent Sample Pack, Cat # 10552-016 from Gibco Life-Technologies, Gaithersburg, MD, USA).

(b) Transfection via electroporation

About 10 ⁷ cells were harvested and suspended in 1 ml cell culture medium. Electroporation with 5 μg plasmid DNA was carried out at room temperature under the following conditions:

# 1 # 2 # 3

Voltage [V] 250 300 400

Current [A] 3.9 3.3 3.2

Capacitance [μF] 125 125 125

At # 1 and # 2, the cells survived quantitatively.

(c) Transfection using Ca? POa

The cells were grown to 50% confluence in 10 cm diameter petri dishes. The medium was changed 2 hours before the transfection. Two solutions were made:

A: 2 x HBS (50 mM Hepes, 280 mM NaCI, 1, 5 mM Na ₂ HPO ₄ , pH: exactly 6.95)

B: 2.5 M CaCl ₂ , 20 μg DNA, made up to 500 μl with water.

Solutions A and B were mixed dropwise and vortexed after each drop. After 20 minutes at room temperature, the mixture including the Ca precipitates was added to the medium. The medium was changed again after 16 hours. (II) Culturing the cells

Lung carcinoma cells (LCLC103H), HeLa and Cos7 cells were used to establish stably transfected cell lines. Permanently transfected cells were obtained by selection with G418 (800 μg / ml) for at least 1 week and these were kept in RPMI-1640 culture medium with 10% FCS and 6 mM glutamine at 37 ° C. with 5% CO ₂ .

(III) Fluorescence microscopy and quantification of cellular fluorescence

(a) Fluorescence microscopy

One day after transfection, the cultures of the living cells were analyzed with a Zeiss "Axiovert S100 TV" microscope using 10x, 20x and 40x objectives for the phase contrast and fluorescence mode. The fluorescence signals were analyzed using single excitation filters for ECFP (436/10, Omega, Brattleboro, VT05302, USA) and EYFP (515/10, Omega), a two-band emission filter (470 / 30-555 / 40, Omega) and a two-band beam splitter (475/565, Omega The images were stored using a cooled digital CCD camera (Hamamatsu C4742-95) and evaluated on a Macintosh computer using the "OpenLab" cell imaging software (Improvision, Warwick, UK).

(b) Quantification of cellular fluorescence

After nine days of G418 selection, the cells were seeded in dishes with a diameter of 10 cm at a suitable density (approx. 10 ⁶ cells per 20 cm ² ) for the statistical analyzes. Three images (calibrated to the brightest intensities) were taken at a given time: a phase contrast image with 30 ms exposure time, the ECFP channel at 500 ms and the EYFP channel at 1 min. And at 30% amplification for signal amplification. After background subtraction, the images of the two fluorescence channels (cut with a basic intensity threshold of approximately 5%) were cut (with this only the histone GFP signals could be measured due to the lower signal intensity of the CB-GFP expression) , Then the fluorescence channels connected. The binary mask was applied to both channels and the mean gray values of the nuclear signals were measured and plotted against each other.

Table 1

Quantification of the conversion of H2A-CFP to H2A-YFP using different constructs, cell lines and transfection methods

Construct cell line transfection conversion conditions [+/-] and [%]

H2A-CFP CLC103H FuGene 6 - 0.0

CB-YFP LCLC103H FuGene 6 - 0.0

H2A-CFP + CB-YFP LCLC103H FuGene 6 / simultaneous + 4.0 ± 0.2

H2A-CFP + DsRed LCLC103H FuGene 6 / simultaneous - 0.0

H2A-CFP + pure GFP LCLC103H FuGene 6 / simultaneous +> 0.1

H2A-CFP + CB-YFP LCLC103H FuGene 6 / separated +> 0.1 mixture + simultaneous

H2A-CFP + CB-YFP LCLC103H FuGene 6 / separate - (+) 0.0 (7.3)

Mixed + 4 hours

delay

H2A-CFP + CB-YFP LCLC103H FuGene 6 / - 0.0.

Over-transfection of the stable line

H2A-CFP + CB-YFP LCLC103H FuGene 6 / 5x DNA- + n.b. Conc.

H2A-CFP + CB-YFP LCLC103H FuGene 6 / 10x DNA- + n.b. Conc.

H2A-CFP + CB-YFP LCLC103H FuGene 6 / linearized + 3.4

H2A-CFP + CB-YFP LCLC103H FuGene 6 / linearized + +++ 10.3 96 ° C

H2A-CFP + CB-YFP CLC103H Dmrie-C / simultaneous + 3.8

H2A-CFP + CB-YFP LCLC103H cellfectin / simultaneous +> 1, 0

H2A-CFP + CB-YFP LCLC103H lipofectin / simultaneous + 2.4 H2A-CFP + CB-YFP LCLC103H GibcoPlus / simultaneous +> 1.0

H2A-CFP + CB-YFP LCLC103H electroporation / + approx. 1.0 simultaneously

H2A-CFP + CB-YFP LCLC103H Ca phosphate / simultaneous + approx. 1.0

H2A-CFP HeLa FuGene 6 - 0.0

H2A-CFP + CB-YFP HeLa Fugene 6 / simultaneous + n.b.

H2A-CFP Cos-7 FuGene 6 - 0.0

H2A-CFP + CB-YFP Cos-7 FuGene 6 / simultaneous + n.b.

Example 3 Determination of the nucleotide sequence

(a) Lvsa extraction and purification of the genomic DNA

Individual H2A cell clones with converted fluorescence properties were grown to confluence in cell culture bottles (25 cm ² , approximately 10 ⁷ cells). 5 ml lysate buffer (1% SDS, 10 mM EDTA, 50 mM Tris) and 0.1 mg / ml (final concentration) Proteinase K were added and then at 37 ° C above sea level. incubated. The extracted lysate was purified twice by adding 5 ml of phenol / chloroform (1: 1, v / v.), Followed by vortexing (1 to 3 min.) And centrifugation for 5 minutes at 3,000 rpm. The DNA was precipitated by adding NaCl (0.1 M final concentration) and 10 ml 10% ethanol. The genomic DNA was removed from the solution using a Pasteur pipette and resuspended in TE buffer (10 mM Tris, 1 mM EDTA). The remaining DNA fragments were incubated at -20 ° C. and centrifuged at 15,000 rpm for 45 min. The pellet obtained was washed with ethanol and also resuspended in TE buffer.

(b) Determination of the nucleotide sequence

The extracted DNA containing the recombined XFP sequences was checked by PCR with various primers, either for XFP or Vector sequences had been designed. The PCR was carried out on a "minicycler" from Biozym Diagnostik (Hess. Oldendorf) with a preheating step for 1 min at 94 ° C. and 35 cycles (denaturation: 30 seconds at 94 ° C.; elongation: 210 seconds at 65 ° C. End annealing: 1 min at 65 ° C. The same primers were used for direct sequencing of the genomic DNA.

Direction of restriction site Sequence of sequence within primer length

Restriction site of the histone and the XFP

Forward EcoRI CTTCGAATTCTG ATGCCTGAACCA 27 bp

GCT

Reverse Kpnl GGGTACC CTTGTACAGCTC 30 bp

GTCCATGCCGA

(c) Comparison of Segments

The homology studies for nucleic acid sequences were carried out using the sequence comparison program “AlignPlus” (Scientific & Educational Software) and MACAW from Greg Schuler (version 2.0.5). The results are shown in FIG.

Claims

claims

1. A method for determining recombination events, the method comprising the following steps:

(a) Production of a vector or a plurality of vectors which contain a gene coding for a first detectable protein (NP ^ gene and a gene coding for a second detectable protein (NP ₂ ), both genes having regions which are homologous to one another and wherein before the recombination event to be detected (1 ) no or only one NP is detectable or (2) the two NPs are detectable in different cell compartments or (3) their fluorescence properties have changed;

(b) transfecting the desired cell and culturing the cell; and

(c) Determination of the presence and / or concentration of the proteins NP _T and NP ₂ , a changed presence, concentration and / or subcellular localization of the proteins NPi and NP _{2 being} an indication of a recombination event that has taken place.

2. The method of claim 1, wherein NP * ι is a first fluorescent protein (XFP ^ and NP _{2 is} a second fluorescent protein (XFP ₂ ) and wherein both proteins differ in their fluorescence.

3. The method of claim 2, wherein the fluorescent protein is BFP, (e) CFP, (e) YFP, RFP or GFP.

4. The method according to any one of claims 1 to 3, wherein the NPi to be detected after the recombination event is not originally expressed.

The method of claim 4, wherein the NPi-encoding gene is operably linked to a promoter and is expressed, and the NP _2- encoding gene is not operably linked to a promoter or an inactive promoter and is not expressed.

6. The method according to any one of claims 1 to 3, wherein NP _{2 is} a mutated form of NPi (or vice versa) and wherein the mutation leads to a protein whose biological function is changed or inactivated.

7. The method of claim 6, wherein NPi is an XFP- * and NP _{2 is} a mutated form of XFPi (or vice versa) and wherein the mutation results in a no longer fluorescent protein.

8. The method of claim 6 or 7, wherein the NP ₂ coding gene is functionally linked to a promoter and is expressed, but is inactive due to a mutation, and the NPi coding gene is not linked to any promoter or an inactive promoter and is not expressed becomes.

9. The method according to any one of claims 1 to 3, wherein

(a) the genes encoding NPi and NP _{2 are} each functionally linked to a promoter and are expressed;

(b) NPi and NP ₂ are expressed as fusion proteins, the fusion partners differing in such a way that the fusion proteins are located in different cell compartments; and

(c) a change in the localization in the different cell compartments indicating a homologous recombination event.

10. The method of claim 9, wherein the NP fusion partner is a polypeptide for nuclear localization and the NP ₂ fusion partner is a polypeptide for Localization in the Golgi apparatus and / or endoplasmic reticulum (or vice versa).

11. The method according to claim 10, wherein the NP fusion partner is human histone H2A.1 and the NP ₂ fusion partner is human catepsin B protein.

12. The method according to any one of claims 1 to 11, wherein the vector or vectors for the investigation of sequence-dependent recombination effects still contain the NPi and / or NP ₂ encoding genes flanking sequences.

13. The method according to any one of claims 1 to 12, wherein the vector (s) also contain a further genetic marker as an internal standard with regard to transfection and expression efficiency.

14. A method for producing a desired nucleic acid sequence from partial sequences, the method comprising the following steps:

(a) producing a vector or a plurality of vectors which contain a first partial sequence and a second or more partial sequence (s), the partial sequences having regions which are homologous to one another;

15. A method for separately amplifying several DNA sequences in vivo while avoiding recombination events between individual DNA sequences, the method comprising the following steps: (a) Production of a vector or several vectors which contain more than one DNA sequence, the DNA sequences having such a low degree of homology to one another that no recombination between the individual DNA sequences can occur;

(b) transfecting the desired cell and culturing the cell;

16. The method according to claim 15, wherein the DNA sequences encode a first detectable protein (NP1) and / or a second detectable protein (NP2) as defined in one of claims 1-9.

17. Vector or vector mixture containing the elements defined in claims 1 to 16.

18. Kit containing the vector or the vector mixture according to claim 17 and optionally detection means for NP! and / or NP ₂ .

19. A cell transfected with a vector or vector mixture according to claim 14.

20. The cell of claim 19, which is eukaryotic or prokaryotic.

21. Use of the cell according to claim 19 or 20 for the investigation of homologous recombination events.