WO2011042181A1 - Cell-tracking model system, transgenic for marker variants - Google Patents

Abstract

The present invention relates to a cell-tracking model system comprising - a donor harbouring a marker ("donor marker") or being capable of expressing said donor marker, said donor harbouring said donor marker or cells of said donor capable of expressing the donor marker being transferable to a recipient animal; and - a recipient animal which is tolerant against a marker which is an immunologically neutral variant of the donor marker ("recipient marker"), said recipient marker being encoded in the genome of the recipient animal in a form being capable of expression of said recipient marker and said recipient marker being physicochemically or biologically distinguishable from the donor marker.

Description

CELL-TRACKING MODEL SYSTEM, TRANSGENIC FOR MARKER VARIANTS

The present invention relates to diagnostic models for cell therapy and cell based gene therapy.

Cell and gene therapy are thought to revolutionise tissue repair in various organs, cancer therapy, and therapy of genetic diseases in the near future. Therefore, cell and gene therapy holds great promise to cure or ameliorate a large variety of diseases of various organ systems. Cell therapy involves the syngeneic (genetically identical), allogeneic (different indi^¬ viduals of the same species), or xenogeneic (between species) transplantation of cells for therapeutic purposes.

Potential therapeutic targets^' for cell and gene therapy are:

• Liver diseases (for example cell therapy of liver cirrhosis or other severe liver diseases)

Lung diseases (for example cell-based gene therapy of cystic fibrosis )

Skin diseases (for example cell therapy of skin defects with epidermal cells)

Cardiovascular diseases (for example cell therapy of

myocardial infarction with mesenchymal cells, gene therapy of cardiomyopathies, for example with vascular growth factors )

• Kidney diseases (for example cell therapy or cell-based gene therapy of glomerulonephritis or of renal failure)

Orthopaedic diseases (for example cell therapy of osteoarth ritis, disc degeneration, ligament injuries, or cell-based gene therapy of genetic myopathies)

• Diabetes mellitus (for example cell therapy of diabetes by transplantation of autologous beta cells grown from precursor cells extracted from pancreas, liver, or bone marrow)

• Diseases of the central nervous system (for example cell

therapy or cell-based gene therapy of neurodegenerative diseases such as Parkinson' s disease or amyotrophic lateral sclerosis, cell therapy of cerebral infarction (palsy) , cell therapy of multiple sclerosis)

• Cancer (for example cell therapy with ex vivo conditioned autologous immune cells, cell-based gene therapy with autologous cells expressing for example immunostimulatory cytokines such as interleukin 2) It is clear that any thorough evaluation of the efficacy and applicability of cell and gene therapy methods requires animal models that allow tracing the fate of individual transplanted donor or manipulated cells in the host organism.

Animal models that allow tracing the fate of native or ma^¬ nipulated donor cells in the host are essential for a vast num^¬ ber of questions ranging from cell linage experiments to regenerative medicine approaches and cancer research. Tracing of cells requires labelling. Cell labelling can be done in a varie^¬ ty of ways. The most widely used approach to label cells is to introduce marker genes into the genome of the cells under inves^¬ tigation by transducing cells ex vivo with a marker gene, or by using cells from transgenic donor animals with a stable genetic marker. The advantage of a stable genetic marker is that it persists in whole progeny of a specific cell. Therefore, use of cells from transgenic donor animals is probably the most robust way of exploring the long-term fate of transplanted cells.

Most of the currently used cell markers such as green fluo^¬ rescent protein (GFP; e.g. Hadj antonakis et al., BMC Biotech- nol., 2 (2002) :11; Brazelton et al., Stem Cells, 23 (2005):1251- 1265), the bacterial enzyme LacZ, firefly luciferase, Herpes simplex thymidine kinase (HStk) , or human placental alkaline phosphatase (ALPP) are foreign, immunogenic proteins. During recent years, it has become increasingly clear that membrane or even intracellular expression of any foreign protein, and, thus, of any marker protein, will elicit immune-mediated rejection of transplanted cells carrying the marker gene in an immunocompetent recipient. Therefore, immune-mediated rejection of genetically altered cells is a general and very significant problem in transplantation studies using cells labelled with any marker gene, and also in gene therapy protocols. The problem has severely hampered the utility of cell tracking models in regenerative and tumour medicine, especially in long-term studies. For example, until recently it was not possible to perform unbiased long-term preclinical safety studies using cells labelled with any immunogenic marker protein in normal immunocompetent animals .

The problem of immune-mediated rejection of cells labelled with marker proteins has been circumvented by the use of immuno- deficient animals such as SCI D (severe combined immunodeficien- cy) mice or drug-based immunosuppression. It is obvious that these strategies create artificial systems that may not be predictive of the situation in a human patient. An intact host immune system is a very important component in the course of many, if not all, diseases, especially cancer. Another approach to bypass immune-mediated rejection of labelled cells is to use non- immunogenic markers. However, non-immunogenic markers such as DNA markers or magnetic particles have several important limitations. They are either difficult to detect (DNA markers), diluted by cell divisions (e.g. magnetic particles or membrane dyes), or may leak out of cells (e.g. magnetic particles) .

In a further approach, the host can be treated with immunosuppressive drugs such as cyclosporins or glucocorticoids. It is clear that with this approach it is difficult to tell whether immune-mediated phenomena were actually absent or not. In addition, immunosuppressive drugs have to be introduced into the experimental system, possibly influencing the outcome of the experiments.

In WO 2006/113962 Al the use of marker tolerant animals for use in a cell-tracking system is disclosed. Marker tolerance was a novel in vivo technology for studying labelled cells in the complete absence of immune-mediated rejection in immunocompetent hosts. This technology has a very broad applicability. The idea of this invention was to overcome the problem of immune-mediated rejection by inducing specific tolerance to the marker protein in immunocompetent hosts. There are several ways provided to induce tolerance to a foreign protein in normal animals. For example, tolerance can be induced by injection of marker-carrying, irradiated cells into normal animals of the same inbred strain directly after birth, i.e., before immunological self-non-self recognition is completed. As proof-of-principle , normal neonatal Fischer 344 rats were tolerised with cells from transgenic donors of the same F344 inbred strain which over-express ALPP under the ubiquitous ROSA26 promoter. It was shown that this strategy results in lifelong tolerance to cells and tissues labelled with ALPP. Although this technique to induce tolerance works very well, it is laborious, has to be confirmed in each animal, and is, therefore, difficult to handle in practice, especially for a large number of animals in serial testing or for providing standardised protocols which are useable in different laboratories. Such induction of specific marker tolerance by neonatal exposure to the foreign antigen, i.e., the marker protein, therefore requires birth control, harvesting and irradiation of transgenic donor cells, and verification of marker tolerance by skin grafts in each animal. In addition, absence of background staining needs to be confirmed in each animal by flow cytometric analysis of peripheral blood (Odorfer et al . , BMC . Biotechnol. 7 (2007), 30; Unger et al., Histochem. , Cell. Biol. 127 (2007) : 669-674; Odorfer et al., J. Cell. Mol . Med., 12 (2008) : 2867-2874 ) . Taken together, although working well, this procedure is quite laborious and time-consuming.

French et al. (Diabetes, 46 (1997) discloses that transgenic expression of mouse proinsulin II prevents diabetes in nonobese diabetic mice. Ogawa et al. (Gene Ther., 11 (2004 ): 292-301) reports on the induction of immune tolerance to a transplant- implications of gene therapy for haemophilia.

Accordingly, there is still an urgent need for a more con^¬ venient model system for observing and studying the performance and effectiveness of a cell based therapy based on genetic molecular markers which would not be biased by unnatural effects, such as immunologic side effects. Such a model could mainly serve as a research tool and should allow the exploitation of the idea of marker tolerance in a cell tracking system in a more reproducible, standardisable and simple way.

Therefore, the present invention relates to a cell-tracking model system comprising

- a donor harbouring a marker ("donor marker") or being capable of expressing said donor marker, said donor harbouring said donor marker or cells of said donor capable of expressing the donor marker being transferable to a recipient animal; and

- a non-human recipient animal which is tolerant against a marker which is an immunologically neutral variant of the donor marker ("recipient marker") , wherein "immunologically neutral" means that said donor marker and said recipient marker are not distinguishable by the immune system of the recipient animal, said recipient marker being encoded in the genome of the recipient animal in a form being capable of expression of said recipient marker and said recipient marker being physicochemically or biologically distinguish- able from the donor marker.

The present invention provides an improved model system for cell tracking which allows the study and tracking of cells which contain a suitable marker without any bias with respect to such markers, i.e. without that the marker would have a negative effect or otherwise influence the reaction of the model animals. The model is easy to establish and to standardise and therefore allows a broad applicability in various fields for observing cells and their movement inside the body of animals. The recipient animals are characterized by a normal immune system ("normal" in terms of being immunologically analogous to the non- tolerant or wild-type animal, and there is no need for immunosuppression or other measures which reduces the reliability of the model system. The recipient animals can be transgenic animals which have the recipient marker as transgene and are therefore tolerant to this marker, because these animals recognise the transgene as "self" (if the transgene has been appropriately expressed during the critical period in the development of the immune system of the recipient animal); the recipient marker can also be an endogenous protein whereto the donor marker is a (n immunologically neutral) variant of this endogenous protein which is biochemically or physicochemically distinguishable so that the prerequisite of the present invention (that the recipient marker is an immunologically neutral variant of the donor marker) is fulfilled. In the first case, the donor harbours or is capable of expressing an immunologically neutral variant of the transgene; in the latter case, the recipient animal can be a wild type animal; the donor then harbours the immunologically neutral variant of the endogenous protein as donor marker or is capable of expressing this donor marker. The recipient animals can also be made tolerant against the recipient marker by usual ways of induction of tolerance (e.g. described French et al., Diabetes 46(1) (1997), 34-39; Xu et al., Clin. Immunol. 111(1) (2004), 47-52; Ogawa et al., Gene Therapy 1(3) (2004), 292-301; EP 1 142 473 Al (tolerance induction in animals for testing whether a gene therapy may be successfully applied) or WO 2006/113962 A (system for cell tracking)).

In the preferred embodiment of the present invention wherein transgenic animals with the recipient marker as transgene are used as recipient animals, stable innate tolerance to the recip- ient marker is induced in the recipient animal by transgenic expression of a mutated donor marker (as recipient marker) which is an immunologically neutral variant of the donor marker. According to the present invention, marker tolerant recipient animals are preferably generated by establishment of transgenic vertebrate (mammal, bird, fish; especially mouse, rat, rabbit, pig, frog, zebrafish, chicken) lines, expressing the recipient marker under the control of a ubiquitous, tissue-specific, or inducible promoter. Regarding the choice of promoter, the only prerequisite for the current invention is that the expression of the recipient marker protein occurs in such a way that the expression results in recognition of the recipient marker protein by the immune system of the recipient animal as self.

According to a preferred embodiment, the present model system is based on a rodent (preferably mouse, rat, and rabbit) or pig system, especially on a system of two inbred mouse/rat /rabbit lines, a transgenic donor mouse/rat /rabbit line and a transgenic marker tolerant recipient mouse/rat /rabbit line. However, also other vertebrate systems can be based on the present invention, especially in those model systems which allow the provision of transgenic animals, such as Xenopus, Zebrafish (or other fish, such as salmonids, carps and tilapias), chicken, and other mammals, such as goat, sheep, cow, etc.) . Based on this technology, novel marker tolerant, immunocompetent animal models can also be generated for multi-modality cell tracking. The model according to the present invention significantly improves the ability to track labelled cells in immunocompetent hosts. The present invention is a breakthrough technology for cell tracking in life sciences, especially in regenerative medicine, because such models are very important to assess the efficacy and safety of novel treatment approaches, especially for investigating the therapeutic targets mentioned above.

Since the present invention provides for an animal model, it is clear that the term "animal" exclusively refers to non-human animals as far as the model animal is concerned. It is further noted that any treatment of the animals referred to herein is exclusively related to non-therapeutic treatment, since the aim of the model is to analyse the movement of the cells which - at least terminally - is generally observed in tissue analysis af- ter sacrifice of the recipient animal. Anyway, in the animal model, final termination of the model animal is mandatory, even if cell tracking is made in the living organism.

The system according to the present invention is, however, also suitable to be used in cell tracking of cell therapy, both in the model system and in humans (of course, in humans, the re^¬ cipient marker is always an endogenous protein and the donor marker is an immunologically neutral variant thereof which is biochemically or physicochemically distinguishable from the en^¬ dogenous marker protein chosen) . It is important that the donor and recipient marker as used for the present invention are also as in all other uses of the present invention - non- therapeutic marker in such systems.

The donor according to the present invention can be any kind of unit harbouring the donor marker, or any kind of unit capable to transfer the donor marker to (cells of) the recipient animal. The expression "harbouring" or "capable to transfer" includes the constitutive expression of the donor marker in a cell of the donor to be transferred, the induced expression of the donor marker in the cells to be tracked after application of an induction stimulus, the ability (e.g. of a gene therapy vector) to express the donor marker in an expression system (e.g. a cell) in the recipient, or nano-vehicles or nano-containers delivering the protein marker to cells of the recipient. For example, the donor can be a gene therapy vector, nano-vehicles, nano- containers, transduced cells, expression vectors in liposomes, transgenic cells or tissue of a transgenic animal. Preferably, the donor is a cell or tissue which expresses the donor marker (or wherein the expression of the donor marker can be induced) , especially preferred as a donor is a transgenic animal expressing the donor marker (or wherein the expression of the donor marker can be induced) .

The donor, especially cells or tissues of a donor animal, can be transferred to the recipient animal wherein the donor marker is or can be expressed (e.g. under the control of a given promoter which can be an inducible promoter (in this case, ex^¬ pression of the donor marker in the recipient animal has to be actively induced) ) .

The recipient according to the present invention is always a vertebrate animal, preferably a mammal, especially a rodent . If the recipient is a transgenic animal, the recipient is characterized by life-long, innate tolerance to the recipient marker, and, because recipient and donor marker are immunologically neutral, the respective matched donor marker.

Donor marker and recipient marker have to be immunologically neutral, i.e. that the two markers are not discriminated (are not distinguishable) by the immune system of the recipient. The immunological neutrality in the recipient can be tested by a tolerance test using skin transplants. According to a preferred embodiment of the present invention, the rodent skin transplantation system as disclosed in Hedrich, "Genetic Monitoring of Inbred Strains of Rats" (Hedrich ed., G. Fischer, Stuttgart (1990), pages 102-114 (Chapter 4.1.3. Testing for Isohistogenei- ty (Skin Grafting) ) ) is used for determining immunological neutrality according to the present invention. Even more preferred, the skin transplantation test described in the example section herein is used for this purpose. Presence of no statistical significant difference in leukocyte numbers in transplants between wt —> wt and donor marker — recipient marker proves immunological neutrality.

On the other hand, the donor marker and the recipient marker have to be physicochemically/biologically distinguishable, i.e. the two markers can be distinguished by a physicochemical or biological method. The corresponding markers (donor marker and matched modified recipient marker) usually differ with respect to their amino acid sequence. Possible alterations in the amino acid sequence in the recipient relative to the donor marker can be amino acid substitutions by point mutations, insertions, or deletions. The amino acid difference causes a functional and/or structural difference, e.g. a difference in enzyme activity, in light or radiation absorbance/excitation, in colour generation (luminescence, fluorescence, phosphorescence, dye processing, etc.), or a difference in binding affinity of monoclonal antibodies, however, the differences do not amount to immunological differences in the recipient; this means that the difference is not recognised by the immune system of the recipient animal (i.e. the distinguishing feature is immunologically neutral as the immune system of the recipient animal is concerned) .

The "variant" of the donor marker according to the present invention generally refers to an immunologically neutral, structurally differing marker compared to the donor marker which is - due to this structural difference - distinguishable in any of its physicochemical or biological properties from this donor marker, but not distinguishable by the immune system of the recipient animal. Preferably the recipient marker is a mutant of the donor marker (or vice versa) . This usually enables a immunological neutrality between the two marker forms. In any way, immunological neutrality between marker pairs (donor marker in recipient animal) can always be determined by the tolerance test using skin transplants as described above.

Preferably, the donor or recipient marker is a fluorogenic or chromogenic marker, especially Escherichia coli lacZ, green fluorescent protein (GFP) , luciferase, human placental alkaline phosphatase (ALPP) , or Herpes simplex virus 1 thymidine kinase (HSVl-tk) .

According to a preferred embodiment, the system of the present invention is characterised in that the donor marker has a higher/varying heat stability, a higher enzymatic activity, different sensitivity to enzyme inhibitors, different fluorescence or luminescence characteristics, or a different acid stability than the recipient marker. Other physicochemical/biological differences include: higher/lower stability with respect to acids/bases or chaotropes; higher/lower enzyme activity with respect to ionic strength; sensitivity with respect to enzyme inhibitors or cofactors; however, preferred marker pairs should be distinguishable by clear-cut differences, such as distinctive features, such as different emission/extinction spectra, different substrate or antibody affinities; one member of the marker pair is heat /acid/base labile, the other is stable; one member of the enzyme marker pair is inhibited by a specific inhibitor, the other is not; one member of the marker pair is recognised by a specific (monoclonal) antibody, the other is not (and/or is recognised by a different (monoclonal) antibody; etc.) . A typical example for the latter (difference in binding properties of antibodies) is disclosed in Hoylaerts et al. (Eur. J. Biochem. 202 (1991), 605 to 616) with respect to alkaline phosphatase.

Another preferred embodiment of the system according to the present invention is characterised by a different substrate specificity of donor and recipient marker. For example, if the marker is an enzyme, donor and recipient marker can differ (e.g. through a point mutation) in specificity with respect to a given substrate (e.g. by a different rate of enzymatic turnover) or with respect to two given substrates (e.g. the donor marker is able to process substrate A and the recipient marker is able to process substrate B; each without significant or relevant ability to process the other substrate) .

Accordingly, the present invention provides "pairs" of markers which can act as donor and recipient marker being immunologically neutral in the recipient animal, i.e. the recipient animal which has an intact immune system cannot distinguish immunologically between the two markers. Preferred "pairs" as donor/recipient markers according to the present invention are selected from the group consisting of: human placental alkaline phosphatase (ALPP) and a heat labile ALPP mutant, especially ALPP^{E 29G}; green fluorescent protein and a GFP mutant with altered fluorescence or shifted spectra, especially GFP , GFP or _GFpY66H. fi_refiy luciferase mutant S284T and firefly luciferase; HSVl-tk and a HSVl-tk mutant with altered substrate specificity or two HSVl-tk mutants with different substrate specificity, es^¬ pecially HSVl-R176Q-sr39tk and HSVl-A167Y-sr39tk.

As a "proof-of principle"-experiment for the present invention, several mouse lines tolerant to ALPP by site-directed mutagenesis were established relating to single amino acid substi^¬ tutions within the ALPP molecule. ALPP was used for such pilot experiments, because this marker was shown to be developmentally neutral in transgenic rats and mice, and because it is one of the best markers to date for histological detection of labelled cells. ALPP is a heat resistant enzyme which survives paraffin and methylmethacrylate embedding. This specific system according to the present invention consists of two transgenic mouse lines, representing donor and recipients animals. Donor animals ubiquitously express the marker enzyme ALPP under a fragment of the murine ROSA26 promoter (Zambrowicz et al., PNAS 94 (1997), 3789- 3794), whereas recipients are transgenic for a mutated derivative of ALPP, named ALPP^E429G (recipient marker), which is also expressed under the same fragment of the ROSA26 promoter. The rationale behind this strategy is a single amino acid substitution in the ALPP enzyme which results in the loss of its heat resistant properties, a difference in physicochemical properties which is easily detectable. Because ALPP is virtually identical to its derivative ALPP^E429G, recipients (recognising the recipient marker as self) are also tolerant to donor ALPP. Therefore, the recipient marker is an immunologically neutral mutant of the donor marker. Due to the heat sensitivity of the mutated ALPP^E429G the presence of the mutated enzyme does not interfere with de^¬ tection of normal donor ALPP after heat pretreatment.

This very elegant model system based on ALPP/ALPP^E429G already shows the very broad applicability of the technology ac^¬ cording to the present invention which is easily transferable to other pairs of immunologically neutral marker proteins. Similar approaches, e.g. single amino acid substitution at crucial posi^¬ tions in the different marker proteins that allow discerning wild-type from mutated protein can be utilized to generate animals tolerant to established laboratory marker proteins, such as GFP, luciferase, HSV-tk and LacZ . In brief, a preferred strategy for making recipient mouse lines tolerant to lacZ (E. coli β- galactosidase ) will be to modify the lacZ transgene in such a way that the enzyme activity is abolished. For fluorescent and bioluminescent proteins advantage of the fact can be taken that only slight modifications within the amino acid sequence of GFP and luciferase can lead to a significant shift in excitation and emission spectra. Accordingly, different derivates of the same marker can be used that allow discrimination between endogenous and transplanted cells within the organism due to their different excitation/emission wavelengths. Because in all cases where only one amino acid is different between original and mutated marker protein, the recipient animals (e.g. mouse lines) can be expected to be tolerant to the original marker. Usually, the marker variant which is easier to detect is used as donor marker, however, the two individual markers of each "pair" can in principle be used either as donor or recipient marker.

It is also possible to use more than one "pair" of markers in order to have two, three, four or even more marker "pairs". This allows a multi-modality in vivo cell tracking system which can, for example be established on multicistronic transgenic constructs. The multi-modality in vivo cell tracking systems allows the combination of e.g. fluorescent and bioluminescent imaging with excellent histological tracking capabilities in marker tolerant hosts. The use of fluorescent or bioluminescent marker such as GFP and luciferase permits optical non-invasive in vivo imaging of transplanted cells. Therefore, the present invention allows the combination of the advantages of the different marker proteins in a multi-modality cell tracking model. Suitable marker pairs which can be tracked by different (monoclonal) antibodies are described in Hoylaerts et al . , 1991 cited above. For example, substitution in wt placental AP of Glu429 for Gly429 or even for His429 (found at this position in tissue-nonspecific alkaline phosphatase) and Ser429 (found in the intestinal alkaline phosphatase) induced a general decrease in affinities as detected by 16 of the 18 antibodies tested. Therefore, specifically wt placental AP and its mutants [Met38]PLAP, [Thr67]PLAP, [Phe68]PLAP, [Ser84]PLAP, [Vall33 ] PLAP, [Arg209] PLAP, [His2 1 ] PLAP,

[Leu254] PLAP, [Leu297 ] PLAP, [His429] PLAP, [Ser429]PLAP and [Gly429]PLAP (as described by Hoylaerts et al.); wt germ cell AP, etc. may be combined to suitable marker pairs which can be differentiately detected by the monoclonal antibodies Fll, D10, C2, H7, BIO G10, B2, H5, A3, F6, E5, C4, 17E3, 7E8, 327, 151, 130 and E6 (as referred to in Hoylaerts et al.).

Further preferred embodiments of marker pairs of mutant/wild type marker protein or of two different mutant marker proteins are the following:

LacZ/ -galactosidase (β-gal)

The β-galactosidase from E. coli has been extensively studied, and represents one of most widely used enzymes in molecular biology. Based on sequence homologies, site-directed mutagenesis studies, and the elucidation of its three-dimensional structure, three highly conserved amino acid residues (Glu-461, Glu-537 and Thy-503) are considered to be essential for stability and catalytic activity. Therefore, amino acid substitutions at one or more of these positions · result in loss of catalytic activity of β-galactosidase .

Additionally, substitutions of the amino acid positions His- 357 and His-540 result in reduced stability to heat (52°C), which provides an alternative possibility to pursue a strategy analogous to ALPP.

GFP (green fluorescent protein) GFP, a protein isolated from the jellyfish Aequorea victoria, emerged as one of the most often used fluorescent marker proteins for in vitro as well as in vivo cell labelling. Due to the widespread usage of GFP, different mutants have been engineered, resulting in altered fluorescence intensity and shifted spectra. In particular, the widely used EGFP variant is a very suitable mutated marker for the present invention. Due to two amino acid substitutions (F64L and S65T) this variant of wild- type GFP is shifted to lower excitation energies (excitation GFP/EGFP 395/488 nm; emission 509 nm for both) . EGFP is optimized for brighter fluorescence (35-fold increase) and higher expression in mammalian cells. It has been shown that an additional amino acid substitution (Y66H) results in blue-shifted excitation at 382 nm and emission maxima at 458 nm (Blue fluorescent protein, BFP) . Therefore, animals, especially mice, transgenic for BFP should also be tolerant to EGFP. In this system, e.g. EGFP transgenic mice can serve as donors, while BFP transgenic mice can serve as marker tolerant recipients. In addition, BFP displays less fluorescence intensity compared to EGFP due to low extinction coefficients and quantum yields, and undergoes rapid photobleaching . The shifted spectrum of BFP allows discrimination between endogenous cells of the recipient (BFP expression) and transplanted donor cells (EGFP expression) , as excitation at 488 nm will not induce BFP fluorescence neither is BFP emission at 458 nm able to excite EGFP, as the excitation wavelength of EGFP is 488 nm. Alternatively, blue shifted emission by introducing Y66F can be achieved, or a non-fluorescent variant by introducing Y66L.

Of course, due to a vast number of known GFP derivates, other combinations are also further preferred: Especially the amino acids forming the internal fluorophore (Ser65, Tyr66 and Gly67) as well as amino acids interacting with them (96, 143, 146, 148, 203, 205, 222) are sensitive for mutational changes in terms of excitation and emission wavelengths as well as (thermo) stability and fluorescence intensity. Further residues also have been proven to influence performance of GFP when mutated: 46, 47, 68, 69, 70, 72, 87, 145, 153, 163, 167, 175, 206, 208, 224, 231, 234, and are therefore putative targets for marker modification by single or multi-amino acid substitution.

Additionally, fluorescent proteins from other species than Aequorea victoria can be used according to the present invention. For instance, variants from GFPmx (GFP from Aequorea macrodactyla) has been shown to produce stronger fluorescence intensities than EGFP at 37°C, show partially red-shifted emission spectra, and can also be expressed in mammalian cells. Further relevant amino acid substitutions will also result in changed emission spectra (CN 1321689) , rendering possible the differentiation between donor marker and its derivative by their specific emission spectra.

It is well known that for in vivo fluorescent imaging red- emitting GFP variants above 600 nm are preferable to minimize absorption and quenching by tissue. However, EGFP is preferred for donor cell labelling in order to be able to combine fluorescent and bioluminescent imaging. In this context it is advantageous if excitation and emission spectra of the fluorescent and the bioluminescent marker (discussed in the next section) do not interfere .

Luciferase

Bioluminescent imaging (BLI) represents a very good tool for in vivo imaging, because animal, especially mammalian tissue completely lacks intrinsic bioluminescence . Furthermore, BLI shows a very good signal-to-noise ratio, because it does not require an external excitation light source but depends on ATP- driven conversion of luciferin to the light-emitting product ox- yluciferin. Up to now many luciferase genes from different spe^¬ cies have been isolated and sequenced, but the strong tissue- absorbance of blue-green light still hampers in vivo monitoring of luciferase-expressing cells. Therefore, a lot of effort is still put in the development of the "ideal BLI luciferase", having an emission wavelength as far red as possible, at least comparable enzyme activity to wild-type luciferases, long intracellular half-life and thermostability. Amino acid positions responsible for emission wavelength, pH (in) sensitivity, heat stability, enzyme activity, and emission intensity have been studied intensely.

One of the most prominent luciferases originates from the firefly Photinus pyralis (FLuc) . It requires ATP and Mg²⁺ as co- factors to function, and oxidizes D-luciferin, its substrate, to release energy in the form of photons, producing a light emis- sion peak at 562 nm.

Recently, a S284T mutant of the Phorinus pyralis luciferase was introduced as an improved version for BLI, especially for imaging in deeper tissues, due to a shift in emission wavelength from 557 nm to 616 nm (pH 7,8). Other mutations like insertion of an arginine analogous to the red light-emitting luciferase of Phrixotris railroad worm at the position Arg356 results in red shift to 608 nm (similar to E354R/Arg356 in L. tur- kestanicus). Similarly, the mutations Q283R red shifts to 603 (pH 7,0), S284G to 609 nm (pH 7,0).

A further improvement of this red-shifted luciferase is the integration of thermostabilizing mutations. For instance the single mutation E354K improved thermostability as well as the combination of the following amino acid substitution Thr214Ala, Ala215Leu, I-le232Ala, Phe295Leu, and Glu354Lys increased thermostability of WT and S284T luciferase mutant.

Besides P. pyralis luciferase also several other enzymes seem convenient for this approach and have been evaluated for the possibility to shift their green light emission to the near infrared. Lampyris turkestanicus mutation E354R/Arg356 red shifts from 555 to 602 nm (pH 7,8) and S284T from 555 to 618 nm (pH 7,8) .

Mutations I288A and S286N in Luciola cruciata (Japanese Gen- ji-botaru) luciferase result in red shifts from 560 nm to 613 and 605 nm, respectively. In Ragophthalmus ohbai substitution of T226 to phenylalanine or other amino acids changes emission from 548 to 590 nm, whereas substitution of asparagine at position 230 in Pyrocoelia miyako luciferase to serine or threonine provokes a shift from 562 to 616 nm (pH 7) and from 547 to 605 nm (pH 8) . The mutation T217I in Lampyris cruciata significantly improved thermostability.

A preferred embodiment of the present invention refers to a combination of mutations resulting in maximum emission wavelength shift and intensity, thermostability, pH insensitivity and enzyme activity for the marker and non-interfering emission spectrum for the derivative. Additionally, it is advantageous to avoid any interfering emission with the fluorescent marker ( GFP ) , because this might be a confounding factor in multimodal- ity tracking models. HSVl-tk

Wild-type HSVl-tk recognizes a variety of pyrimidine and acycloguanosine nucleoside analogues and is widely used as a reporter gene for nuclear imaging (positron emission tomography,

PET) with a variety of derivates (e.g. 29-deoxy-29-fluoro-5- iodo-l-b-D-arabino-furanosyluracil (FIAU) , 2 ^' -fluoro-2 ^' -deoxy-1- b-D-arabinofuranosyl-5-ethyluracil (FEAU) , 2 ^' -deoxy-2 ^' -fluoro-5- methyl-l-b-D-rabinofuranosyluracil ( FMAU) , 2^'-fluoro-2^'-deoxy-5- fluoro -ethyl-l-b-D-arabinofuranosyluracil or 9- [ 4 -fluoro-3- (hydroxymethyl ) butyl ] guanine (FHBG)) differentially labelled with either ¹³¹I, ¹²³I, ¹²⁴I, ¹²⁵I or ¹⁸F.

Recently, two mutants of HSVl-sr39tk were disclosed. HSV1- sr39tk differs from HSVl-tk by seven nucleotide substitutions leading to five different nonpolar . amino acids. HSVl-sr39tk shows enhanced activity relative to HSVl-tk. The two mutants of HSVl-sr39tk carry one additional substitution each, either R176Q or A167Y. The two derivatives HSVl-R176Qsr39tk and HSV1- A167Ysr39tk exhibit nucleoside specificity to acycloguaniosine- based and pyrimidine-based radiotracers, respectively. In this way, discrimination between both enzymes can be achieved by employing different tracers. Immunologically, HSVl-R176Qsr39tk and HSVl-A167Ysr39tk are expected to be neutral.

Alternatively, expression of an inactive mutant of HSVl-tk in recipient animals would also meet the requirements. Random mutagenesis by several groups have identified putative essential amino acid residues for thymidine or thymidilate kinase activity of HSVl-tk: residues 165-177 and 155-165, 159-173 especially D162, R163, H164, L17057, Q125, H58, 128, Y172 and E225. More over, complete loss of activity has been shown for the substitution M128F.

Multidetection models

A preferred embodiment of the present invention is an immunocompetent, marker tolerant animal (especially mouse) model which can be used at the same time for histological, fluorescent, and bioluminescent cell tracking. Therefore, multicistron- ic transgene constructs are used, where AL PP , EGFP , and lucifer- ase are driven by the same ubiquitous ROSA promoter. It is clear that such a multi-modality tracking model can also be obtained by interbreeding the separate donor and recipient models. Howev- er, when the separate lines are interbred, differential integration effects can occur, so that expression of the different markers may not be uniform in certain types of cells. This problem can be ruled out by de novo generation of an animal model based on a multicistronic transgene construct. However, both approaches can be followed according to the present invention.

Simple interbreeding of various combinations of marker and marker-derivate expressing mouse lines can also be performed to permit the possibility to visualize putative cell fusion events post cell or tissue transplantation. For this purpose, for instance luciferase transgenic mice can be interbred with tg (ALPP^E429G) mice to achieve luciferase/ALPP^E429G-expressing mice, which can be transplanted with ALPP positive cells. Eventually, fusion between endogenous (luciferase expressing) and exogenous (ALPP expressing) cells can be monitored, as double positive cells will only be available due to fusion events. Single ALPP positive cells on the other hand are then definitely of exogenous origin. Analogous to this example various combinations with involvement of two or more transgenes are possible, e.g. donor: ALPP, recipient: ALPP^E429G + GFP, ALPP^E429G + Luc, ALPP^E429G + lacZ or ALPP^E429G + HStk; donor: GFP, recipient: A^GFP + ALPP, A^GFP + Luc, A^GFP + LacZ, or A^GFP + HStk; donor: Luc, recipient: A^Luc + ALPP, A^GFP + GFP, A^GFP + lacZ or A^GFP + HStk; donor: lacZ, recipient:

_AlacZ _{+ AL}pp_{? A}lacZ ₊ pp^ ^la Z _{+ Luc Qr A}lacZ _{+ HStk;} d_{onor :} ALPP +

GFP, recipient: ALPP^E429G + A^GFP + Luc, ALPP^E429G + A^GFP + LacZ or _ALPpE 29G _{+ A}GFP _{+ HStk; etc >} (i._e. and all further combinations possible; Δ denotes immunologically neutral variant of the superscript marker protein) .

System applications

Central to the applications for using the system according to the present invention is the transgenic marker tolerant animal as recipient, independent of the mode of employment as well as of the marker-pair used.

The vertebrate animal to be used according to the present invention is not critical in principle, however, animals, especially mammals, which are already established in laboratory practice or which are known as animal models for other scientific or industrial purposes. Specifically preferred animals to be used as recipients (and optionally as donors) are rabbits, rodents, especially hamster, mice, guinea pigs or rats; primates, especially chimpanzees; or pigs. According to a preferred embodiment, donor and recipient are inbred animals. In case the provision of inbred animals is difficult or impossible, autologous cells can be modified ex vivo to receive the donor marker and then act as donor cells which may be transplanted to the recipient .

Depending on the model or on the cells to be studied, the marker is controlled by a specific promoter. A preferred promoter is an inducible promoter (reviewed by Romano, Drug News Per- spect 17 (2004), 85-90; Clackson, Gene Therapy 7 (2000), 120- 125; Pollock et al . , Current Opin Biotechnol 13 (2002), 459- 467). In other preferred embodiments, the promoter is a ubiquitous constitutive promoter, especially ROSA26 (R26) promoter, β- actin promoter, especially human, rat, or chicken β-actin promoter, or β-actin promoter with cytomegalovirus enhancer, cytomegalovirus promoter, ubiquitin promoter, or SV40 promoter. In other preferred embodiments, the promoter is a cell- or tissue- specific promoter, especially a constitutive promoter. A specific advantage of a constitutive promoter is that all cells derived from a genetically labelled cell express the marker regardless of differentiation status. A specific advantage of an inducible promoter and a cell- or tissue-specific promoter in the current invention is that marker gene expression can be limited in space and/or time. As mentioned above, primary, ubiquitous expression is favoured for both donor and recipient transgene expression, e.g. by using either Rosa26Promoter (full length or truncated (Zambrowicz et al . PNAS 94(1997), 3789- 3794)) or CMV (cytomegalovirus; US 5,168,062) will be used. Concerning the R26 promoter, knock-in strategies instead of blastocyst injection for transgene introduction are alternatively preferred .

Regarding donor transgene expression in principle any promoter can be employed, advantageous choices are mainly dependent on the question to be addressed by the model:

1) cell type or tissue specific expression

2) spatio-temporal expression

3) inducible expression by various strategies e.g. tet- systems, cre-ERT systems, ...

4) promoters, truncated promoters, mutated promoters for promoter/reporter studies

5) any promoter suitable for specific additional transgene expression in conjunction with marker expression e.g. via IRES

Application and tracking the fate of cells as well their performance in all possible aspects of cellular and molecular biology, can be carried out in various different designs depending on the question addressed. Therefore, the present invention also relates to the use of the system according to the present invention for tracking cells; it also relates to a method for tracking cells wherein the donor with the donor marker is trans^¬ ferred to the recipient animal, whereafter the donor marker (in cells) is tracked in the recipient animal. According to the present invention, a "donor" may be e.g. an animal, tissue or cell that expresses at least one marker (donor marker) suitable for detection within the compatible recipient post administration that does not induce immune mediated rejection in the recipient.

"Recipient" can be (i) an animal expressing compatible marker-derivate ( s ) to the chosen marker (s) of donor, being therefore tolerant to the chosen donor's marker (s) or (ii) an animal expressing compatible marker-derivate ( s ) to the chosen marker (s) of donor, being therefore tolerant to the chosen donor's marker (s) and marker (s) suitable for detection and different from the marker (s) expressed by the donor, to allow monitoring of fusion events of endogenous and exogenous cells.

In this context the system(s) are convenient e.g. for the following applications:

1) Cell/tissue transplantation from donor to recipient.

2) Administration of a cell line derived from a donor animal to a recipient.

3) Administration of cells transformed in vivo and/or ex vivo with additional transgene constructs to recipient.

4) Administration of cells from compatible inbred strains, ex vivo transformed with compatible marker (s) to recipient.

5) Administration of cells from compatible inbred strains, ex vivo transformed with compatible marker (s) and additional modifications to recipient.

6) Administration of cells from compatible inbred strains, ex vivo transformed with compatible marker (s) used as reporters.

7) Administration of cells from compatible inbred strains, ex vivo transformed with compatible marker (s) in combination with another transgene to monitor expression/function of the other transgene.

8) other donors, such as complete tissue or organs, tissue- engineered constructs, vectors, liposomes, nanovehicles , etc..

The model according to the present invention is the "next generation" of in vivo cell tracking models, including the preferred embodiments, combines several important advantages: (i) stable genetic labelling of cells of interest, (ii) unlimited source of genetically tagged cells, (iii) immunocompetent hosts, and (iv) most importantly, absence of immune-mediated rejection of labelled cells, allowing valid long-term studies. An additional advantage of the approach according to the present invention is - in the embodiment that also the donor is an animal of the same kind as the recipient (e.g. both are mice) - that the heterozygous transgenic donor and recipient can be interbred with gene-targeted knockout or knock-in animals, e.g. of the same strain. Thus, labelled cells from homozygous gene-targeted mice can be used for cell tracking. Such a system allows addressing very specific biological questions in many different research areas. Specifically preferred areas are: liver diseases (for example cell therapy of liver cirrhosis or other severe liver diseases), lung diseases (for example cell-based gene therapy of cystic fibrosis), skin diseases (for example cell therapy of skin defects with epidermal cells), cardiovascular diseases (for example cell therapy of myocardial infarction with mesenchymal cells, gene therapy of cardiomyopathies, for example with vascular growth factors), kidney diseases (for example cell therapy or cell-based gene therapy of glomerulonephritis or of renal failure) , orthopaedic diseases (for example cell therapy of osteoarthritis, disc degeneration, ligament injuries, or cell-based gene therapy of genetic myopathies), diabetes melli- tus (for example cell therapy of diabetes by transplantation of autologous beta cells grown from precursor cells extracted from pancreas, liver, or bone marrow) , diseases of the central nervous system (for example cell therapy or cell-based gene therapy of neurodegenerative diseases such as Parkinson's disease or amyotrophic lateral sclerosis, cell therapy of cerebral infarction (palsy) , cell therapy of multiple sclerosis) , or cancer (for example cell therapy with ex vivo conditioned autologous immune cells, cell-based gene therapy with autologous cells expressing for example immunostimulatory cytokines such as interleukin 2).

The present invention is further described by the following examples and the drawing figures, yet without being restricted thereto .

Fig. 1 shows the homology of human alkaline phosphatases. (A) Heat properties of human APs . (B) The sequences of GCAP and PLAP differ in only seven amino acids. (C) An exchange of the C- terminal part of GCAP results in a single amino acid substitution at position 429 and in resistance to heat of mutant C5;

Fig. 2 shows histochemical ALPP detection in tissue sections of different organs after heat inactivation at 72°C for the in^¬ dicated time. Tissues from ALPP transgenic donors show strong staining, whereas, similar to wild-type mice, no enzyme activity can be detected in tissues of ALPP^E429G mice; and

Fig. 3 shows that tg (ALPP^E429G) recipients are tolerant to the marker ALPP. (a) Histochemistry of skin grafts 6 months postsurgery. Grafts from tg(ALPP) mice were rejected by sex matched as well as by sex mismatched wild-type recipients, demonstrated by the decrease in ALPP positive cells within the grafts. In tg (ALPP^E429G) recipients, ALPP positive grafts are still fully intact, 6 months post surgery. (b) Evaluation of leukocyte infiltration in skin transplants 6 month post surgery. CD45R represents a transmembrane protein expressed on leukocytes and is used here to monitor immune-mediated rejection of skin transplants. In case of wt —> wt and auto-transplants (donor = recipient) no infiltrate could be detected comparable to ALPP —» ALPP^E429G transplants. In contrast, significant infiltrate was detected in ALPP —> wt transplants.

EXAMPLES :

The aim of the present examples is to generate marker tolerant mouse models as novel tools for regenerative medicine, cancer research, and cell tracking studies in general. In these in vivo cell tracking models, labelled cells can be tracked for long periods of time in the complete absence of immune-mediated rejection of labelled cells. The first example is based on the ALPP/ALPP^E429G mouse model. Further embodiments involve the gener^¬ ation of donor and marker tolerant recipient mouse lines for the most widely used protein markers GFP, lacZ, luciferase, and HSV- tk, as well as a mouse model for multi-modality cell tracking. All these marker proteins are non-toxic at appropriate doses/expression levels and developmentally neutral in transgenic animals .

Each example involves generation of a marker tolerant animal model for a specific marker protein (a "pair" of donor and recipient marker) or for multi-modality cell tracking (more than one "pair") . Design of the constructs is followed by in vitro testing in cell culture. Thereafter, appropriate constructs are used for generation of transgenic animals. Tolerance of recipient lines is tested by skin transplantation. In the examples, site-directed mutagenesis is used for generation of the models. This strategy is taking advantage of the detailed structure- function information already available for all the marker proteins used in the present example section.

As a matter of convenience and because of its advantageous character, site-directed mutagenesis in the present examples is restricted to a single amino acid substitution, if possible, or alternative to as few mutations as necessary to meet the crite^¬ ria for physico-chemical or biological differentiation of donor marker and recipient marker, but maintain immunological neutrality between donor and recipient marker.

The present invention is based on a dual marker system consisting of a donor and a corresponding marker tolerant recipient. In a mouse model, this can preferably be accomplished by a mouse line on the same inbred background (e.g. C57BL/6) : The donor line expresses the wild-type marker protein and serves as an unlimited source for genetically labelled cells and tissues, whereas the recipient line expresses a mutated form of the marker which can be easily distinguished from the wild-type form by its physicochemical or biological properties. The mutation can preferably be introduced into the wild-type form by site- directed mutagenesis of one amino acid. If only one amino acid is different between wild-type and mutated form of the marker, the immune system of the recipient will usually not recognize donor cells labelled with the wild-type marker as foreign. In other words, the recipient mouse line will be tolerant to the wild-type marker. As proof-of-principle human placental alkaline phosphatase is provided in the present example, which is based on an established neonatal tolerization model making use of this marker (ALPP is known to be a superb marker for histological detection of labelled cells) .

All four human alkaline phosphatases (APs) are highly homologous (Fig. 1) . However, only placental alkaline phosphatase is heat resistant. It was demonstrated that one particular amino acid is mainly responsible for the different sensitivity of mam^¬ malian APs to heat. It. was further shown that germ cell AP can be made heat resistant by substitution of the C-terminal part of the molecule by the corresponding ALPP sequence which differs only in one amino acid (Fig. 1). For the purposes of the present invention, advantage was taken of this unique feature of ALPP. By site-directed mutagenesis, a heat sensitive form of ALPP was generated by changing glutamic acid 429 to glycin in the C- terminal part of the enzyme. Thus, the heat-sensitive derivative _ALppE429G _{is v r}tually identical to normal ALPP, and differs only in one amino acid. Accordingly, a transgenic animal expressing _ALPpE 29G _{does not} recognize ALPP as a foreign protein (ALPP^{E 29G} is "immunologically neutral" compared to ALPP) .

Initially the heat inactivation characteristics of ALPP and ALPP^E429G were tested in transfected fibroblast cultures in vitro. These experiments confirmed that ALPP^E429G was heat sensitive. Next, eight donor and twelve recipient mouse lines were generated on C57BL/6 inbred background by pronuclear injection and random integration of the transgene. Only lines with a single integration site were used for further experimentation. Both the ALPP and the ALPP^{E 29G} transgenes were driven by a truncated form of the ROSA26 promoter, leading to ubiquitous expression of the transgene. Donor mouse lines were selected for high ALPP expression, whereas ALPP^E429G-expressing recipients were selected for low expression to facilitate ALPP^E429G heat inactivation. For tracking of ALPP-labelled cells in an ALPP^E429G-transgenic recipient it is essential to be able to discriminate ALPP from ALPP^E429G. Figure 2 shows that heat inactivation destroys the enzyme activity of ALPP^E429G, but not of ALPP. Therefore, ALPP can easily be discriminated from ALPP^E429G by histochemical staining, and ALPP^E429G transgenic mouse lines do not show background staining after heat inactivation, which in any case is necessary to inactivate endogenous APs. These data show that the model system according to the present invention works very well.

Of course, the crucial question was whether the ALPP^E429G transgenic mice are tolerant to ALPP. Proof-of-concept experiments are conducted to verify persistent marker tolerance. Survival of skin grafts was used as the most robust way to assess long-term tolerance (Hedrich et al., 1990). Evaluation of acute and chronic rejection was performed by regular inspection of grafts as well as by quantification of CD45R positive cells infiltrating the transplant 24 weeks post surgery. Additionally, tg(ALPP) derived transplants were stained for the marker to examine the remain of exogenous tissue after 24 weeks. Skin grafts from sex-matched wild-type to wild-type mice did not show signs of acute or chronical rejection and were accepted in all cases. However, when skin was transplanted from ALPP transgenic mice to wild-type mice, hair loss, granulation and size reduction of the transplant were observed, which resulted in partial or total rejection of transplants and scar formation. Similar results, but earlier onset of evidence for rejection, were obtained when skin was transplanted from male to female, where basically the male- specific minor histocompatibility antigen H-Y is responsible for rejection. Importantly, no evidence was found for immune- mediated rejection of ALPP positive skin transplants transferred to ALPP^E429G mice by transpant inspection.

These data were confirmed by histological analysis since chronic rejection is difficult to assess and might escape detection as outlined by Hedrich et al . In skin grafts from tg(ALPP) donors transplanted into sex matched wild-type mice, ALPP positive cells decreased with time, suggesting immune-mediated rejection of marker-expressing cells in non-tolerant recipients

(Fig. 3a) . Similar results were obtained when skin was transplanted from male tg(ALPP) to female C57BL/6 or tg (ALPP^E429G) (sex mismatched; smm) , where the male-specific minor histocompatibility antigen H-Y is responsible for rejection. However, skin grafts from ALPP transgenic donors transplanted into tg (ALPP^E429G) mice showed strong ALPP staining, 6 months post-transplantation

(Fig. 3a), demonstrating long-term and persistent tolerance of tg (ALPP^{E 29G}) animals to ALPP.

Examination of immune cell infiltration in transplants by detection of CD45R-positive cells in histological sections, 6 months post surgery, further confirmed these data, as no infiltrate was detected in wt —> wt and auto-transplants (donor = re- cipient) as well as in ALPP → ALPP^E429G transplants whereas ALPP -» wt transplants showed increased leukocyte infiltration (Fig. 3b) . These data clearly show that ALPP^{E 29G} transgenic mice are tolerant to skin transplants from ALPP transgenic mice as for leukocyte numbers in wt → wt and ALPP —> ALPP^E429G transplants no statistical significant difference was detected whereas leukocyte accumulation in ALPP —> wt transplants was highly significant compared to wt —> wt transplants. Presence of no statistical significant difference in leukocyte numbers in transplants between wt —> wt and ALPP —» ALPP^E429G proves immunological neutrality.

Taken together, these results show that a very elegant and convenient long-term in vivo cell tracking system in immunocompetent mice was successfully established by application of the present invention to a mouse model.

Methodological approaches

Cloning ALPP-wt and ALPPE429G mutant

R26 Promoter was amplified via PCR from genomic DNA extracted from tg(hPLAP) F344 rats (Kisseberth et al., Developmental Biology 214, 128-138 (1999) and genomic ALPP sequence including 3^'UTR was amplified from human whole blood genomic DNA. PCR products (fori 5 ^' -GTCGACTAGATGAAGGAGAGC-3 ^' and revl 5^'- GAGCCACATATGGGAAGCGGT-3 ^' , for2 5 ' -ATGCCCAGAATTCCTGCCTCG-3 ^' and rev2 5 ^' -GAAAGGAGCCTGCCTGGTACC-3 ^' ; for3 5 ^' -GGTACCAGGCAGGCTCCTTTC- 3^' and rev3 5 ^' -GAGGCAGAATCTCGCTCTGTC-3 ^' ) where cloned into pCR2.1 or pCR-4-TOPO® (Invitrogen, Carlsbad, USA) and sequenced to verify mutation-free amplification. For E429G substitution splice overlap extension was performed using the following primers, forward 5 ^' -TAGCACGTGGGAGACACTCCA-3 ^' and reverse 5'- GTGCCCCTGGACGGAGAGACCCACGCAGGCGAGGAC-3 ' ; forward 5'-

TGCGTGGGTCTCTCCGTCCAGGGGCACTGCTGACTG-3 ' and reverse 5'-

GAAAGGAGCCTGCCTGGTACC-3 ' . R26P and ALPP genomic sequence segments (wt for ALPP and SOE-mutated for ALPP^E429G) were eventually assembled and cloned into a modified pEGFP-N3 vector, where CMV promoter was removed by ASCI and Nhel digest, and EGFP was removed by Kpnl and Xbal digest. Complete R26P-ALPP and R26P- ALPP cassettes where sequenced to verify sequences. Both cassettes were then excised from vector backbone with Sail and Aflll digest, separated by agarose gel electrophoresis, isolated from gel using gel extraction kit (Qiagen, Hilden, Germany) following the manufacturers instructions.

In vitro inactivation test of ALPP^E429G

Modified pEGFP-N3 vectors containing either R26P-ALPP or R26P-ALPP^E429G cassettes where transfected in mouse 3T3 fibroblasts using Effectene (Qiagen, Hilden, Germany) following the manufacturers instruction. Stable transfected cells where selected by G418 (300 pg/ml and 600 pg/ml; Sigma, Munich, Germany) treatment und subsequently single cell clones where obtained by limiting dilution, plating 1 cell per well in 96 well plates. Clones were expanded in RPMI 1640 medium supplemented with 10% FCS and Pen/Strep. For inactivation time course cells where grown on 6-well plates, fixed with pre-chilled Acetone/Methanol (30:70), 3 minutes at -20°C. Inactivation was performed in water bath for 0, 35, 45, 60 and 75 min at 65°C and 72°C. Fixed cells where then incubated overnight in TNM buffer (0.1M Tris-HCl, pH 9.5, 0.1M NaCl, 5 mM MgCl₂) containing 0.17 mg/ml of the substrate 5-bromo-4-chloro-3-indolyl phosphate (BCIP, Sigma) and and nitrotetrazolium blue chloride at RT and embedded in Mowiol. In comparison to wt-ALPP, that showed stable enzyme activity up to one hour heat inactivation, ALPP^E429G activity was abolished by incubation at 72°C.

Pronuclear injection was performed as described previously (T. Rulicke. Pronuclear injection of mouse zygotes. In: Methods in Molecular Biology. Germ cell protocols. Volume 254/2: Schatten H. (Ed.) The Humana Press Inc. 165-194 (2004)). Briefly, the R26P-ALPP and R26P-ALPPE429G cassettes, driven by a truncated R26 promoter were excised from vector backbone with Sail and Aflll digest, separated by agarose gel electrophoresis and recovered from gel using gel extraction kit (Qiagen) following the manufacturers instructions, and injected into the pronucleus of fertilized oocytes derived from a C57BL/6 mating. The purified DNA constructs were injected into a pronucleus at a concentration of 2.0 ng/μΐ injection buffer. Injected zygotes were transferred at the same day into pseudo-pregnant surrogate mothers. To identify transgenic founder animals the genotype of the offspring was tested by Southern blot and PCR analysis using primers R26f 5 ' -TGAATTCCTGCCTCGCCACTGT-3 ' and E2r 5^'- AAGGCCTGGCTCACTCACCATC-3 ' and U3f 5 ' -GATGGAGACCATCCTGGCTAAC-3 ^' and U4r 5 ' -GATCTAGTAACGGCCGCCAGTG-3 ' amplifying products of 350 bp and 210 bp, respectively, for the transgene allele.

Southern Blot

To identify transgenic founders, genomic DNA was. isolated from tail biopsies by phenol/chloroform extraction and isopropa- nol precipitation and 10 g was digested by restriction enzyme BamHI, and separated by agarose gel electrophoresis, wt genomic DNA was used as negative control. DNA was transferred to Nylon membrane (Hybond N+; Amersham Pharmacia Biotech, Uppsala, Sweden) and hybridized with two different probes. Probes where gained by restriction end nuclease digest of R26P-ALPP cassette and subsequent isolation of agarose gel. Probe A by Blnl digest

(846 bp) and probe B by SacI/EcoRI digest (1427 bp) . Probe labeling was performed using PRIME-It-II-Random Labeling Kit

(Stratagene, La Jolla, USA) . Membranes where then washed repeatedly and exposed to X-ray film with an intensifying screen at - 80°C for 1 to 6 days.

ALPP and ALPPE429G expression

To test transgene expression in several tissues of 7 tg(ALPP) and 12 tg (ALPP^E429G) mouse lines, mice where sacrificed by exsanguination from V. cava under Ketamin/Xylazin anaesthesia (70/7 mg/kg i.p.) and tissue samples from liver, kidney, lung, spleen, heart, brain, duodenum, jejunum, intestine, stomach, pancreas, thymus, uterus, over, testis, aorta, lymph node, gallbladder, skin, muscle, tibia, femur and calvaria where harvested and fixed in 40% EtOH, dehydrated and embedded in paraffin and where subject to histological analysis.

Immunoblotting

Frozen lung samples were homogenized in 0.25 M Tris-HCl pH 6.8 + 0.4% SDS for 2 x 60 seconds at 6500 rpm and cooling at - 20 °C for 2 min between the runs in the MagNA Lyser Instrument (Roche) and homogenates were diluted 1:1 with 6% SDS. 1.25 pg for tg(ALPP) and 50 \iq protein for tg (ALPP^E429G) and wild-type derived samples were separated by SDS-polyacrylamide gel electrophoresis and immunoblotting was performed. For immunological detection of ALPP and ALPP^{E 29G} rabbit monoclonal-anti-hPLAP Clone SP15 (Thermo Fisher Scientific) and ECL Plus (GE Healthcare) was used.

RNA isolation

For RNA isolation tissue was harvested, immediately shock frozen in liquid nitrogen, and stored at -80°C until RNA isolation. Frozen tissue was homogenized in TRI Reagent (Ambion) for 60 sec at 6500 rpm in the agNA Lyser Instrument (Roche) . RNA was extracted with l-Bromo-3-chloropropane (Sigma) and precipitated using isopropanol. RNA purity and quality was determined spectrophotometrically (BioPhotometer ; Eppendorf) as the A260/A280 ratio showed expected values between 1.8 and 2 and the A260/A230 ratio values were 1.8 or greater. Additionally the absence of RNA degradation was verified via Agarose gel. Reverse transcription of RNA was performed using iScript™ cDNA Synthesis Kit (Bio-Rad) following the instructions of the manufacturer.

Full length expression of transgene was determined by several PCR assays using primers spanning the entire transcript: E2f and E5r 5 ^' -GCTACGCAGCTCATCTCCAA-3 ^' , E5f 5 ^' -GCTACGCAGCTCATCTCCAA- 3^' and E9r 5^'- CTCTCAATGGCGTCGTCGAA-3 ^' , E8f 5^'- GATCCACCGA- GACTCCACACT-3 ^' and Ellr 5^'- AGGCCATGACGTGCGCTATGAA-3 ^' , U3f 5^'- GATGGAGACCATCCTGGCTAAC-3 ^' and U4r 5 ^' -GATCTAGTAACGGCCGCCAGTG-3 ^' . As a control, genomic DNA from wild-type C57BL/6 was used as template .

Genotyping

PCR was performed with U3f and U4r primers on genomic template DNA isolated from tail biopsies.

Skin transplantation

Recipient mice (C57BL/6, tg(ALPP) and tg (ALPP^E429G) were either immunised four weeks pre surgery i.p. or not with 100 μΐ freshly harvested whole blood of donor mice (C57BL/6 or tg(ALPP)). Coagulation of whole blood was prevented by adding 10 μΐ trisodium-citrate-dihydrate-solution (0,136 molar) per 100 μΐ blood.

For surgery mice were anaesthetised with 2-4% isoflurane. 0,5 x 0,5 cm full skin allografts were transplanted from donors to recipients. One donor mouse was used for 3-10 recipients of different groups. In any case an auto-graft served as control. Grafts were protected from self-destruction by bandaging for the first week and with applying a ruff for another week. Oral met- amizol application was used for analgesia administered pre- surgery and every 6 hours post-surgery for 24h. For post-surgery infection control Enrofoxacin (Baytril®; 10 mg/kg) was administered s.c. daily for 3 days. Photographs were taken after one week and subsequently from the 3^rd week at an interval of 3 weeks until mice were sacrificed 24 weeks post surgery by exsanguina- tion from V. cava under Ketamin/Xylazin anesthesia (70/7 mg/kg i.p.) For evaluation of immune mediated rejection sections where stained with anti-CD54R and number of positive cells where counted per tissue section.

Table 1

Statistical analysis

Statistics were computed using SPSS for Windows 16.0 (SPSS). Normal distribution of data from CD45R quantification was con^¬ firmed by Kolmogorov-Smirnov test and further analyzed using one-way analysis of variance (ANOVA) , followed by Student- Newman-Keuls multiple comparison test. P values of less than 0.05 were considered significant. Data are given as means ± s . e . m.

Histological analysis

Skin samples where fixed in 40% Ethanol, dehydrated and embedded in paraffin. Five-pm-thick paraffin sections were cut with a HM360 microtome (Microm, Walldorf, Germany) , and were mounted on slides pre-treated with 3-aminopropyltriethoxy-silane (APES, Sigma-Aldrich, Deisenhofen, Germany, rehydrated and heated at 72°C for 30 min in deionxsed water to block endogenous alkaline phosphatase activity.

For detection of enzyme activity, sections were incubated in TN buffer (0.1 Tris-HCl, pH 9.5, 0.1 M NaCl, 5 mM MgC12) containing 0.17 mg/ml of the substrate 5-bromo-4-chloro-3-indolyl phosphate (BCIP, Sigma) and nitrotetrazolium blue chloride at room temperature overnight. Subsequently, sections were counter- stained with nuclear fast red (Sigma) , dehydrated, and mounted using Vectamount (Vector, Burlingame, CA, USA) .

For evaluation of immune-mediated rejection sections were stained with anti-CD54R and the number of positive cells was counted per transplant .

Claims

Claims :

1. : Cell-tracking model system comprising

- a donor harbouring a marker ("donor marker") or being capable of expressing said donor marker , said donor harbouring said donor marker or cells of said donor capable of expressing the donor marker being transferable to a recipient animal; and

- a non-human recipient animal which is tolerant against a marker which is an immunologically neutral variant of the donor marker ("recipient marker"), wherein "immunologically neutral" means that said donor marker and said recipient marker are not distinguishable by the immune system of the recipient animal, said recipient marker being encoded in the genome of the recipient animal in a form being capable of expression of said recipient marker and said recipient marker being physicochemically and/or biologically distinguishable from the donor marker.

2. : System according to claim 1, characterised in that said donor or recipient marker is a fluorogenic or chromogenic marker, especially Escherichia coli lacZ, green fluorescent protein (GFP) , luciferase, or human placental alkaline phosphatase (ALPP) ; or Herpes simplex virus type 1 Thymidine kinase (HSV1- tk) .

3. : System according to claim 1 or 2, characterised in that said donor marker has a higher heat stability, a higher enzymatic activity, a higher fluorescence or luminescence intensity, different excitation/emission spectra, different substrate or antibody affinities, or a higher acid stability compared with said recipient marker.

4. : System according to any one of claims 1 to 3, characterised in that said donor/recipient marker are selected from the group consisting of: human placental alkaline phosphatase (ALPP) and a heat labile ALPP-mutant, especially ALPP^E429G; green fluorescent protein and a GFP mutant with altered fluorescence or shifted spectra, especially GFP^F64L'^S65T or GFP^Y66H; firefly luciferase mutant S284T and firefly luciferase; Herpes Simplex Virus Thymi- dine kinase with altered substrate specificity, especially HSV1- R176Qsr39tk and HSVl-A167Ysr39tk, or with loss of function mutation, especially 128F.

5. : System according to any one of claims 1 to 4 , characterised in that said animal is a rabbit, a rodent, especially a hamster, a mouse, a guinea pig, a pig or a rat; a primate, especially a chimpanzee .

6. : System according to any one of claims 1 to 5, characterised in that said marker is controlled by an inducible promoter.

7. : System according to any one of claims 1 to 5, characterised in that said marker is controlled by an ubiquitous constitutive promoter, especially R26, β-actin or β-actin with cytomegalovirus enhancer.

8. : System according to any one of claims 1 to 5, characterised in that said marker is controlled by a cell- or tissue-specific promoter .

9. Use of a system according to any one of claims 1 to 8 for tracking cells, preferably for liver diseases, especially cell therapy of liver cirrhosis or other severe liver diseases; lung diseases, especially cell-based gene therapy of cystic fibrosis; skin diseases, especially cell therapy of skin defects with epidermal cells; cardiovascular diseases, especially cell therapy of myocardial infarction with mesenchymal cells, gene therapy of cardiomyopathies, especially with vascular growth factors; kidney diseases, especially cell therapy or cell-based gene therapy of glomerulonephritis or of renal failure; orthopaedic diseases, especially cell therapy of osteoarthritis, disc degeneration, ligament injuries or cell-based gene therapy of genetic myopathies) ; diabetes mellitus, especially cell therapy of diabetes by transplantation of autologous beta cells grown from precursor cells extracted from pancreas, liver, or bone marrow; diseases of the central nervous system, especially cell therapy or cell- based gene therapy of neurodegenerative diseases, especially Parkinson's disease or amyotrophic lateral sclerosis, cell therapy of cerebral infarction (palsy) , or cell therapy of multiple sclerosis) ; or cancer, especially cell therapy with ex vivo conditioned autologous immune cells, cell-based gene therapy with autologous cells expressing immunostimulatory cytokines, especially interleukin 2.

10. Method for tracking cells wherein a donor harbouring a donor marker as defined in any one of claims 1 to 8 is transferred to a recipient animal as defined in any one of claims 1 to 8 where^¬ after said donor marker is tracked in said recipient animal.