WO2007069062A2 - Cassette system for expression control and cell differentiation by inducible rna interference and uses thereof - Google Patents

Abstract

A regulatory system comprising an integration system with a recombinase target site, such as frt, a marker, such as GFP, and a vector that can integrate into the recombinase target site is disclosed. A regulatory nucleic acid, such as shRNA targeting the production of a certain protein, can be inducibly expressed via the so integrated vector from the site defined by the recombinase target site. The sytem can be used to manipulate regulatory processes in a cell, including cell differentiation.

Description

CASSETTE SYSTEM FOR EXPRESSION CONTROL AND CELL DIFFERENTIATION BY INDUCIBLE RNA INTERFERENCE AND USES THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. provisional application no. 60/750,374, filed December 15, 2005, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention is directed at vectors and eukaryotic cells, in particular eukaryotic stem cells, for the inducible expression of regulatory nucleic acids and the use of these vectors and cells.

BACKGROUND AND INTRODUCTION

In recent years, interest in the stable expression of genes in mammalian cells from specific genomic loci has grown. Systems have been developed that allow for the introduction of an Flp-recombination-target (FRT) site into the genome of a mammalian cell line. Cell lines containing an integrated FRT site can be selected via antibiotic resistance conferred by a respective gene introduced with the FRT site. An expression vector containing a gene of choice can be integrated into the genome via FIp recombinase-mediated DNA recombination at this FRT site, allowing stable expression of the gene of interest from the genomic locus defined by the original FRT site. A hygromycin resistance gene that is only transcribed upon integration of the gene carrying vector provides a positive selection marker of successful integration of this vector. The SV40 promoter allows for potent expression of protein in differentiated eukaryotic cells (Invitrogen, Corp., CA).

Certain of these systems allow for the generation of stable mammalian cell lines exhibiting tetracycline-inducible expression of a protein of interest from a specific genomic location via the SV40 promoter. The vector containing the gene for the protein of interest is integrated into the genome via FIp recombinase-mediated DNA recombination at the FRT site and the production of the protein of interest is inducible by the addition of tetracycline (FIp-In T-REx System™, Invitrogen, Corp., CA). RNA interference (RNAi) is a process where introduction of dsRNA into a cell causes destruction of RNA in a sequence-specific manner. RNAi has been observed in a wide variety of organisms and data show that double-stranded (ds) RNA serves as the initial trigger of RNA interference, which, upon recognition, is processed into short fragments. Such short interfering (si) RNAs are then incorporated into a dsRNA-induced silencing complex (RISC) for specific RNA degradation. Mirco (mi) RNAs undergo a similar processing in the cell as (si)RNAs. However, while siRNAs are the result of transposons, viruses or endogenous genes expressing long dsRNA, miRNAs are the products of endogenous, non-coding genes whose precursor RNA transcripts can form small stem-loops from which they mature into miRNAs. miRNAs are encoded by genes distinct from the mRNAs whose expression they control.

The publications and other materials, including patents, used herein to illustrate the invention and, in particular, to provide additional details respecting the practice are incorporated herein by reference. For convenience, the publications are referenced in the following text by author and date and are listed in the appended bibliography.

Two approaches have gained popularity for expressing siRNAs: (1) The sense and antisense strands constituting the siRNA duplex are transcribed by individual promoters or (2) siRNAs are expressed as fold-back stem-loop structures that give rise to siRNAs after intracellular processing. The endogenous expression of siRNAs from introduced DNA templates is thought to overcome some limitations of exogenous siRNA delivery, in particular the transient loss-of-phenotype (see U.S. Patent publication 20060212950).

siRNA and miRNAs have been widely embraced by the scientific community as a new research tool and their potential applications are vast. However, while being highly specific, stability and targeting remain some of the major obstacles in the use of such RNAs.

To address stability issues, siRNA, miRNA as well as other "interfering" RNAs such as antisense RNA (asRNA) can be encoded via vectors and cells may be transformed with those vectors. However, while isolated cells in culture can be transformed with vectors without triggering any adverse reaction, cells or tissue of a multicellular organism usually recognize any foreign genetic material and mount an immune response against it. Direct delivery of vectors that lead to longer transcripts can also result in a dramatic increase in interferon concentrations that may in turn trigger destruction of the foreign DNA or RNA or the cell, or tissue that was transformed and is expressing the foreign gene undergoes apoptosis.

Van de Wetering et al. (2003) reported the stable integration of a doxycycline inducible siRNA that allowed for specific downregulation of β-catecin. The group produced cell lines that stably expressed the Tet repressor (by using blasticidin selection). A modified H1 promoter was then used to create siRNA expression constructs, which were introduced into the Tet repressor-expressing cells. Cells that had the siRNA expression constructs stably integrated into their chromosome were then selected using Zeocin selection and used to inducibly inhibit gene expression.

There is a need for a system allowing inducible expression of regulatory nucleic acids from a defined locus of a genome of a eukaryotic cell. There is also a need for a system that allows the expression of regulatory nucleic acids in undifferentiated cells. There is furthermore a need for a system that allows one to induce, at any stage of cellular development and in any cellular context, the production of one or more regulatory nucleic acids that target specific protein of interest. There is also a need for a system that allows one to initiate cell differentiation by switching on one or more regulatory nucleic acids that up- or downregulates the expression of one or more proteins involved in the differentiation of stem cells.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a schematic view of doxycycline-inducible shRNA expression.

(A) The Tet repressor (tetR) binds to the Tet operator (TO) blocking RNA polymerase III. Addition of doxycycline (Dox) removes tetR and shRNA is produced and processed intracellular^ to 21 nt siRNA as described by van de Wetering (2003).

(B) A modified Flip-In™ system is used to recombine a vector containing the shRNA of interest into a defined locus marked by GFP and flanked by the Flp-recombinase-target

(frt) and the neomycin resistance gene. After successful recombination following cotransfection with plasmids encoding flipase and frt-containing Tet-shRNA, cells lose both GFP expression and G418 resistance after dislocation from the promoter but acquire hygromycin resistance and inducible shRNA expression. Frt is marked by an asterisk.

Figure 2 shows the effects of LIF removal in CCE and CGR8 cells. (A) Both cell lines (CCE and CGR8) were grown for 3 days in medium with or without LIF.

Cells grown with LIF show the dome-like colony morphology of undifferentiated cells, whereas cells kept in medium without LIF show a flat, spread-out morphology characteristic of differentiated ES cells. (B) The left panel shows a Brf1 (Butyrate response factor 1 , Zfp36L1) Western blot of CGR8 cells grown with or without LIF. Samples were taken at indicated days, α-tubulin served as loading control. The right panel shows a Northern blot with β-actin as loading control. Note that protein and RNA levels of Brf1 drop after LIF removal.

Figure 3 shows that Brf1 expression is controlled by LIF and Stat3.

(A) LIF removal experiment. CCE cells were kept for 4 days either with or without LIF as indicated. Protein levels of Brf1 , AUF1 , HuR and α-tubulin are shown.

(B) BrM mRNA levels monitored by Northern blot, GAPDH served as loading control.

(C) Effect of Stat3 siRNA. Cells were plated with or without LIF and, as indicated, treated for 48h with Stat3 or β-globin (control) siRNA. Expression of Stat3 and Brf1 was monitored by Western blotting.

Figure 4 depicts the inducible downregulation of Stat3.

(A) After 7 days in culture (+LIF; -/+Dox) RNA from indicated cells was isolated and processed for Northern blotting using a 5'-labeled Stat3 oligonucleotide probe. Markers shown on the right include synthetic 21 nt Stat3 siRNA and 58nt Stat3 shRNA. (B) Time-course experiment over 7 days in presence or absence of LIF and doxycycline as indicated. Lysates from F3-1 control cells and the two Stat3 shRNA clones F3-1-S2 and F3-1-S3 were analyzed by Western blot against Stat3. GAPDH served as loading control.

Figure 5 illustrates the downregulation of Brf1 by LIF removal or shRNA induction. After 7 days in culture in presence or absence of LIF and with doxycycline as indicated, Western blot analysis for Stat3 and Brf1 was performed for F3-1 control cells and three independent Stat3 shRNA clones F3-1-S3, F3-1-S6 and F3-1-S8.

Figure 6 shows a DNA microarray analysis.

RNA from indicated cells and conditions was extracted at d3 and subjected to Affymetrix DNA microarray analysis.

(A) Shown are levels of Stat3 and Brf1 from F3-1 (control), F3-1-S2 (Stat3 shRNA) and F3-1-B14 (Brf1 shRNA) all grown with and without LIF and with LIF in presence of doxycycline as indicated.

(B) Nanog levels of F3-1 and F3-1-S2 cells under conditions as in (A).

Figure 7 shows the induction of differentiation by Stat3 shRNA.

(A) After 7 days in culture (+LIF/-Dox; -LIF/-Dox; +LlF/+Dox), F3-1-S2 and F3-1-S3 cells were inspected for morphological changes after doxycycline addition. F3-1 cells served as control. Colony morphology of undifferentiated cells is compact (left panels), that of differentiated cells spread-out and extended. Magnification is 40-fold.

(B) At the indicated time of culture (+LIF/-Dox; -LIF/-Dox; +LIF/+Dox) RNA from parallel cultures shown in (A) was extracted and processed for Northern blotting. Northern blots probed for three stem cell markers (Fgf-4, Oct4, Rex-1 ) are shown on the left and quantification on the right. RNA levels were normalized against β-actin; the RNA levels of cells at day 3 +LIF/-Dox were set as 100%.

Figure 8 shows the doxycycline-inducible downregulation of Brf1. (A) After 7 days in culture (+LIF; -/+Dox) RNA from indicated cells was isolated and processed for Northern blotting using a 5'- labeled Brf 1 oligonucleotide probe. Markers shown on the right include synthetic 21 nt BrM siRNA and 58nt BrM shRNA. (B) After 3 days in culture in the presence of LIF (-/+Dox) as indicated, BrM was examined by Western blot analysis, α-tubulin served as loading control.

Figure 9 shows the embryoid body morphology.

Morphology of EBs at d10 from F3-1 and F3-1-B14 cells either kept with or without doxycycline from day 0 to day 18. Additionally, F3-1-B14 EBs were either kept with doxycycline during the first four days or from day 4 until the end of the experiment. Figure 10 depicts the increase of beating areas within EBs.

Embryoid bodies from F3-1 and F3-1-B14 cells were plated with or without doxycycline into 24 well plates. For each cell and condition around 720 EBs were plated and beating areas were counted at day 18.

Upper panel: Shown in red are beating areas. Note that a single EB may contain more than one beating area, and beating areas may become confluent with time. Numbers indicate beating areas from 720 bodies plated.

Lower panel: The size of beating areas from 3 experiments was quantified, averaged and is shown with standard error of the mean.

Figure 11 shows that Brf1 downregulation is correlated with stimulation of cardiomyocyte formation.

(A) Control cells (F3-1) and Brf1 shRNA cells (F3-1-B14) were cultured in the absence of LIF with or without doxycycline for 18 days. Western blot analysis was performed for

Nkx2.5, Gata4 and GAPDH. Also shown are undifferentiated F3-1 and F3-1-B14 cells grown in LIF and mouse heart as controls.

(B) Semi-quantitative RT-PCR of the cardiac marker α-cardiac actin at indicated days after LIF removal. GAPDH served as control. (C) F3-1 and F3-1-B14 EBs were plated into 48-well plates at two bodies per well and cultured with or without doxycycline. EBs were checked daily for onset of beating and scored. The data is shown as percentage of beating bodies to total plated. Upper panel: F3-1 and F3-1-B14 EBs cultured with doxycycline for the total length of the experiment. For each time point at least 3 experiments are averaged and standard errors of the mean are shown.

Lower panel: As above, but doxycycline was present where indicated only from day 0 to day 4. For each time point the average and standard errors of the mean of at least 3 experiments are shown.

Figure 12 shows the results of a semi-quantitative PCR analysis.

(A) RT-PCR analysis of myogenin, Gata4 and Nkx2.5 at indicated days after LIF removal. 18S rRNA served as loading control. Numbers of PCR cycles are shown on the right. (B) Variation of cycles: Analysis of α-cardiac actin, Gata4, Nkx2.5 and GAPDH using different cycle numbers as indicated.

Figure 13 shows Brf1 overexpression. CCE cells were transfected with Brf1 wildtype (plRES Brf1 wt) or Brf1 zinc-finger mutant (plRES Brf1 mut) (Stoecklin et al, 2002) and selected for 10 days with puromycin. Notabe is the drastic inhibitory effect of wildtype Brf1 compared with the zinc-finger mutant, which is unable to bind mRNA.

SUMMARY OF THE INVENTION

The present invention is directed towards a regulatory system/a kit comprising

(a) an integration system comprising at least one first recombinase target site, at least one sequence encoding at least one marker having an expression status, wherein integration of a sequence into the recombinase target site changes said expression status of the at least one marker, and

(b) a vector comprising at least one second recombinase target site compatible with the first recombinase target site of (a), and at least one inducible expression cassette.

A sequence encoding a regulatory nucleic acid, such as, but not limited to, shRNA, miRNA or antisense RNA, may be inserted into the inducible expression cassette of vector (b) so that the regulatory nucleic acid is inducible expressed subsequent to integration of the vector of (b) into the system of (a) in an eukaryotic cell.

The regulatory system/kit may also comprise (c) a vector expressing a recombinase mediating the integration of the vector in (b) into the system of (a) via the first and second recombinase target site and/or (d) a vector expressing a repressor repressing transcription via the inducible expression cassette.

The integration system of (a) may be a genome of a modified eukaryotic cell and, upon introduction of the vectors of (b), (c) and, optionally (d), expression of the regulatory nucleic acid may be inducible in the modified eukaryotic cell. The modified eukaryotic cell may be a stem cell and the marker may be under the control of a wide spectrum promoter system such as, but not limited to, CAG and EF1α.

In certain embodiments, the expression cassette is inducible by a chemical inducer acting on a repressor such as, but not limited to, doxycycline, which acts on a tet repressor.

The first and second recombinase target sites may be a flp-recombinase-target site or a loxP site, while the recombinases mediating the integration of the vector of (b) into the system of (a) may be a f Ip- recombinase or a Cre-recombinase.

A kit according to the present invention may, in a separate container, have instructions for the use of (a) and (b) to insert a sequence encoding said regulatory nucleic acid via the vector of (b) into the system of (a).

The present invention is also directed towards a genetic construct for expressing at least one regulatory nucleic acid in stem cells comprising

(a) a sequence encoding at least one regulatory nucleic acid, such as, but not limited to, shRNA or miRNA, wherein the regulatory nucleic acid directly or indirectly up- or down regulates production of at least one protein, and

(b) an induction cassette, wherein expression of the regulatory nucleic acid is under the control of the induction cassette and wherein the regulatory nucleic acid, upon induction in stem cells via said induction cassette, up- or down regulates said at least one protein.

The genetic construct may be a modified stem cell, such as a human stem cell, that also comprises at least one gene encoding the at least one protein that is being up- or down regulated. This protein may, in certain embodiments, be a posttranscriptional regulator, such as Brf1 that regulates, in cis, mRNA turnover. The regulatory nucleic acid may downregulate Brf1 , contribuing to cardiomycotes formation.

A chemical inducer, such as, but not limited to doxycycline, estrogen or dexamethason, may induce said induction cassette. The invention is also directed towards a method for producing a system for inducible up- or down regulation of a protein comprising: constructing a genetic system comprising at least one first recombinase target site, at least one sequence encoding at least one marker having an expression status, wherein integration of a sequence into the recombinase target site changes said expression status of said at least one marker, wherein a sequence for a regulatory nucleic acid that up- or down regulates the protein and which is integrated into at least one inducible expression cassette of a vector comprising at least one second recombinase target site compatible with said first recombinase target site, is expressed upon integration of the vector into the genetic system by at least one recombinase mediating recombination via the first and second recombinase target site, and induction of the inducible expression cassette via an inducer removing repression of transcription of said regulatory nucleic acid. The sequence encoding the marker may be expressed via an broad sprectrum promoter system.

In certain embodiments, the genetic system is integrated into a genetically modified stem cell or at least one cell of a transgenic non-human animal. Also within the scope of the present invention are genetically modified stem cells and transgenic non-human animals produced according to the method described above and/or comprising cells described herein.

The present invention is also directed at polynucleotide sequences that have substantial homology or substantial identity with the disclosed polynucleotide sequences. The terms "substantial homology" or "substantial identity", when referring to a polynucleotide sequence of the present invention, indicate that the polynucleotide sequence in question exhibits at least about 30% identity with the respective portion of the disclosed polynucleotide sequence, usually at least about 70% identity, more usually at least about 80% identity, preferably at least about 85% or at least about 90% identity, more preferably at least 95% or 96%, even more preferably at least 97% or 98%, and most preferably at least 99% identity with any sequence mentioned herein. The percentage of sequence identity for polynucleotides is calculated by aligning the sequences being compared, and then counting the number of shared residues at each aligned position. No penalty is imposed for the presence of insertions or deletions, but they are permitted only where required to accommodate an obviously increased number of amino acid residues in one of the sequences being aligned. When one of the sequences being compared is indicated as being "consecutive", then no gaps are permitted in that sequence during the comparison. The percentage identity is given in terms of residues in the test sequence that is identical to residues in the comparison or reference sequence.

DETAILED DESCRIPTION OF VARIOUS AND PREFERRED EMBODIMENTS OF THE

INVENTION

DEFINITIONS

"Brf1" in the context of the present invention stands for Butyrate response factor 1 , also known as Zfp36L1. A human Brf1 is available under Swiss Prot accession no. Q07352, whose February 1, 1996 version is incorporated herein by reference in its entirety. However, non-human Brfis, in particular any mammalian Brf1, more in particular any rodent Brf1 , is also within the scope of the present invention.

A "recombinase target site" is any site that allows recombination between a first and a second nucleic acid molecule that contains a compatible recombinase target site. A recombinase target site is "compatible" with another recombinase target site, if, upon exposure of the first and second nucleic acid to a single or multiple "recombinases" recombination between the two nucleic acids may proceed. However, further auxiliary factors might be required to allow such recombination to occur. One recombinase useful in the context of the present invention is flp-recombinase that can mediate recombination of two nucleic acid molecules each containing an flp-recombinase target (frt) site. Another recombinase useful in the context of the present invention is the Cre recombinase that can mediate recombination of two nucleic acid molecules each containing a loxP site.

A "marker" is, in the context of the present invention, any protein that can be used to assess the integrity of the sequence mediating expression of or encoding the marker. Typically, when those sequences are intact, the marker is expressed, while when their integrity is destroyed, for example, by insertion of an additional sequence, the marker ceases to be expressed. However, this is only one way to change the expression status of a marker. A wide variety of alternatives are well known in the art and within the scope of the present invention. As the person skilled in the art will appreciate, positive and negative markers are within the scope of the present invention. For example, a positive marker, that is a marker, that is only expressed upon integration of, e.g., the vector (b) into the system of (a) of the regulatory system described herein is within the scope of the present invention. Such a positive marker will render it easier to ensure that the desired recombination event took place. Within the scope of the present invention are a variety of markers, including, but not limited to, antibiotic resistance markers, that are markers that confers resistance against antibiotics (e.g. Hygromycin, Puromycin, Neomycin, Blasticidin, Zeocin), fluorescent protein markers, that are any fluorescent protein (FP) (e.g., Green FP (GFP), Red FP (RFP), Blue FP (CFP), Yellow FP(YFP)) as well as markers based on chemoluminescence (e.g., luciferase) and markers such as beta- galactosidase (lacZ). Not expressed surface markers are also within the scope of the present invention. Here selection is accomplished by, e.g., immuno-absorbance to magnetic beads that are pre-coated with the antibody against the surface marker.

An "expression cassette" is a nucleic acid sequence comprising one or more restriction sites for inserting an additional nucleic acid sequence into it as well as one or more elements that allow transcription of this additional nucleic acid sequence. An "inducible expression cassette" ensures that transcription does not proceed in a cell prior to exposure to an inducer. Transcription is induced, which may take the form of derepression, by an inducer, such as a protein or other molecule (e.g., Cadmium²⁺ (or other divalent cation). In cases in which repressors are involved, upon addition of this inducer, transcription of the RNA starts. Repression can be achieved, for example, via a Tet-repressor based system, including systems based on the Tet-repressor (TetR) itself or the tTR-KRAB system which uses a TetR that is fused to the KRAB domain of human Kox1. Inducible expression cassettes often include specific elements to which, e.g., a repressor may bind. Upon exposure to an inducer the repressor may dissociate from the element allowing transcription to proceed. An "induction cassette" according to the present invention comprises parts of an inducible expression cassette. In particular, such an induction cassette includes elements for expression which is induced via, e.g., a chemical or physical inducer, of a sequence that is under the control of such an induction cassette. Preferred inducers of the present invention are chemical inducers such as, but not limited to, antibiotics of the tetracycline family, including the tetracycline analog doxycycline as well as estrogen or dexamethason.

A "stem cell" according to the present invention is a cell from an embryo, fetus or adult that has, under certain conditions, the ability to reproduce itself for long periods or, in the case of adult stem cells, throughout the life of the organism. It also can give rise to specialized cells that make up the tissues and organs of the body. Stem cells according to the present invention include for example, but are not limited to, pluripotent stem cells, embryonic stem cells, embryonic germ cells and adult stem cells.

A "broad spectrum promoter system" is a system comprising at least a promoter that provides for detectable expression (via the protocol described by Chung et al. in Stem Cells 20(2): 139-45 (2002)) at different stages of cell development, including in particular the "embryonic stem cell" stage, of a nucleic acids that are under its control as well as other stages, such as the "adult stem cell" stage. Thus, any nucleic acid under the control of such a broad spectrum promoter system will be detectably expressed in embryonic stem cells. In that they differ from commonly used promoters of viral origin such as the CMV and SV40 promoters, which often do not show detectable expression in embryonic stem cells. CAG (also CBA or CB), a fusion of the CMV immediate early enhancer and a modified chicken β-actin promoter, and EF1 α (Elongation factor 1 α) are non limiting examples of promoter systems that fall under the definition of broad spectrum promoter system according to the present invention.

A "regulatory nucleic acid" is any kind of nucleic acid that, when acting on one of the entities involved in, e.g., the expression of a gene, affects the level of expression of a protein encoded by the gene. Antisense RNA, for example, blocks translation of an m- RNA. However, many regulatory RNAs rely on RNA Interference (RNAi), a phenomenon in which small RNAs (referred as small interference RNAs (siRNAs) or mico RNAs (miRNAs)) can induce efficient silencing of gene expression. SiRNA is hereby a double stranded form of RNA that causes RNA degradation making use of the cell's mechanism to combat double stranded virus RNAs. MiRNA is encoded by cells and regulates the expression of genes by binding to the 3'-untranslated regions (3'-UTR) of specific mRNAs.

A "vector" is a plasmid, cosmid, phagemid or phage DNA or other DNA molecule which is able to replicate autonomously in a host cell, and which is characterized by one or a small number of restriction endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without loss of an essential biological function of the vector, and into which DNA may be inserted in order to bring about its replication and cloning.

The invention is directed at a regulatory system that allows for an inducible expression of regulatory nucleic acids from a specific locus in the genome of a eukaryotic cell. After a first recombination target site is created in a eukaryotic cell (a "modified eukaryotic cell") such as a stem cell, a vector comprising a sequence encoding a regulatory nucleic acid that is inserted into an inducible expression cassette of the vector, is integrated into this recombination site via the action of a suitable recombinase. Thus, expression of the regulatory nucleic acid from a specific location defined by this first recombination site can be accomplished by providing an inducer. Such a system has a wide array of uses. As will be described in more detail, using the posttranscriptional regulator Brf1 (Butyrate response factor 1 , Zfp36L1) as an example, the regulatory nucleic acid, e.g., shRNA that is processed to siRNA that targets the mRNA of Brf1 is inserted into said vector. Subsequent to the integration of the vector into the genome, the expression of said shRNA can, in the example, be induced by doxycycline. However, as the person skilled in the art will appreciate, inducers may vary widely and will depend on the inducible expression cassette chosen. In particular, it will depend how, the expression cassette is kept "silent" prior to induction. For example, the expression, may be repressed by a repressor, a term which includes in the context of the present invention any agent that prevents transcription of the nucleic acid that is under its control. In the example described in more detail below, the expression of shRNA for regulating the Brf mRNA turnover, in this case, degrading the mRNA and thus causing a decrease in the production of the Brf protein, is repressed via the tet repressor (TetR) that binds to a tet. operator (TO). The TetR bound to the TO will prevent transcription from, here, the polymerase III dependent (Pol III) promoter H1 , which allows the production of transcripts carrying only a few non-homologous bases at their 3' ends. The term "expression" of a sequence is, in the context of the present invention, used broadly and is not confined to the "expression" of proteins. Thus, the term is used to describe the production of nucleic acids such as shRNAs, siRNAs and miRNAs. The regulatory system of the present invention will now be explained using the nonlimiting example depicted in Fig. 1.

Fig. 1 A depicts an inducible cassette for the expression of shRNAs. A repressor, namely the Tet repressor (tetR) binds to the Tet operator (TO) blocking RNA polymerase III. Addition of a suitable inducer, namely doxycycline (Dox) removes tetR and shRNA is produced and processed introacellularly to 21 nt siRNA. (van de Wetering, 2003). The upper part of Fig. 1 B is a schematic showing an integration system of the present invention. Here the integration system is part of the genome of an eukaryotic cell, e.g., a stem cell. The integration system shown can be used to integrate a vector comprising, e.g., a shRNA of interest, into a defined locus of such a eukaryotic cell via site specific recombination. The integration system that allows for such recombination may be created by inserting a vector comprising a recombinase target site, such as the FIp- recombinase target site shown, and a gene for a marker, such the Green-fluorescent protein (GFP) or any other, preferably, non-toxic marker, into the eukaryotic cell of interest. In a preferred embodiment, the marker is under the control of a broad spectrum promoter system. The integration system might, for example, integrate into the genome of a eukaryotic cell randomly. As can be seen from the Figure, in the embodiment depicted, not only GFP is expressed, but a second marker, here, neomycin phosphotransferase, providing the experimentator with an additional level of control. The person skilled in the art will appreciate, that as with the first marker, wide variations are possible with regard to the nature of this the second marker. However, generally, substantially non-toxic markers, that are markers that allow for extended propagation of the modified eukaryotic cell without causing cell death are preferred. In yet another preferred embodiment, the marker does not require cell lysis, thus cell death, to assess its expression as it is the case for β-galactosidase or luciferase. In one embodiment of the present invention, the recombinase target site and the marker are, as shown, part of the genome of a eukaryotic cell, such as a stem cell. Such a modified eukaryotic cell provides a system that can be readily transformed to one that expresses a regulatory nucleic acid of interest. In certain embodiments of the invention, a cell line expressing a regulatory nucleic acid of interest can, starting out with such an eukaryotic cell, be created in less than 14 days, preferably in less than 10 days. Even if screening is performed to confirm downregulation and regulatory nucleic acid expression, the time involved may be less than 5 weeks, preferably less than 4 weeks. A homozygous mouse expressing such a regulatory nucleic acids may, in certain embodiments of the present invention, be created in less than 25 weeks, preferably less than 24 weeks, more preferably about 23 weeks or less. This time frame includes the production of chimeras and two rounds of mating to obtain the homozygous mouse. Depending the inducible system that regulates the nucleic acid of interest, the cell in which the regulatory nucleic acid is to be expressed might express, e.g., a repressor such as the tet-repressor (TetR), preventing induction of the regulatory nucleic acid of interest prior to exposure to the respective inducer. In one preferred embodiment, the eukaryotic cell is stably transformed with a vector expressing the repressor, e.g., the TetR.

The lower part of Fig. 1 B that, upon flp- recombinase mediated integration of a vector comprising a shRNA of interest, expression of the marker(s)of the integration system cease. In the embodiment shown, the regulatory system is also set up so that upon successful integration of such a vector into the recombinase target site, a marker, such as hygromycin phosphotransferase, is expressed. Such a positive selection marker of integration is part of some embodiments of the present invention and provides the experimentator yet with an additional level of control.

In the vector comprising the regulatory nucleic acid, here the shRNA, this regulatory nucleic acid is inserted into an inducible expression cassette. In the embodiment shown inducibility is accomplished by a TO that binds the TetR and thus prevents transcription of the sequence under its control, here the shRNA. The TetR dissociates from the TO upon addition of doxycycline (DOX). Thus, to allow inducible expression of the shRNA, a repressor, in the embodiment shown, TetR must be present in the cell of interest prior to its transformation with the vector comprising, in this instance, the shRNA under the control of the TO. Not shown in this Figure is the expression cassette for the TetR. The TetR might also be constitutively expressed by eukaryotic cells. This can be accomplished, for example, via a vector comprising the TetR or, depending on the induction system used, another repressor,that integrates into the genome, e.g., randomly. The vector comprising the regulatory nucleic acid also comprises a recombination target site that is compatible with the recombination target site of the integration system that is integrated or integratable into the genome of the cell of interest. In the embodiment shown in Fig. 1 B, such a compatible recombinase target site is another frt site. Recombination of the vector into the integration system described above is mediated by a recombinase. In the embodiment shown, the recombinase is a FIp- recombinase (Flipase), which can be expressed by a separate vector that is, in a preferred embodiment, co-transfected with the vector comprising the regulatory nucleic acid into the cell containing the integration system described herein.

As the person skilled in the art will appreciate, any regulatory nucleic acid can be inducibly expressed using the system described herein. Creating a regulatory system that allows the expression of regulatory nucleic acids targeting specific proteins of interest, will allow one to assess the function of the protein at a particular stage. For example, if the expression of the protein is shut down at a specific time in the development of a cell and the cells behavior changes, this change can be attributed to the specific protein whose expression was shut down. If this change in behavior is a desirable one, it may be of interest to produce genetic constructs that express this specific regulatory nucleic acid. In a preferred embodiment of the present invention, a protein up- or downregulated by the regulatory nucleic acid of the present invention is involved or suspected to be involved (candidate protein) in the differentiation of stem cells. Accordingly, the respective regulatory nucleic acid may be introduced into a stem cell and inducibly expressed therein. The downregulation of protein of interest may lead to changes in the stem cell that by itself or in combination with other factors or events, lead to its differentiation of the stem cell into a differentiated cell such as a cardiomycotes. This renders the system and cells, in particular, stem cells described herein valuable for the production of a certain type of differentiated cell having therapeutic use in tissue reconstitution and regeneration.

For example, embryonic stem cells that have been differentiated according to the present invention can be used for tissue reconstitution or regeneration in mammals in need thereof. The cells are generally administered in a manner that permits them to graft to the intended tissue site and reconstitute or regenerate the functionally deficient area.

Neural stem cells have been transplanted directly into parenchymal or intrathecal sites of the central nervous system. Grafting was performed using single cell suspension or small aggregates at a density of 25,000-500,000 cells per μl (U.S. Pat. No. 5,968,829). The efficacy of neural cell transplants was assessed in a rat model for acutely injured spinal cord as described by McDonald et al. (1999).

The efficacy of cardiomyocytes produced according to the present invention can be assessed in animal models for cardiac cryoinjury, which causes 55% of the left ventricular wall tissue to become scar tissue without treatment (Li et al, 1996; Sakai et al, 1999 [1], Sakai et al, 1999 [2]). Successful treatment will reduce the area of the scar, limit scar expansion, and improve heart function as determined by systolic, diastolic, and developed pressure. Cardiac injury can also be modeled using an embolization coil in the distal portion of the left anterior descending artery (Watanabe et al,1998), and efficacy of treatment can be evaluated by histology and cardiac function. Cardiomyocyte preparations embodied in this invention can be used in therapy to regenerate cardiac muscle and treat insufficient cardiac function (U.S. Pat. No. 5,919,449 and WO 99/03973).

Any Graft Versus Host Disease (GVHD) that is associated with grafting of cells produced according to the present invention may be suppressed by different well known methods, one of which includes administering a modulator of Notch signaling (see U.S. patent publication 20060140943).

A non-human animal model that expresses, preferably in each of its cells, regulatory nucleic acid of interest is a desirable tool for science and medicine. Using the inducible expression system of the present invention, the regulatory nucleic acid can be switched on in particular tissues and the function of the protein targeted by the regulatory nucleic acid can be assessed in the particular tissue and, if desirable, at a specific stage.

The transgenic non-human animal of the present invention may be produced by a variety of techniques for genetically engineering transgenic animals, including those known in the art.

As used herein, the term "transgenic non-human animal" refers to a genetically- engineered non-human animal, whose genome has been altered by introduction of a transgene. As further used herein, the term "transgene" refers to any nucleic acid, coding or not coding, introduced into the non-human animal by genetic manipulation, wherein the particular introduced nucleic acid is not endogenous to the animal, but might be a duplicate or modified version of an endogenous nucleic acid. Such a transgenic non-human animal may be produced by several methods, including, but not limited to, introduction of a transgene into an embryonic stem cell, newly fertilized egg, or early embryo of a non-human animal; integration of a transgene into a chromosome of the somatic and/or germ cells of a non-human animal; and any of the methods described herein.

In one embodiment, the genome of transgenic animal of the present invention comprises, as a result of genetic engineering, the components of the integration system described herein. The transgenic animal may also comprise a regulatory nucleic acid which has been inserted into an inducible expression cassette as described herein.

A transgenic non-human animal according to the present invention might be created as follows: (a) generating an embryonic stem cell comprising the desired components described herein, e.g., an integration system; (b) introducing the so created embryonic stem cell into a blastocyst of a non-human animal, to produce a treated blastocyst; (c) introducing the treated blastocyst into a pseudopregnant non-human animal; (d) allowing the transplanted blastocyst to develop to term; (e) identifying a transgenic non-human animal whose genome comprises the desired features; and (f) breeding the transgenic non-human animal to obtain a transgenic non-human animal having the desired genetic make-up.

The non-human animal may be any suitable animal (e.g., cat, cattle, dog, horse, goat, rodent, and sheep), but is preferably a rodent. More preferably, the non-human animal is a rat or a mouse.

The use of the systems and vectors of the present invention is now described using a specific non-limiting example. In particular, the role of post-transcriptional forms of regulation in ES differentiation using the vectors and systems described herein.

Embryonic stem (ES) cell lines provide an attractive system to study the basically unresolved question of how stem cells decide between self-renewal and differentiation (Smith, 2001). From a clinical perspective, they provide a promising tool for the emerging field of regenerative medicine. The pluripotency of cultured murine ES cells is maintained by the cytokine leukemia inhibitory factor (LIF), which restrains ES cells from differentiation and acts via LIF-receptor-dependent activation of the transcription factor Stat3 (Williams et al, 1988; Smith et al., 1988; Matsuda et al, 1999; Niwa et al, 1998). How the Stat3 targets maintain pluripotency and why loss of Stat3 activation leads to differentiation is not known, although recent work assigns a key role to c-myc (Cartwright et al, 2005). In addition to Stat3, other transcription factors including Oct4, nanog, Sox2 and the BMP4 regulatory protein also play important roles in maintaining the pluripotent state of ES cells (Nichols et al, 1998; Niwa et al, 2000; Shimozaki et al, 2003; Chambers et al, 2003; Mitsui et al, 2003; Ying et al, 2003; Qi et al, 2004). Recent results from human ES cells indicate that promoter regions of 353 genes, including Stat3, are co- occupied by Oct4, nanog and Sox2 (Boyer et al, 2005). This is consistent with a model where a hierarchical system of transcription factors controls the balance between self- renewal and differentiation and where subtle changes may be sufficient for triggering differentiation (Smith, 2001 ; Boiani et al, 2005). Thus, while it has been well established that the differentiation of pluripotent embryonic stem cells is restricted by a hierarchy of transcription factors, little is known whether posttranscriptional mechanisms similarly regulate early embryoid differentiation.

The systems and vectors of the present invention were used to address the question whether post-transcriptional forms of regulation also play a role in ES differentiation. In this context, the control of mRNA turnover of transcripts containing an AU-rich element (ARE) in their 3'-untranslated region (3'UTR) is of particular interest, as this element is present in many transcription factors, cytokines, chemokines and other regulators (Chen et al, 1995; Bakheet et al, 2001). ARE-binding proteins such as AUF1 , TTP or Brf1 (Zfp36L1), which promote ARE-dependent mRNA decay (Zang et al, 1993; Lai et al, 1999; Lai et al, 2000; Stoeklin et al, 2002), have been identified, while HuR acts as a stabilizer (Ma et al, 1996). These proteins regulate access of decapping enzymes and RNases including deadenylases and exosomal enzymes to the transcripts. The presence of common signals such as the ARE on many transcripts has suggested the concept of a "posttranscriptional operon" (Keene et al, 2002), and it may well be that a similar form of regulation also operates in ES cells and embryogenesis. First, it was assessed whether the expression of the posttranscriptional ARE-dependent regulators Brf1 , AUF1 and HuR would change under conditions at which ES cells are triggered to differentiate. When LIF was removed, CCE cells lost the compact dome-like colony morphology characteristic of undifferentiated cells and assumed the morphology of differentiated cells (Fig. 2A). In parallel, Brf1 protein levels dropped progressively, whereas expression of HuR and AUF1 was not affected (Fig. 3A). Northern blot analysis suggested that this regulatory effect on Brf1 expression occurred at the mRNA level (Fig.3B). The same general conclusion was obtained when a second ES line, CGR8, was similarly examined (Figs. 2A and B). As LIF supports the pluripotent phenotype by activating the transcription factor Stat3, we tested whether this effect would be mimicked by siRNA downregulation of Stat3. Indeed, treatment with Stat3 specific siRNA led to a concomitant downregulation of both Stat3 and Brf1 protein levels, assessed at day 2 (Fig. 3C). As it appears that Brf1 is a target gene of the LIF-gp130-Stat3 pathway, we hypothesized that a reduction of its expression may be linked to a role in ES cell differentiation.

Thus, the target of further investigation was Brf1 , originally discovered as an immediate- early gene (Gomperts et al, 1990) and a member of a small family of RNA-binding proteins with a conserved and characteristic CCCH zinc-finger domain recognizing AREs in the 3'UTR and promoting mRNA decay. The mRNA decay promoting activity of Brf1 is negatively regulated by phosphorylation via PKB, which promotes complex formation to 14-3-3 (Schmidlin et al, 2004). Target mRNAs of Brf1 are not known but play a role in development, as mice lacking both alleles die at d11 (Stumo et al, 2004).

For this specific experiment, we used a specific embodiment of the system and vectors described above. The system was set up in CCE cells. The small hairpin RNA (shRNAs) were inducibly expressed using the system shown in Fig. 1A. The Tet repressor protein binds to the Tet operator within the H1 promoter and acts as a 'roadblock' for the RNA polymerase III. Following abrogation of repressor binding by addition of doxycycline, the downstream shRNA sequence is transcribed by RNA polymerase III, and terminated by a run of five T residues (Brummelkamp et al, 2002). CCE cells were first transfected with a plasmid encoding the Tet repressor (Gossen et al, 1992; Yao et al, 1998), and after puromycin selection, a stable clone was selected which maintained Tet repressor expression well over the time required for embryoid body formation (data not shown). This clone was further transfected with a construct where GFP, under control of a CAG promoter, is flanked by a frt recombination site (O'Gorman et al, 1991) and the neomycin resistance gene. A defined locus, marked by frt-GFP-neo was thus created as a target for a Flp-recombinase integration of an inducible shRNA. The GFP expressing clone F3-1 , which is resistant to G418 and expresses GFP well past day 10 after induction of differentiation (data not shown), served as the host system for a flipase-encoding vector together with the shRNA vector containing a frt site and the selectable marker hygromycin B phophotransferase (hph). After successful recombination, the GFP gene of F3-1 was displaced by hph and inducible shRNA could be expressed via the H1 promoter together with hygromycin resistance gene that was expressed via the CAG promoter (Fig. 1 B).

The system and vectors of the present invention were first tested by introducing a shRNA targeting Stat3, since successful downregulation of Stat3 would be expected to trigger differentiation (Niwa et al, 1998) and hence could be easily monitored. Selected were two F3-1 -derived Stat3-shRNA clones (F3-1-S2; F3-1-S3), which displayed the expected profile: GFP-negative, G418 insensitive, hygromycin resistant, and correct recombination as verified by PCR (data not shown). When cells were treated for 7 days with doxycycline, both clones displayed strong siRNA induction with negligible background expression (Fig. 4A). Notably, Stat3 protein levels were downregulated in both clones tested, while doxycycline had no effect on F3-1 control cells (Fig. 4B). The effect was detectable after 1-2 days, and was more pronounced in F3-1-S3 cells, apparently reflecting clonal variation. In contrast, LIF removal downregulated Stat3 in all 3 clones including F3-1. Consistent with the result from Fig. 3, which supported that Brf 1 is controlled at least in part by LIF-Stat3, we observed that addition of doxycycline to clones F3-1-S2, F3-1-S3 and in additional Stat3 shRNA expressing clones also led to downregulation of Brf1 (Figs. 5 and 6A). As with Stat3 expression, we observed some clonal variation (not shown).

Next it was investigated whether addition of doxycycline would trigger the morphological changes characteristic for differentiation. As shown in Fig. 7A, both shRNA clones displayed vigorous morphological differentiation upon addition of doxycycline despite the presence of LIF (right panels). To corroborate this finding, we assessed three known markers of undifferentiated ES cells (Oct4, Fgf-4, Rex-1). As expected, their levels dropped following LIF removal in all three clones tested, while addition of doxycycline reproduced the same effect as LIF removal in the two shRNA containing clones, but not in F3-1 control cells (Fig. 7B). The stem cell marker nanog, measured at day 3, was reduced by LIF removal, but not by doxycycline in both F3-1 and F3-1-S2 cells (Fig. 6B), consistent with the fact that nanog is not controlled by Stat3 (Chambers et al, 2003). Together these data indicated that the morphological changes induced by shRNA were accompanied by reprogramming gene expression of established regulators and show that the frt-GFP locus functions reliably as an acceptor of the shRNA cassette with the expected doxycycline inducible response. It was concluded that the system can be used as a tool to investigate other genes with suspected roles in differentiation and concentrated our further studies on Brf1.

The same strategy as with Stat3 was used and clones containing doxycycline-inducible Brf1 shRNA were isolated. Shown in Fig. 8A are Northern blots from two representative clones clones F3-1-B9 and F3-1-B14. Again, siRNA is strongly induced in both clones, with negligible background expression and no signal in control cells. A parallel Western blot at day 3 showed that doxycycline led to downregulation of Brf1 protein (Fig. 8B). However, treatment with doxycycline for up to seven days produced no morphological changes (data not shown). It wa also assessed whether Brf1 shRNA might affect differentiation at later stages. Thus, EBs were produced and further cultured by allowing them to attach. Unexpectedly, EBs from F3-1-B14, but not from control cells, displayed a markedly altered morphology when cultured with doxycycline. Shown in Fig. 9 are representative examples photographed at day 10. While EBs from control cells appeared as compact cellular masses surrounded by a halo of outgrowing cells, doxycycline treatment of F3-1 -B14 cells led to an apparent loosening of the central mass, increased outgrowth and formation of satellite "microbodies". Interestingly, these changes were observed when doxycycline was present until day 4, but not when added after day 4. These data argue that early but not later changes in Brf1 levels affect the architecture of an embryoid body.

It is well established that cardiomyocyte formation occurs spontaneously in cultured EBs, easily recognizable under microscopy as "beating areas", or detectable biochemically by measuring cardiac specific markers such as the transcription factor Nkx2.5 (Komuro et al, 1993; Sachinidis et al, 2003). Surprisingly, it could be observed that induction of Brf1 shRNA by doxycycline led in F3-1-B14 cells to a substantial increase in number and size of beating areas (Fig. 10). Noticable was also the elevated background in these cells (140 areas) compared to the F3-1 control cells, which may reflect some leakiness. Addition of doxycycline had also a low, but reproducible effect on F3-1 control cells, an effect that is, however, negligible compared to the very strong doxycycline effect in Brf1 shRNA expressing cells. To substantiate this effect of cardiomyocyte formation, we examined the expression of various markers. 18 days following addition of doxycycline, Nkx2.5 and Gata4 expression was weak in controls but strong in F3-1-B14 cells as shown by Western blot and PCR analysis (Fig. 11 A and Fig. 12B). The skeletal muscle marker myogenin was not expressed in ES cells (Fig. 12A). Additionally, monitored the cardiac marker α-cardiac actin by semi-quantitative RT-PCR at days 4 and 18 and observed weak induction in F3-1 at day 18 and higher levels in response to doxycycline in F3-1-B14 (Fig. 11B, Fig. 12B). These findings are in agreement with the microscopic data and indicate that the appropriate change in gene expression took place.

It was further investigated whether downregulation of Brf1 would affect both the kinetics and/or the magnitude of cardiomyocyte formation and if doxycycline has to be present throughout the experiment. To that end, two EBs were plated into each well of a 48-well plate, which allowed daily inspection of individual beating bodies and scoring of the percentage of beating. As shown in Fig. 11C (upper panel), beating body formation in doxycycline-treated F3-1-B14 cells was first observed on day 9 and reached a plateau at around day 14. In the absence of doxycycline, beating body formation was observed in the range of control cells (In these experiments the number of beating bodies was scored rather than beating areas as in Fig. 10). The difference between the two stimulation indices is explained by the fact that a given beating body generally contains more than one beating area that is also of larger size. As with the morphological changes (Fig. 9), this effect on beating bodies required the presence of doxycycline early (dθ-d4) in the experiment (Fig. 11C, lower panel), and no effect was seen when the drug was added from day 4 onwards (data not shown).

Materials and Methods

Cell culture. CCE ES cells (Robertson et al, 1986; Keller et al, 1993) were cultured on gelatin-coated dishes in 250U/ml LIF (Chemicon) containing medium, consisting of high glucose DMEM (Sigma) supplemented with 15% fetal calf serum (Invitrogen), 2mM L- glutamine (Stem Cell Technologies), 0.1 mM non-essential amino acids (Stem Cell Technologies), 1mM sodium pyruvate (Stem Cell Technologies) and 100μM monothioglycerol (Sigma). Cells were frozen in medium containing 50% FCS, 40% culture medium and 10% DMSO (Sigma). Experiments with CCE-TR-FRT cells (see below) were performed in ES medium containing 100U/ml LIF in the presence or absence of 2μg/ml doxycycline (Dox).

To generate cardiomyocytes from EBs, ES cells were cultured for two days in hanging drops, followed by a two-day suspension culture. Then, 30 or 2 EBs were plated into either gelatin-coated 24- or 48-well plates, respectively, and cultured for 24 hours in maintenance medium containing IMDM (Sigma), 20% FCS (Gibco), 2mM L-glutamine (Stem Cell Technologies), 0.1 mM non-essential amino acids (Stem Cell Technologies) and 100μM monothioglycerol (Sigma) to allow attachment of EBs to the culture dish. Thereafter, cells were kept for 48 hours in starvation medium consisting of maintenance medium supplemented with only 0.2% FCS, followed by culture in supplemented medium, corresponding to maintenance medium containing SRM2 (Sigma) instead of FCS.

Plasmids. Tet repressor plasmid (pCAG-TR-IRESpuro3): pCAG and plRESpuro3 (Clontech) plasmids were digested (Spel and EcoRI) and the IRESpuro3 fragment ligated into pCAG. Digestion of pcDNA6/TR (Invitrogen) vector (AfIII, blunt ending, Notl) releases the tetR-IVS insert. This fragment was cloned into pCAG-IRESpuro3 digested by Notl.

To generate the FRTd2EGFP plasmid (pCAG-FRTd2EGFP-IRESneo3), the d2EGFP was amplified without the start codon from pd2EGFP-N1 (Clontech) with BgIII and Notl linkers. The CAG promoter was cut from pCAG with EcoRI and Spel and ligated with an oligo containing an ATG, a FRT site (McLeod et al, 1986), EcoRI and BgIII linkers to the d2EGFP fragment. This insert was finally inserted into plRESneo3 (Clontech) digested with Spel and Notl.

To generate shRNA plasmids (pTER-shRNA-FRT) the shRNAs (Stat3, Brf1) were cloned into pTER-Ni (van de Wetering et al, 2003) using BgIII and Notl. Plasmids were opened with Nsil and Sapl and blunted, followed by insertion of an FRT-Hygro-SV40pA fragment from pcDNA5/FRT (Invitrogen) digested with Pvull. To generate the flipase plasmid (pCAG-Flipase), flipase was PCR amplified from the pOG44 vector (Invitrogen) and the product digested with Bsal and blunt ended (IVS- Flipase-pA) and was then inserted into pCAG vector opened with Hindlll and blunt ended.

Transient and stable transfection of CCE cells. All transfections were done using Lipofectamine 2000 according to the standard protocol from Invitrogen; however, cells were incubated with liposome/plasmid complexes for only 3 h at 37°C/5% CO₂.

For generation of cells exhibiting Dox inducible expression of shRNAs, CCE ES cells were first transfected with the pCAG-TR-IRESpuro3 vector and selected with 1μg/ml puromycin (Calbiochem). Clones were identified by Western blot with mouse anti-tetR monoclonal Antibody Mix (MoBiTec). A high expressing clone (TR8) was chosen for further transfection with pCAG-FRT-EGFP-IRESneo3. Selection was done with Geneticin (Gibco) at a concentration of 600 μg/ml.

Recombination of shRNA was done by co-transfection of the flipase containing vector (pCAG-Flipase) and the vector containing the shRNA (pTER-sh RNA-FRT). Cells were then selected with hygromycin (Calbiochem) at a concentration of 165 U/ml. Cells were then further screened for loss of both GFP and G418 resistance.

shRNA, siRNA and primers

Murine Stat3 specific oligonucleotides (5' GAT CTG AGT CAC ATG CCA CGT TGG TTC AAG AGA CCA ACG TGG CAT GTG ACT CTT TTT A 3', and 5' AGC TTA AAA AGA GTC ACA TGC CAC GTT GGT CTC TTG AAC CAA CGT GGC ATG TGA CTC A

3') murine Stat3 siRNA: 5' GAG UCA CAU GCC ACG UUG G (XM_109608) control siRNA (human β-globin): 5' CAA GAA AGU GCU CGG UGC C (V00497.1) murine Brf1 specific oligonucleotides (5' GAT CTG TCC GAA TCC CCT CAC ATG TTC AAG AGA CAT GTG AGG GGA TTC GGA CTT TTT A 3', and 5' AGC TTA AAA AGT CCG AAT CCC CTC ACA TGT CTC TTG AAC ATG TGA GGG GAT TCG GAC A 3') murine Stat3 primer 5' AGT CAC ATG CCA CGT TGG T 3' murine α-cardiac actin PCR primers (forward 5' GCT TTG GTG TGT GAC AAT 3' GG₁ reverse 5' GTG ATA ATG CCA TGT TCA ATG G 3') murine Nkx2.5 PCR primers (forward 5¹ CGG AAC GAC TCC CAC CTT TAG G 3', reverse 5' GGA ATC CGT CGA AAG TGC CC 3') murine Gata4 PCR primers (forward 5' CGA GAT GGG ACG GGA CAC T 3', reverse 5' CTC ACC CTC GGC CAT TAC GA 3') murine myogenin PCR primers (forward 5' ACA AGC CAG ACT CCC CAC TC 3', reverse 5' GCA CTC ATG TCT CTC AAA CGG T 3') murine GAPDH PCR primers (forward 5' CAC CAC CAA CTG TTA GCC 3', reverse 5' CCT GCT TCAC CAC CTT CTT G 3') murine 18S rRNA primers (forward 5' CGG CTA CCA CAT CCA AGG AA 3', reverse 5' GCT GGA ATT ACC GCG GCT 3')

Northern blot. Total RNA was harvested using Trizol (Invitrogen). To detect Brf1 , Oct4, Rex-1 , Fgf-4 and β-actin, Northern blots were hybridized overnight with [α³²P]-dCTP labeled PCR fragments generated from cDNA of the aforementioned genes (Brf 1 : nt 945-1328 #M58566; GAPDH, nt 589-1246, #M33197; β-actin: nt 516-1144 #NM008085; Oct4 nt 731-1101 #NM_013633; Rex-1 nt 687-1059 #NM_009556; Fgf-4 nt 250-583 #NM_010202) (Raineri et al, 2004). To analyze expression of shRNA in F3-1-Stat3 and F3-1-Brf1 clones, 30μg of total RNA were separated on 15% polyacrylamide gels containing 8M urea (Anamed). Gels were stained with ethidium bromide to check for equal loading before RNA was transferred by electroblotting onto Hybond-N+ (Amersham) membranes. After UV-crosslinking, filters were hybridized at 45⁰C in 0.5M sodium phosphate buffer pH 7.2 containing 1% BSA (Fraction V, Sigma), 7% SDS and 5mM EDTA using a [γ³²P]-ATP labeled Stat3 or Brf 1 specific oligonucleotide of 19 nucleotides. Blots were analyzed using the Personal Molecular Imager^® FX (Biorad) and the Quantity One^® software.

Western blot. The Western blot protocol employed and generation of Brf1 antibodies has recently been described (Raineri et al, 2004). A monoclonal anti-tetR antibody mix (MoBiTec) was used to detect expression of the Tet repressor. A monoclonal antibody against α-tubulin (Clone 236-10501 , Molecular Probes) was used. Stat3 (Cell Signaling), Nkx2.5 (Clone N-19, Santa Cruz Biotechnology, Inc.), Gata4 (Clone H-112, Santa Cruz Biotechnology, Inc.) and HRP-coupled GAPDH (Abeam) polyclonal antibodies were utilized. Alkaline phosphatase-coupled goat-anti-rabbit IgG (Southern Biotechnology Associates Inc.) and horseradish peroxidase-coupled goat-anti-mouse IgG (DAKO) and rabbit anti-goat IgG (Southern Biotechnology Associates Inc.) were used as secondary antibodies. Development was performed using CDP-Star (Roche) or ECL Advance (Amersham).

DNA microarray analysis. 2μg of total RNA was purified using the RNeasy Mini Kit (Qiagen) and reverse transcribed and purified using the Affymetrix one-cycle cDNA synthesis kit (Affymetrix, CA, USA) as per the manufacturer's instructions. Labeled cRNA was produced using the Affymetrix IVT Labeling Kit (Affymetrix). Nucleic acid concentrations were quantified using Nanodrop and RNA quality was determined with an Agilent 2100 Bioanalyzer. 15μg of fragmented cRNA were loaded onto Affymetrix MOE430v2 GeneChips and scanned using an Affymetrix GeneChip 3000 7G scanner. The CEL files were quality controlled using Genedata's Refiner 3.1 program (Genedata AG, Basel, Switzerland). Expression values and detection P-values were estimated using Genedata's implementation of GCRMA (Wu etal.; Nat Biotechnol, 2004, 656-8) in Refiner 3.1. Data analysis was performed using Analyst 3.1 (Genedata AG). Experiments were performed in triplicate and for a gene to be considered it had to have a detection P-value < 0.04 in at least 2/3 of the replicates of one or more condition. Genes had to pass a 1 -way ANOVA (P < 0.01 ) and were clustered using a 24-group Self Organizing Map. Final gene lists were then checked for interactions using Pathway Studio (Ariadne Genomics).

Microscopy. A Nikon Eclipse TE200 microscope equipped with a Hamamatsu digital camera (C4742-95) was used, all pictures were made at a 4OX magnification. Beating areas were marked under live-microscopy using Openlab 2.2 Software (Improvision) allowing quantification.

Discussion The system and vectors of the presented invention were tested using a Tet-based inducible RNA interference system, which allowed the downregulation of any transcript of choice following flp recombinase mediated DNA recombination in ES cells. The power of the system was documented in experiments in which Stat3, the known regulator of ES cell differentiation, was targeted. In addition, it allowed assignment of a novel function to the posttranscriptional regulator Brfϊ in ES cell-derived cardiomyocyte formation. Clone F3-1 , carrying both the Tet repressor and an frt-GFP-marked locus that integrated into genomic regions favorable for long-term gene expression (even long after onset of differentiation), was of preferred for this embodiment of the invention. Establishment of the doxycycline-inducible shRNA system in ES cells was also of significance in view of the reported difficulties in establishing the Tet system in ES cells, thought to be due to a tendency in ES cells towards gene silencing. The efficiency of successful recombination assessed by the three selection markers GFP, neomycin and hygromycin resistance, and confirmatory PCR was remarkably high and approached about 50% with Stat3 and 70% with Brf1 plasmids, respectively (data not shown). With Stat3, strong doxycycline- dependent induction of 21 nt siRNA formation, negligible background and a strong morphological and biochemical response including signs of differentiation and appropriate changes of the stem-cell markers Oct4, Fgf-4 and Rex-1 , were observed. Niwa et al. (1998) have previously described an ES system where tetracycline induction triggered differentiation via the formation of a dominant-negative form of Stat3 protein expressed from a chromosomal site. The system and vectors of the invention compared well with this dominant-negative system. Thus, the RNAi approach is suitable to probe the possible function of other suspected regulators such as Brf1. That Brf1 is controlled at least in part by Stat3 was suggested first by the LIF removal experiment where Brf1 levels dropped (Fig. 2), and supported by transient transfection of Stat3 siRNA, which led to concomitant downregulation of Stat3 and BrM (Fig. 2C). In several Stat3 shRNA clones tested, doxycycline led to downregulation of Brf1 protein and transcripts (Figs. 5 and 6A). These results are consistent with DNA microarray data of others who reported Brf1 (Zfp36L1) downregulation after LIF removal (Palmqvist et al, 2005; Sekkai et al, 2005). That the effect in some clones was less intense than that following LIF removal suggests that not all effects on gene expression by LIF removal are mediated by Stat3. While a Stat3 site in the promoter region of Brf 1 has been reported, support for additional control elements was provided in a paper on the human ES cell transcriptome where chromatin immunoprecipitation was combined with DNA microarray analysis to identify promoters of transcripts expressed in stem cells that are co-occupied by Oct4, nanog and Sox2. Interestingly, both Stat3 and Brf1 were amongst the 353 transcripts which fulfilled these criteria (Boyer et al, 2005). Oct4 and Sox2 bind the Brf1 promoter 2497nt upstream of the transcription initiation site, and nanog at position -1733. When testing the effects of Brf1 shRNA induction in ES long-term cultures, it was unexpectly discovered that the morphology of EBs, the number of beating areas within an embryoid body and also the total number of beating areas was dramatically altered. A time-course revealed that down regulation of Brf1 did not change the kinetics of cardiomyocyte formation, but rather amplified the spontaneous formation known to occur in cultures of murine and human EBs (Sachinidis et al, 2003). It is significant that both effects on general EB morphology and on cardiomyocyte formation do not occur when Brf1 is downregulated only at day4 or later in EB cultures. This supports that a transient early change in Brf 1 levels may set the stage for downstream processes executed by Brf1 target genes. Based on the data gathered, the following model was established: Brf 1 , a target of Stat3, is a suppressor of differentiation expressed in undifferentiated pluripotent stem cells. Reducing its levels physiologically via reduction of LIF-Stat3 signaling or by inducible RNA interference leads to changes in embryoid body architecture and enhancement of cardiomyocyte formation. As Brf 1 regulates ARE- dependent mRNA turnover, the model predicts the existence of ARE-containing transcripts favoring cardiomyogenesis. The systems and vectors of the present invention provides a tools for testing this model. Overexpression of Brf1 would be expected to suppress cardiomyogenesis, which, however, could not be tested, as transfected Brf1 is toxic to ES cells (Rg. 13). In human ES cells, the protocol described here may prove useful in regenerative cardiology for replacement therapy. In addition, transgenic mice generated from the lines described here might circumvent the lethality of, e.g., Brf1 knockouts (Stumpo et al, 2004) and allow the downregulation of Brf1 and other proteins in adult tissue to reveal further functional aspects of this and others, e.g., posttranscriptional regulators.

Bibliography

Bakheet T, Frevel M, Williams BR, Greer W, Khabar KS. ARED: human AU-rich element-containing mRNA database reveals an unexpectedly diverse functional repertoire of encoded proteins. Nucleic Acids Res. 2001;29(1):246-254.

Boiani M, Scholer HR. Regulatory networks in embryo-derived pluripotent stem cells. Nat Rev MoI Cell Biol. Nov 2005;6(11):872-884.

Boyer LA, Lee Tl, Cole MF, et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell. Sep 23 2005; 122(6) :947-956.

Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science. 2002;296(5567):550-553. Li RK, Jia ZQ, Weisel RD, et al. Cardiomyocyte transplanta-. tion improves heart function. Ann Thorac Surg 1996;62:654.

Cartwright P, McLean C, Sheppard A, Rivett D, Jones K, Dalton S. LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism. Development. Mar 2005; 132(5) :885-896.

Chambers I, Colby D, Robertson M, et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell. May 302003; 113(5) :643- 655. Chen CY, Gherzi R, Ong SE, et al. AU binding proteins recruit the exosome to degrade ARE-containing mRNAs. Cell. Nov 162001 ;107(4):451 -464.

Chen CY, Shyu AB. AU-rich elements: characterization and importance in mRNA degradation. Trends Biochem Sci. 1995;20(11):465-470.

Gao M, Wilusz CJ, Peltz SW, Wilusz J. A novel mRNA-decapping activity in HeLa cytoplasmic extracts is regulated by AU-rich elements. Embo J. Mar 1 2001 ;20(5):1134- 1143. Gomperts M, Pascall JC, Brown KD. The nucleotide sequence of a cDNA encoding an EGF-inducible gene indicates the existence of a new family of mitogen-induced genes. Oncogene. Ju1 1990;5(7):1081 -1083.

Gossen M, Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A. Jun 15 1992;89(12):5547- 5551.

Keene JD, Tenenbaum SA. Eukaryotic mRNPs may represent posttranscriptional operons. MoI Cell. Jun 2002;9(6):1161-1167.

Keller G, Kennedy M, Papayannopoulou T, Wiles MV. Hematopoietic commitment during embryonic stem cell differentiation in culture. MoI Cell Biol. Jan 1993;13(1):473-486.

Komuro I, Izumo S. Csx: a murine homeobox-containing gene specifically expressed in the developing heart. Proc Natl Acad Sci U S A. Sep 1 1993;90(17):8145-8149. Lai WS, Carballo E, Strum JR, Kennington EA, Phillips RS, Blackshear PJ. Evidence that tristetraprolin binds to AU-rich elements and promotes the deadenylation and destabilization of tumor necrosis factor alpha mRNA. MoI Cell Biol. Jun 1999; 19(6) :4311- 4323.

Lai WS, Carballo E, Thorn JM, Kennington EA, Blackshear PJ. Interactions of CCCH zinc finger proteins with mRNA. Binding of tristetraprolin-related zinc finger proteins to Au-rich elements and destabilization of mRNA. J Biol Chem. Jun 9 2000;275(23): 17827- 17837.

Li RK, Jia ZQ, Weisel RD, et al. Cardiomyocyte transplantation improves heart function. Ann Thorac Surg 1996;62:654. Lykke-Andersen J, Wagner E. Recruitment and activation of mRNA decay enzymes by two ARE-mediated decay activation domains in the proteins TTP and BRF-1. Genes Dev. Feb 1 2005;19(3):351-361.

Ma WJ, Cheng S, Campbell C, Wright A, Furneaux H. Cloning and characterization of HuR, a ubiquitously expressed Elav-like protein. J Biol Chem. Apr 5 1996;271 (14):8144- 8151.

Matsuda T, Nakamura T, Nakao K, et al. STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells. Embo J. Aug 2 1999;18(15):4261- 4269.

McDonald JW, Liu XZ, Qu Y, Liu S, et al. Transplanted embryonic stem cells survive, differentiate and promote recovery in injured rat spinal cord. Nat Med. 1999;5:1410- 1412.

McLeod M, Craft S, Broach JR. Identification of the crossover site during FLP-mediated recombination in the Saccharomyces cerevisiae plasmid 2 microns circle. MoI Cell Biol. Oct 1986;6(10):3357-3367. Mitsui K, Tokuzawa Y, ltoh H, et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell. May 30 2003; 113(5):631 -642.

Nichols J, Zevnik B, Anastassiadis K, et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell. Oct 30 1998;95(3):379-391.

Niwa H, Burdon T, Chambers I, Smith A. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev. Ju1 1 1998;12(13):2048-2060.

Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet. Apr 2000;24(4):372-376.

O'Gorman S, Fox DT, Wahl GM. Recombinase-mediated gene activation and site- specific integration in mammalian cells. Science. Mar 15 1991 ;251 (4999):1351 -1355. Palmqvist L, Glover CH, Hsu L, et al. Correlation of murine embryonic stem cell gene expression profiles with functional measures of pluripotency. Stem Cells. May 2005;23(5):663-680. Qi X, Li TG, Hao J, et al. BMP4 supports self-renewal of embryonic stem cells by inhibiting mitogen-activated protein kinase pathways. Proc Natl Acad Sci U S A. Apr 20 2004;101 (16):6027-6032.

Raineri I, Wegmueller D, Gross B, Certa U, Moroni C. Roles of AUF1 isoforms, HuR and BRF1 in ARE-dependent mRNA turnover studied by RNA interference. Nucleic Acids Res. 2004;32(4):1279-1288.

Robertson E, Bradley A, Kuehn M, Evans M. Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector. Nature. Oct 2-8 1986;323(6087):445-448.

Sachinidis A, Fleischmann BK, Kolossov E, Wartenberg M, Sauer H, Hescheler J. Cardiac specific differentiation of mouse embryonic stem cells. Cardiovasc Res. May 1 2003;58(2):278-291.

Sakai T, Li RK, Weisel RD, et al.: Fetal cell transplantation: A comparison of three cell types. J Thorac Cardiovasc Surg 1999;118: 715-725. [1]

Sakai T, Li RK, Weisel RD, et al. Autologous heart cell transplantation improves cardiac function after myocardial injury. Ann Thorac Surg. 1999; 68: 2074-2080. [2]

Schmidlin M, Lu M, Leuenberger SA, et al. The ARE-dependent mRNA-destabilizing activity of BRF1 is regulated by protein kinase B. Embo J. Dec 8 2004;23(24):4760- 4769.

Sekkai D, Gruel G, Herry M, et al. Microarray Analysis of LIF/Stat3 Transcriptional Targets in Embryonic Stem Cells. Stem Cells. Nov 2005;23(10): 1634-1642.

Shimozaki K, Nakashima K, Niwa H, Taga T. Involvement of Oct3/4 in the enhancement of neuronal differentiation of ES cells in neurogenesis-inducing cultures. Development. Jun 2003;130(11):2505-2512.

Smith AG. Embryo-derived stem cells: of mice and men. Annu Rev Cell Dev Biol. 2001 ; 17:435-462.

Smith AG, Heath JK, Donaldson DD, et al. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature. Dec 15 1988;336(6200):688-690.

Stoecklin G, Colombi M, Raineri I, et al. Functional cloning of BRF1 , a regulator of ARE- dependent mRNA turnover. Embo J. Sep 22002;21 (17):4709-4718.

Stumpo DJ, Byrd NA, Phillips RS, et al. Chorioallantoic fusion defects and embryonic lethality resulting from disruption of Zfp36L1 , a gene encoding a CCCH tandem zinc finger protein of the Tristetraprolin family. MoI Cell Biol. JuI 2004;24(14):6445-6455. van de Wetering M, Oving I, Muncan V, et al. Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector. EMBO Rep. Jun 2003;4(6):609-615. Watanabe E, et al. Cardiomyocyte transplantation in. a porcine myocardial infarction model. Cell Transplant. 1998;7:239-246.

Williams RL, Hilton DJ, Pease S, et al. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature. Dec 15 1988;336(6200):684- 687.

Yao F, Svensjo T, Winkler T₁ Lu M, Eriksson C, Eriksson E. Tetracycline repressor, tetR, rather than the tetR-mammalian cell transcription factor fusion derivatives, regulates inducible gene expression in mammalian cells. Hum Gene Ther. Sep 1 1998;9(13):1939- 1950.

Ying QL, Nichols J, Chambers I, Smith A. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell. Oct 31 2003;115(3):281-292.

Zhang W, Wagner BJ, Ehrenman K, et al. Purification, characterization, and cDNA cloning of an AU-rich element RNA-binding protein, AUF1. MoI Cell Biol. 1993;13(12):7652-7665.

Claims

We claim:

1. A regulatory system comprising:

(a) an integration system comprising at least one first recombinase target site, at least one sequence encoding at least one marker having an expression status, wherein integration of a sequence into said recombinase target site changes said expression status of said at least one marker,

(b) a vector comprising at least one second recombinase target site compatible with said first recombinase target site of (a), and at least one inducible expression cassette,

(c) optionally, a vector expressing a recombinase mediating the integration of the vector in (b) into the system of (a) via said first and second recombinase target site, and (d) optionally, a vector expressing a repressor repressing transcription via said inducible expression cassette, wherein a sequence encoding a regulatory nucleic acid is inserted into said inducible expression cassette of vector (b) so that said regulatory nucleic acid is inducible expressed subsequent to integration of the vector of (b) into the system of (a) in an eukaryotic cell.

2. The regulatory system of claim 1 , wherein said integration system of (a) is a genome of a modified eukaryotic cell and wherein, upon introduction of the vectors of (b), (c) and, optionally (d), expression of said regulatory nucleic acid is inducible in said modified eukaryotic cell, said vectors of (c) and (d) being part of the system of claim 1 or being separately provided.

3. The regulatory system of claim 2, wherein said modified eukaryotic cell is a stem cell and wherein said marker is under the control of a wide spectrum promoter system.

4. A regulatory system according to any of the above claims, wherein said expression cassette is inducible by a chemical inducer acting on the repressor of (d).

5. A regulatory system according to any of the above claims, wherein said first and second recombinase target sites are flp-recombinase-target or loxP sites and said recombinase mediating the integration of the vector of (b) into the system of (a) is flp- recombinase or a Cre-recombinase.

6. A regulatory system according to any of the above claims, wherein said regulatory nucleic acid is shRNA, miRNA or antisense RNA.

7. A kit comprising, in separate containers, at least (a) and (b) of the regulatory system of any preceding claim and in yet another spearate container instructions for use of (a) and (b) to insert a sequence encoding said regulatory nucleic acid into the vector of (b) and subsequent integration of the vector of (b) into the integration system of (a), which is or has been integrated into a genome of an eukaryotic cell.

8. Use of the regulatory system or kit of any of of the preceding claims.

9. A genetic construct for expressing at least one regulatory nucleic acid in stem cells comprising

(a) a sequence encoding at least one regulatory nucleic acid, wherein said regulatory nucleic acid directly or indirectly up- or down regulates production of at least one protein,

(b) an induction cassette, wherein expression of said regulatory nucleic acid is under the control of said induction cassette and wherein said regulatory nucleic acid, upon induction in stem cells via said induction cassette, up- or down regulates said at least one protein.

10. The genetic construct of claim 9, wherein said genetic construct is a genetically modified stem cell and wherein said genetically modified stem cell further comprises at least one gene encoding said at least one protein.

11. The genetic construct of claim 10, wherein said stem cell is a human stem cell.

12. A genetic construct according to claim 9 or any subsequent claim, wherein said regulatory nucleic acid is shRNA or miRNA.

13. A genetic construct according to claim 9 or any subsequent claim, wherein said at least one protein is a posttranscriptional regulator that regulates in cis mRNA turnover.

14. A genetic construct according to claim 13, wherein the posttranscriptional regulator is butyrate response factor 1 (Brfϊ) and wherein said regulatory nucleic acid downregulates Brfϊ , contribuing to cardiomycotes formation.

15. A genetic construct according to claim 9 or any subsequent claim, wherein a chemical inducer induces said induction cassette.

16. A system according to claim 4 or a genetic construct according to claim 15, wherein said chemical inducer is doxycycline, estrogen or dexamethason.

17. Use of the genetic contruct according to claim 9 or any subsequent claim.

18. A method for producing a system for inducible up- or down regulation of a protein comprising: constructing a genetic system comprising at least one first recombinase target site, at least one sequence encoding at least one marker having an expression status, wherein integration of a sequence into said recombinase target site changes said expression status of said at least one marker, wherein a sequence for a regulatory nucleic acid that up- or down regulates said protein and which is integrated into at least one inducible expression cassette of a vector comprising at least one second recombinase target site compatible with said first recombinase target site, is expressed upon integration of said vector into said genetic system by at least one recombinase mediating recombination via said first and second recombinase target site, and induction of the inducible expression cassette via an inducer removing repression of transcription of said regulatory nucleic acid.

19. The method of claim 18, wherein said sequence encoding the marker is expressed via a broad sprectrum promoter system.

20. The method of claim 18, wherein said genetic system is integrated into genetically modified stem cell or at least one cell of a transgenic non-human animal.

21. A genetically modified stem cell or transgenic non-human animal produced according to the method of claim 18.