Tetracycline-controlled transgene activation using the ROSA26-iM2-GFP knock-in mouse strain permits GFP monitoring of DOX-regulated transgene-expression
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Conditional gene activation is an efficient strategy for studying gene function in genetically modified animals. Among the presently available gene switches, the tetracycline-regulated system has attracted considerable interest because of its unique potential for reversible and adjustable gene regulation.
To investigate whether the ubiquitously expressed Gt(ROSA)26Sor locus enables uniform DOX-controlled gene expression, we inserted the improved tetracycline-regulated transcription activator iM2 together with an iM2 dependent GFP gene into the Gt(ROSA)26Sor locus, using gene targeting to generate ROSA26-iM2-GFP (R26t1Δ) mice. Despite the presence of ROSA26 promoter driven iM2, R26t1Δ mice showed very sparse DOX-activated expression of different iM2-responsive reporter genes in the brain, mosaic expression in peripheral tissues and more prominent expression in erythroid, myeloid and lymphoid lineages, in hematopoietic stem and progenitor cells and in olfactory neurons.
The finding that gene regulation by the DOX-activated transcriptional factor iM2 in the Gt(ROSA)26Sor locus has its limitations is of importance for future experimental strategies involving transgene activation from the endogenous ROSA26 promoter. Furthermore, our ROSA26-iM2 knock-in mouse model (R26t1Δ) represents a useful tool for implementing gene function in vivo especially under circumstances requiring the side-by-side comparison of gene manipulated and wild type cells. Since the ROSA26-iM2 mouse allows mosaic gene activation in peripheral tissues and haematopoietic cells, this model will be very useful for uncovering previously unknown or unsuspected phenotypes.
KeywordsOlfactory Bulb Gene Target Mouse Embryonic Stem Cell Clone Conditional Gene Expression ROSA26 Promoter
To understand the detailed function of particular genes we must be able to translate analytical and database information into experimental model systems. In this respect, the laboratory mouse can be used as a higher vertebrate model that permits the application of genetic tools for the in vivo analysis of gene function. Since constitutive, global expression of transgenes is often associated with embryonic lethality or unwanted pleiotropic effects, tight postnatal transgene regulation is the method of choice. Furthermore, correlations between phenotypes and 'ON' and 'OFF' states of the regulated gene can provide reliable cues for understanding gene-function. However, to ensure that the observed phenotypes are correlated with the gene function under investigation the reservoir of genetic tools has to be carefully evaluated.
Over the last decade the tetracycline (tet)-regulatory system has been extremely useful for generating conditional transgenic mouse models (for reviews see [1, 2, 3]). Besides its fundamental appeal as an established reversible regulatory system in mice, tet 'ON/OFF'-systems have attracted considerable interest because of their unique potential for reversible gene regulation, with the additional advantage of allowing adjustment of transgene expression levels through the administration of predetermined amounts of the pharmacological inducer doxycycline (DOX) [4, 5, 6]. The graded response of tet-regulated transgenes thus provides the unique opportunity to analyze gene dosage effects in living animals.
Although a large collection of tet-controlled transgenic mice suitable for studying gene function in many different cell types is available to the scientific community (http://www.zmg.uni-mainz.de/tetmouse for an electronically searchable database), no comprehensive analysis of a tet-regulatory effector line using the ubiquitously expressed Gt(ROSA)26Sor gene locus has been published. Therefore, we generated a tet 'ON' knock-in mouse line (R26t1Δ) containing the iM2 reverse DOX-dependent transactivator coding region  and an iM2 responsive GFP gene at the Gt(ROSA)26Sor gene locus. Analysis of R26t1 mice using different iM2-responsive reporter strains revealed mosaic induction of conditional reporter gene activity in peripheral organs and in hematopoietic cells. No substantial iM2 reporter gene activation was detected in most parts of the adult brain with the exception of the bulbus olfactorius and, to a lesser extent, the striatum.
Generation of the ROSA26-iM2PtetGFP knock-in mouse
The generation of the R26 t1 gene locus by homologous recombination in the genome of ES cells was pre-screened by PCR and confirmed by Southern blotting (Figure 1B). Six independent ES cell clones with insertion of the inducible iM2-PtetGFP module at the Gt(ROSA)26Sor locus were identified (Figure 1B). All six ES cell clones showed DOX-inducible GFP fluorescence (Figure 1B) and ES cell clone 64 was used for blastocyst injections to generate the ROSA26-iM2-PtetGFP mouse line, designated R26t1. Removal of the loxP flanked neo/ura selection cassette was performed by in vivo excision using the deleter-cre strain  thus generating the R26 t1Δ allele in a sub-line (R26t1Δ) lacking the neo/ura genes (Figure 1C).
In embryos, spleen, kidney and liver of R26t1Δ mice rapid amplification of cDNA ends (RACE) revealed that R26 t1Δ transcripts contained 3'-exon1 sequences of ROSA26 transcript variant 2 (Figure 1C). Since the ROSA26 promoter and exon1 of splice variant 2 were not present in the targeting vector, this finding confirms that the recombined R26 t1Δ gene locus drives iM2 expression from the ROSA26 promoter. Thus the presence of splice variant exon1 at the 5'-end of iM2 transcripts provides additional proof for successful iM2 gene module insertion into the Gt(ROSA)26Sor gene locus. Currently, we cannot resolve why the exon1 splice variant 1 was not detected in our analysis. Because sequence alteration at the exon1 splice donor site could be excluded by sequencing it is possible that the presence of a second artificial intron in front of the iM2 coding sequence might influence the maturation of the primary R26t1Δ transcript.
The presence of the neo spacer sequence and the gene dosage effect on the DOX regulated GFP in the ROSA26-iM2-GFP locus
Characterisation of the R26t1Δeffector mouse in hematopoietic tissues
Evaluation of the R26t1Δeffector mouse in the brain
Immunohistochemistry of sagittal brain sections confirmed strong, DOX-dependent GFP expression in the olfactory bulb of R26t1Δ and R26t1Δ/t1Δ mice (Figure 4B). However, the GFP-staining was restricted to the glomeruli formed between axonal projections of olfactory receptor neurons (ORNs) and dendritic processes from mitral cells of the olfactory bulb. Since the cell bodies of mitral cells within the olfactory bulb were negative for GFP immunostaining, the GFP signal in the olfactory bulb is derived from ORNs, which have their cell bodies in the nasal cavity of the olfactory epithelium outside the brain. Hardly any GFP-positive cells were apparent in brain sections of DOX-induced R26t1Δ and R26t1Δ/t1Δ mice; with the exception of sparse GFP-positive cells in the striatum and hypothalamus of homozygous R26t1Δ/t1Δ mice (Figure 4B) directly confirming the immunoblot results (Figure 4A). Thus, as noticed in peripheral tissues, the increased gene dosage of the R26t1Δ/t1Δ allele improved the expression of GFP in the brain. Our findings therefore suggest that in heterozygous R26t1Δ mice, a critical threshold of DOX-activated iM2 for transcriptional activation of Ptet-GFP is not reached in most cells of the brain.
In order to increase the level of active transactivator in the brain, we generated R26t1Δ mice that express the DOX-activated transcription factor tTA in the forebrain by breeding R26t1Δ and TgCamKII-tTA mice . Compared to R26t1Δ and R26t1Δ/t1Δmice, adult R26t1Δ/TgCamKII-tTA animals showed many more GFP-positive cells in brain regions with strong TgCamKII-tTA expression including striatum, cortex and glomeruli of the olfactory bulb thus favouring the hypothesis that detectable brain-specific GFP expression needs a certain threshold of DOX-induced activator molecules (Figure 4C). However, only a few pyramidal cells in hippocamal layer CA1 demonstrated GFP expression (Figure 4C) despite the high activity of tTA in those brain regions [20, 21]. This lack of optimal tTA mediated Ptet-GFP gene activation in the R26 t1Δ locus might be caused by a negative interference of the CamKII promoter expressed tTA and R26t1Δ encoded iM2 forming inactive heterodimers. The formation of inactive heterodimers is supported by mice expressing iM2 and tTA together with a Ptet-GFP responder transgene TgPtetbi-GFP-GluA1/nlacZ . As described by Mack et al., TgCamKII-tTA/TgPtetbi-GFP-GluA1/nlacZ mice show high numbers of GFP positive cells in the forebrain (Figure 4D, left). However, in littermate animals in addition harbouring the R26 t1Δ allele, the GFP signal dramatically dropped (Figure 4D, right) demonstrating reduced activity of the tTA transactivator in presence of the R26 t1Δ allele. In summary, our results indicate that a certain threshold level of iM2 expression is needed to activate Ptet promoters and provide direct evidence that the observed lack of Ptet-mediated GFP activation in DOX induced R26 t1Δ mice might not just be the result of poor penetration of DOX through the blood brain barrier.
Activation of R26t1Δcontrolled expression of Ptet regulated transgenes is DOX-dependent and inhomogeneous
Compound R26t1Δ/TgPtet-Wnt1-IRES-luciferase mice were either treated with DOX in the drinking water or were never exposed to DOX and after luciferin injection bioluminescence levels were recorded (Figure 5A). DOX-exposure during three days induced significant levels of luminescence whereas no bioluminescence signal was monitored in mice never exposed to DOX. Consistent with the previously reported variable levels of transgene expression from the Gt(ROSA)26Sor locus in different adult mouse tissues [16, 17, 24], the luminescence was not uniformly distributed in DOX-induced R26t1Δ/TgPtet-Wnt1-IRES-luciferase mice. This inhomogeneous luciferase activity pattern thus confirms our immunoblot observation that the R26 t1Δ locus leads to highly variable Ptet-transgene expression.
Next, R26 t1Δ induced Ptet-gene activation was visualized on a cellular level in compound R26t1Δ/TgPtetbi-GFP/lacZ mice by X-Gal staining (Figure 5B). As expected, no β-galactosidase (β-gal) expressing cells were detected without DOX treatment (data not shown). In line with the DOX-dependent induction of luciferase expression (Figure 5A), compound R26t1Δ/TgPtetbi-GFP/lacZ animals treated for two weeks with DOX showed lacZ reporter gene activity in skin, tongue, colon, small intestine, pancreas, lung, kidney, testis and liver. Interestingly, β-gal expression in these organs was restricted to subsets of cells. No β-gal+ cells were detected in heart, bladder and stomach (Figure 5B).
Taken together our results indicated that the expression of conditional Ptet transgenes can be tightly regulated with the R26 t1Δ allele and that the R26 t1Δ mouse model implements mosaic Ptet-transgene activation in a number of peripheral tissues.
The promoter of the Gt(ROSA)26Sor locus is frequently used for ubiquitous expression of reporter genes in the mouse. These animal models represent valuable tools for conducting conditional gene expression studies. However, it is debated whether the endogenous promoter of the Gt(ROSA)26Sor locus can direct conditional transgene activation to all tissues of adult mice [16, 17, 18, 19].
Now we provide additional information by a qualitative and quantitative analysis of a gene targeted mouse line (R26t1Δ) that contains the DOX-inducible iM2 transactivator together with an iM2-dependent GFP gene inserted into the Gt(ROSA)26Sor locus. We could show that after gene targeting the ROSA26 promoter drives expression of the inserted transcriptional activator gene in several tissues of R26t1Δ mice. In embryo, liver, spleen and kidney exon1 of the ROSA26 transcript variant 2 was used in iM2 transcripts. If and to what extent exon1 of transcript variant 1 is used in iM2 transcripts cannot be concluded from our data, since the conducted RACE analysis did not provide a quantitative analysis of all transcripts of the ROSA26 promoter in R26t1Δ mice. More importantly, exon1 transcript variant 2 is located outside of our targeting vector providing additional evidence that the Gt(ROSA)26Sor targeting was successful. Since electroporation with our targeting vector provided several randomly inserted GFP expressing ES cell clones, all containing transcriptional start sites in the short arm of the targeting vector (data not shown), the transcript analysis conducted here provides direct experimental proof for the ROSA26 promoter-specific transcription of the iM2 transgene and can be used as a functional test for demonstrating correct gene transcription of the rearranged locus.
In gene targeted mice ROSA26 promoter controlled iM2 activity was evaluated by monitoring (i) the co-inserted downstream Ptet-GFP reporter gene, (ii) the conditional induction of the TgPtet-Wnt1-IRES-luciferase  or the TgPtetbi-GFP/lacZ  transgenes. All three different reporter systems, including the very sensitive luciferase reporter, demonstrated convincingly that the R26 t1Δ allele permits gene induction with no background activity of the iM2-controlled gene in the non-induced state. These results indicate that illegitimate activation of Ptet-GFP in the Gt(ROSA)26Sor locus by read through transcripts or promoter interference does not take place. Our experiments also demonstrate that different iM2 responder genes could be induced by DOX. However, the responder gene expression was moderate and highly variable between and within different tissues. We detected conditional transgene expression in a subset of cells from skin, tongue, colon, small intestine, pancreas, lung, kidney, testis and liver, whereas some organs like heart, bladder and stomach had no detectable transgene induction.
In addition to the localization of conditionally activated cells within peripheral organs, we provide a detailed picture of R26 t1Δ encoded iM2 expression in the hematopoietic system. Although for the initially gene-trapped β-Geo ROSA26 mouse strain the expression of the integrated β-Geo reporter gene in immature red blood cells, lymphoid and myeloid lineages was found  and several ROSA26-driven reporter mouse strains exist and have been used in hematopoietic tissues [8, 10, 13, 14, 15], no detailed information about the potential and tissue-specificity of ROSA26-driven tet 'ON/OFF' mouse systems is available for hematopoietic tissues. To provide this missing information, we analyzed induced R26t1Δ mice by flow cytometry. The results of these experiments indicate that the R26 t1Δ allele is active in different adult blood cell types and also in the lineage negative c-Kit- and Sca-1-expressing (LKS) population, which contains hematopoietic stem cells and progenitors.
In contrast to the peripheral tissues and the hematopoietic system, in cells of the central nervous system the activity of the R26t1Δ encoded iM2 was barely detectable. Olfactory receptor neurons projecting to glomeroli of the olfactory bulb showed the highest DOX-inducible iM2 activity in homozygous R26t1Δ/t1Δ and heterozygous R26t1Δ mice. In whole brain extracts, however, only low GFP protein levels could be detected in mesencephalon, hippocampus and cortex of DOX-induced homozygous R26t1Δ/t1Δ mice. Immunohistochemical staining of brain sections revealed a few, scattered GFP-expressing cells. The low levels of functional iM2 appeared to be one reason for the dysfunction of the R26t1Δ allele in most cells of the central nervous system since an increase of the R26t1Δ gene dosage in homozygous mice provided higher GFP expression levels in several brain regions including the glomeroli of the olfactory bulb. Even more neurons showed R26t1Δ-derived GFP expression when the levels of functional transactivator were increased by forebrain specific tTA expression using a CaMKII promoter driven transgene . However, GFP expression was lower than in other transgenic Ptet-GFP responder mice, most likely due to the presence of the iM2 in R26t1Δ animals. The reduced tTA activity in R26t1Δ genotypes therefore is best explained by the mutual interference of tTA and rtTA (iM2) heterodimers [26, 27] and can be convincingly visualized by the reduction of GFP-GluA1 expression in the CA1 pyramidal neurons of the hippocampus in compound transgenic mice.
Applying a very similar targeting strategy, Bäckman and colleagues recently generated a ROSA26-rtTA knock-in mouse containing a Ptet-Cre responder element inserted downstream of a ROSA26-driven rtTA cassette. Interestingly, DOX-induced adult mice expressed low Cre mRNA levels and therefore failed to activate recombination of a floxed reporter gene suggesting that the endogenous ROSA26 promoter might be too weak for efficiently inducing conditional transgene activation . Besides the low activity of the endogenous ROSA26 promoter, a second reason for the poor iM2 activity in the brain might be the blood brain barrier, which may limit the free accessibility of DOX for neurons in the brain. However, using intra-cerebral DOX injection or rAAV virus mediated tTA gene delivery into the brains of adult R26t1Δ mice, we failed to achieve neuronal GFP expression in the injected cortex or hippocampal areas (data not shown). Thus as described in previous studies  the Ptet promoter might be subject to epigenetic gene silencing when not activated during early stages of development. In this respect it is of note that in all experiments studying the expression of the R26 t1Δ GFP allele in the brain, we already applied DOX to the embryo. Similarly, we used tTA expression to activate the R26 t1Δ encoded GFP in the forebrain since the CamKII promoter of Tg-CamKII-tTA is transcriptionally active in the mouse brain during early development [25, 29] and thus the Ptet promoter region is still open for transcriptional activation.
Currently, we can not formally exclude the possibility that, in addition to other effects, the lack of detectable Ptet activation in the brain might in part be caused by transcriptional interference, which is known to reduce or extinguish transcriptional activity of downstream promoters in double gene constructs [30, 31, 32] and was recently described for the CMV and the ROSA26 promoter in a targeted Gt(ROSA)26Sor locus in ES cells . However, the fact that the R26t1Δ-iM2 activation of transgenic Ptet-responders in trans was limited as well, and that in the SK3 channel the insertion of a very similar linked transactivator and responder gene was operative  strongly argues against promoter interference. In addition, negative expression variation effects of Ptet caused by its 91 bp fragment from the CMV promoter can be responsible for the inhomogeneous activity of the Ptet promoter.
The presented qualitative and semi-quantitative analysis of our R26t1Δ mouse line provides detailed data on the iM2 and Ptet controlled GFP activity, which is a key information for future experiments using R26t1Δ mice. This knowledge can be useful to guide the experimental design of particular research projects using DOX-regulated gene expression. Finally, the here conducted analysis provides valuable information about the potential of the R26t1Δ mouse for activating transgene expression in different tissues and hematopoietic lineages and thus will help to decide if this mouse model is suitable for a particular in vivo experiment.
Construction of the targeting vector
For gene targeting at the Gt(ROSA)26Sor locus we inserted the coding sequence for the iM2 transactivator (rtTA-M2;), containing a codon improved version of the tet-inducible M2 transcription factor , a loxP flanked neo selection marker and a Ptet-controlled GFP followed by a hgh polyadenylation signal into the PacI/AscI site between exon1 and 2 targeting vector pROSA26PA  to generate plasmid pROSA-iM2. In detail: a plasmid containing the tri-TAUBi-AF cassette was used as starting material [35, 36]. The unique KpnI and ClaI sites were removed from phM2-1 by filling in with Klenow polymerase followed by ligation. A 120 bp fragment containing the adenovirus major late transcription splice acceptor sequence (SA) from the intron1/exon2 boundary was amplified by PCR from pSAßGal  using primer SA-F (5'-TTTGGCCATACTGGCCTTAATTAATAGGGCGCAGTAGT CCAGGG-3') and SA-R (5'-TTTACTAGTACTGGAAAGACCGCGAAGAGTT-3') to introduce a PacI site upstream of the SA and was inserted into pCR4 (Invitrogen, Karlsruhe, Germany) by TOPO cloning. The SA fragment was recovered by SfiI and SpeI digestion and inserted 5' relative to the adenoviral tripartite leader sequence  to yield phM2-2. Subsequently, a fragment encompassing the SV40 splice donor-splice acceptor intron together with the N-terminal part of the humanized GFP (GFP ) expression unit  was amplified by PCR using primers GFP1F (5'-AAGCGCGCAAGCTTATCGATACCGTCGACC-3') and GFP1R (5'-GTATTCCAGCTTG TGGCCGAG-3'). A second fragment including the C-terminal portion of GFP and the SV40 polyadenylation signal was amplified by PCR using primer GFP2F (5'-CTCGGCCACAAGCTGGAATAC-3') and GFP2R (5'-AAGGCCGGCCGGCGCGCCCGT AATACGACTCACTATAGGG-3') to introduce an AscI site between the SV40 polyadenylation signal and the FseI site. Both PCR fragments were inserted into pCR4 by TOPO cloning. These two fragments, were excised using BssHI/PflMI and PflMI/FseI digestion respectively and ligated into the AseI/FseI sites of phM2-2 to place the GFP expression unit under the control of the tetracycline-responsive promoter resulting in the phM2-3 plasmid. This plasmid was then digested using PacI and AscI and cloned into PacI and AscI linearized pROSA-PA (kindly provided by S. Srinivas, Department of Physiology, Anatomy and Genetics, Oxford, UK). Plasmid pROSA-PA is a derivative of pROSA26.1  where the unique XbaI restriction site, between exon1 and exon2 is replaced by a PacI/SwaI/AscI polylinker . The resulting pROSA-hM2 plasmid was linearized with KpnI and subsequently used for electroporation.
Generation of R26t1 and R26t1Δknock-in mice
The R1 embryonic stem (ES) cell line  was electroporated with the linearized pROSA-hM2 targeting vector and G-418-resistant clones were screened for homologous recombination by PCR with the GC-Rich PCR system (Roche, Mannheim, Germany) using primers ROSA-IN (5'-CCTAAAGAAGAGGCTGTGCTTTGG-3') and SA-IN (5'-CATCAAGGAAACCCTGGACTACTG-3'). Furthermore, homologous recombination of ES cells was analyzed by Southern blotting using an 80 nucleotide probe  located mainly upstream of the 5' homology region (5' rosa probe) and an 624 nucleotide neomycin probe (neo probe). Correctly recombined ES cell clone 64 was injected into blastocysts to generate chimeric mice. Successful germ-line transmission and correct integration was confirmed by PCR, using primers ROSA-IN and SA-IN. For in vivo excision of the neomycin-resistant cassette, germ-line-transmitting R26t1 knock-in mice were crossed to the deleter-cre strain . Successful excision of the loxP flanked neo/ura cassette was confirmed by PCR. Genotyping was performed using a three primer PCR approach with oligonucleotides ROSA01 (5'-TTCCCTCGTGATCTGCAACTCC-3') as forward primer and oligonucleotides ROSA02 (5'-GCTTCAGATGTGCCTTGCTCTC-3') and ROSA07 (5'-CATCAGACTTCTAAGATCAGG-3') as reverse primers under standard PCR conditions, yielding products of 565 bp for the Gt(ROSA)26Sor allele and 916 bp for the R26t1Δ allele respectively. Gene targeted mice were generated under the licence Az.: 35-9185.81/G-74/07 (Regierungspräsidium Karlsruhe, Germany).
The transgenic mouse lines deleter-Cre, TgPtet-Wnt1-IRES-luciferase, TgCamKII-tTA, TgPtetbi-GFP/lacZ and the TgPtetbi-GFP-GluA1/nlacZ have been described previously [11, 20, 21, 22, 25]. To induce transgene expression animals were administered a solution of doxycycline (DOX, Sigma-Aldrich, Taufkirchen, Germany) in water containing 1% sucrose. For each experiment the exposure times and DOX concentrations used are specified. DOX treatment was performed under the licence 35.-9185.82/A49/06, Regierungspräsidium Karlsruhe. Terminal experiments under the license MPI/T-15/08 and 177-07/981-18 Landesuntersuchungsamt Rheinland-Pfalz.
RNA isolation and RACE
Total RNA from mice tissues was homogenized using an Utra Turrax (IKA, Staufen; Germany) and RNA was isolated with TRI REAGENT® (Molecular Research Center Inc. Cincinnati, OH 45212, USA) followed by phenol/chloroform purification. The 5' RACE was performed using the FirstChoice® RLM-RACE kit (Ambion, Applied Biosystems, Darmstadt Germany) according to the manufacturer instructions. The 5' RLM PCR was performed using in the first reaction the itTA-race out primer (5'-TCTGTAGGCCTGGTGCCCAAGTG-3') and for the nested PCR the itTA-race in primer (5'-CCCTTCCAGAGGGCAGAAGTGGGTG-3') was used. The PCR products were directly subcloned into the pCR4-topo vector using TOPO® cloning technology (Invitrogen, Darmstadt, Germany) and subsequently sequenced. Sequences were analyzed using Lasergene (DNAStar.COM; Madison, Wi, USA). Sequences for the RACE iM2 transcripts of the R26t1Δ allele were annotated to the NCBI database and are available under the accession number HM748862.
In vivobioluminescence imaging
DOX-induced (3 mg DOX/ml for three days) and non-induced compound R26t1Δ/Ptet-Wnt1-IRES-luciferase mice were intraperitoneally injected with luciferin (150 mg/kg body weight, Becton Dickinson, Heidelberg, Germany) and anesthetized by continuous inhalation with isofluorane (Merck, Darmstadt, Germany). After waiting for five minutes to allow distribution of luciferin, the mice were placed in the chamber of an IVIS Lumina optical imaging system (Caliper Life Sciences, Rüsselsheim, Germany) and bioluminescence levels were collected for 15 seconds. The signal intensity was scaled to a pseudocolor image, which was then superimposed on a grayscale photo of the mice using Living Image software v. 3.0 (Caliper Life Sciences, Rüsselsheim, Germany).
Southern and Western blotting
Southern blots were performed employing standard procedures using an outside ROSA26 probe as described previously  and as an internal neomycin-specific (neo) probe, a 624 bp DNA fragment amplified from the neo gene with the primers rspneo4 (5'-GGC TATTCGGC TATGAC TGGGC -3') and rspneo5 (5'-GGGTAGCCAACGC TATGTCC TG-3').
For Westen blot analysis proteins were isolated with an Ultra Turrax in 25 mM HEPES (ph 7,4) containing protease inhibitors (Complete, EDTA-free; Roche, Mannheim, Germany). The homogenates were centrifuged (900 g; 10 min at 4°C) to remove cell debris and 10-25 μg of protein was resolved on a 8-12% SDS-polyacrylamid gel, transfered onto nitrocellulose membranes and probed with anti-GFP (Abcam; ab6556 1:5000, Abcam, Cambridge, UK) and anti-calnexin antibodies (1:3000, Stressgene, Ann Arbor, USA).
Histology and X-Gal staining
For immunohistology mice were anesthetized with isofluoran (Abbott, Ludwigshafen, Germany) and perfused with PBS/4% paraformaldehyde (PFA, Sigma-Aldrich). The brain was isolated and post fixed in PBS/4%PFA for an additional 3 to 12 hours. For immunohistochemistry, 40- to 70-μm thick vibratom slices were used. Antibody staining on floating vibratom sections was performed as described before  using the primary polyclonal anti-GFP primary antibody (Abcam; ab6556 1:5000) together with the anti-rabbit antibody coupled to horseradish peroxidase (Vector Laboratories, 1:600, Vector Laboratories, Eching, Germany) as the secondary antibody. Staining was visualized with 3-3' diaminobenzidine (DAB, Fluka GmbH, Deisenhofen, Germany), mounted on slides and air-dried. DAB-developed slides were coversliped with Eukitt mounting medium (Kindler GmbH, Freiburg, Germany). Staining for X-Gal was performed as previously described . All sections were counterstained with fast red (Sigma-Aldrich, Taufkirchen, Germany). Images were captured using a colour view digital camera running on an Olympus BX50 WI microscope. Images were digitalized using the analySIS software package (Soft Image Systems, Münster, Germany) and imported into Photoshop.
Cells acquisition was performed on a FACSCalibur or LSRII cytometer (BD) and analyzed using the FlowJo software (Ashland, USA). For lineage determination cells were analyzed as previously described . Before flow cytometry, blood cells from DOX-induced (14 days of 3 mg/ml DOX) or non-induced (normal drinking water) ROSA26-iM2 R26t1Δ mice were pre-incubated with PBS and supplemented with 5% rat serum for 30 minutes to reduce nonspecific binding. Dead cells were excluded by 7-amino-actinomycin D (Becton Dickinson, Heidelberg, Germany) staining. For cell staining we used antibodies (all from Becton Dickinson, Heidelberg, Germany) directed against the following: CD4 (GK1.5), CD8 (53-6.7), B220 (RA3-6B2), CD19 (1D3), Gr1 (RB6-8C5), CD11b/Mac1 (M1/70), Ter119, CD71 (R17 217.1.4), CD41 (MWReg30), c-Kit (2B8), Sca-1 (D7). The CD23 antibody was from Southern Biotech, Birmingham, USA. In all cases, the number of GFP-expressing cells was determined in four independent experiments analyzing, each time, a minimum of 5 × 105 cells.
For statistical data analysis the two-tailed t test was applied using the Prism4 package for Windows (GraphPad software, La Jolla, USA).
We thank L.A. Chodosh for providing the Ptet-Wnt1-IRES-luciferase responder mouse strain. We also thank the animal technicians of the Mainz mouse facility for excellent assistance and mouse care. Finally, we are grateful to Peter H. Seeburg for his constant support and to Ari Waisman enabling us to finish the work in his lab. This study was supported by the Deutsche Forschungsgemeinschaft (Forschergruppe 527 „Suszeptibilitätsfaktoren der Tumorgenese"; SFB636/A4), the VW foundation, the Dr. Mildred Scheel Stiftung für Krebsforschung (10-1982-Bo) and the Nationalen Genomforschungsnetzes (NGFN).
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