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 [16–19].
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
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
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, 13–15], 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
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
GFP allele in the brain, we already applied DOX to the embryo. Similarly, we used tTA expression to activate the R26
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 [30–32] 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.