BACKGROUND
controlling gene expression in a temporally and spatially inducible manner is an important aspect of transgenic approaches.
a key development has been the application of gene expression systems based on the tetracycline-resistance  operon of the tn <dig> transposon of e. coli.
in the absence of tetracycline, the tet repressor  dna binding protein binds to a defined dna operator sequence  and suppresses transcription.
addition of tetracycline  causes a conformational change in tetr preventing it from binding to teto  <cit> .
in the tet transactivator  system, tetr was fused to vp <dig>  a strong transcriptional activator, so that it now activates expression upon binding to teto in the absence of tc  <cit> .
it was further improved by developing a reverse tet transactivator  which requires tc for binding teto for transactivation, thus eliminating long-term exposure to tc  <cit> .
also, doxycycline  was introduced as an inducer due to its superior qualities of lower toxicity, longer half-life and high bioavailabilty.
finally, the rtta2s-m <dig> variant generated by mutagenesis constitutes a significant improvement over the original rtta since it is induced at a 10-fold lower dox concentration compared to rtta, it shows no background dna binding and it is also more stable in mammalian cells  <cit> .
application of the rtta system in mice requires two transgenic lines: one line expressing the rtta under an appropriate promoter and a second line carrying the target transgene of interest linked to a tet-responsive element  containing teto sequences.
however, the efficiency of the tet system in transgenic animals is often negatively impacted by the chromosomal site of integration of the transgenes.
this becomes particularly important when ubiquitous induction in several tissues is desirable.
whereas chromosomal position effects can be partly alleviated by the use of gene domain regulatory elements such as locus control regions or insulators  <cit> , these elements are often tissue specific.
by contrast, regulatory elements associated with ubiquitously expressed housekeeping genes are active in all tissues.
for example, methylation-free cpg islands associated with the 5' ends of housekeeping genes are known to be localized in regions of active chromatin  <cit> .
previous evidence from cell transfection experiments has suggested that transgenes containing cpg islands have the potential to protect from position effects, even when integrated in heterochromatin.
for example, large fragments spanning cpg islands from the ubiquitously expressed human tbp-psmb <dig> and hnrnpa2b1-cbx <dig> loci were able to protect from heterochromatic silencing  <cit> .
these regions are structurally similar in that they contain divergently transcribed promoters embedded within an extended cpg island.
in the hnrnpa2b1-cbx <dig> locus the two divergently expressed genes code for heterogeneous ribonucleoprotein a2/b <dig> and for chromobox homolog  <dig>  <cit> .
assays in mammalian cells showed that the hnrnpa2b1-cbx <dig> cpg island gave rise to reproducible, stable and non-variegated expression from the endogenous hnrnpa2b <dig> promoter, even when integrated in centromeric heterochromatin  <cit> .
furthermore, the coupling of the hnrnpa2b1-cbx <dig> cpg island to the cmv promoter gave substantial improvements in the level and stability of expression, resulting in improved production of recombinant proteins in cho cells  <cit> .
these observations suggest that methylation-free cpg islands may harbor dominant chromatin remodeling functions.
in this report, we show that the hnrnpa2b1-cbx <dig> cpg island is sufficient to generate an efficient, ubiquitously expressed rtta inducible system in transgenic mice.
RESULTS
we used an ~ <dig> kb dna fragment encompassing the human hnrnpa2/b1-cbx <dig> gene locus cpg island, which includes the two divergently transcribed promoters .
the rtta2s-m <dig> variant was cloned under the control of the hnrnpa2/b <dig> promoter as shown  and the entire  <dig> kb fragment was purified and microinjected into mouse fertilized eggs.
a total of  <dig> founders transgenic for the hnrnpa2b1-cbx3/rtta2s-m <dig> construct were generated and seven transgenic lines were established through transmission of the transgene.
transgenic founder  <dig> had two integration sites, which separated upon breeding to generate lines 2a and 2b.
integrity of the transgenes was established by southern blotting using the  <dig> kb dna microinjection fragment as probe .
for the majority of transgenic lines, we also determined transgene copy numbers  and chromosomal integration sites using fish .
we next carried out northern blot analysis to determine the tissue distribution and relative expression levels of the rtta2s-m <dig> transgene in the different lines.
line  <dig> is clearly the highest expressing line in all tissues tested, lines 2a and 2b expressed weakly in some tissues, whereas lines  <dig>   <dig>   <dig> and  <dig> barely expressed .
these data show that transgene expression levels are not copy number dependent and are prone to position effects.
this was confirmed by quantitative rt-pcr for lines  <dig>  2a, 2b,  <dig> and  <dig> in heart and liver rna.
plotting normalized levels of expression against transgene copy numbers shows no direct correlation between expression levels and copy numbers, for example, the single copy line  <dig> expresses at much higher levels than the multi copy line 2a .
the chromosomal sites of integration of the transgenes cannot readily account for these results.
for example, line  <dig> carrying  <dig> copies of the transgene in a euchromatic site does not express, whereas single copy line  <dig>  also integrated in euchromatin, expresses at high levels.
these observations contrast with previous work in transfected cells showing that a broader  <dig> kb genomic fragment spanning the hnrnpa2b1-cbx <dig> locus expressed consistently even when integrated in centromeric heterochromatin  <cit> , or that the  <dig> kb hnrnpa2b1-cbx <dig> fragment when linked to a cmv promoter showed consistently detectable, though variable, transgene expression for all integration events  <cit> .
reasons that could account for these differences include the requirement of additional elements for position independent transgene expression which are lacking from the  <dig> kb fragment we used  <cit> , or that the use of antibiotic resistance in cell transfections introduces a bias for open chromatin integration, particularly when using the cmv promoter  <cit> .
we next focused on the utility of the highly expressing line  <dig> as a tool for widespread inducible transgene expression upon dox treatment.
we first obtained further evidence that the rtta2s-m <dig> transgene is indeed widely expressed in this line.
by employing rt-pcr, we detected expression of the transgene in all tissues assayed , thus confirming the ubiquitous expression of the rtta2s-m <dig> transgene driven by the hnrnpa2b1-cbx <dig> cpg island.
we next generated three transgenic lines carrying a tre-gfp reporter construct and crossed them with line  <dig>  analysis of gfp expression in the liver of adult mice treated with dox showed that tre-gfp line  <dig> was induced more efficiently than tre-gfp lines  <dig> and  <dig> .
thus, tre-gfp line  <dig> was used in all subsequent experiments.
because of the high levels of autofluorescence observed in the liver and other tissues , we assayed induction of gfp expression in different tissues by immunohistochemistry with an anti-gfp antibody .
we observed clear induction of gfp expression in the kidney, lung, heart, spleen and liver specifically upon dox treatment of double transgenic mice .
thus, taken together these results show that the rtta2s-m <dig> transgene is expressed in the liver, heart, brain, ovary, kidney, lung, spleen, muscle, thymus and blood  with dox inducibility demonstrated in the liver, kidney, heart, lung, spleen and blood .
to-date there have been few other studies reporting the use of the improved rtta2s-m <dig> variant in transgenic mice, for example, for the tissue specific inducible expression of genes in the retinal ganglia cells or the brain  <cit> .
transgenic mouse line  <dig> represents an important addition to the transgenic tools that are available exploiting the superior properties of the rtta2s-m <dig> system.
despite robust expression of the rtta2s-m <dig> transgene in the brain of line  <dig> , we were unable to detect gfp induction in the brain , even after treatment with  <dig> mg/ml dox for up to  <dig> days .
this is most likely due to the well-known limited penetration of the blood-brain barrier by dox.
the study by michalon et al.
<cit>  has suggested that induction in the brain may require treating mice for longer periods with higher doses of dox.
however, in the case of line  <dig> where the rtta2s-m <dig> transgene is expressed in many tissues, one needs to keep in mind the potential side effects that prolonged treatment with high amounts of dox may have in tissues other than the brain  <cit> .
in order to test the dose response of line  <dig> to dox, we crossed it with a transgenic mouse line expressing the human phospholipid transfer protein  under the control of a tet responsive element , which was created for the purposes of another study .
pltp is a plasma protein with several key roles in lipoprotein metabolism and, potentially, atherogenesis  <cit> .
we used a sensitive assay to measure pltp activity in the serum of double line 9/tre-pltp transgenic mice as a means of quantifying induction with increasing dox concentrations added to the drinking water.
we found a 6- to 7-fold induction in pltp activity for dox concentrations ranging from  <dig>  to  <dig>  mg/ml .
pltp activity appeared to decline with increasing dox dosage , however, these concentrations of dox are much higher than those normally used in mice for induction.
thus, taken together, the data presented in figure  <dig> establish a useful range of dox concentrations for inducing pltp activity and, in addition, demonstrate the superior sensitivity of induction in the serum of the rtta2s-m <dig> variant in transgenic mice with as little as  <dig>  mg/ml of dox.
in order to demonstrate the utility of line  <dig> for dox inducible expression in mouse embryos, we established a sensitive genetic system based on a myc-tagged murine gata- <dig> transgene under the control of a tre.
gata- <dig> is an essential transcription factor with important functions in endoderm differentiation .
importantly, gata- <dig> overexpression in transgenic embryos results in branching defects in the lung, showing that lung development is sensitive to gata- <dig> dosage  <cit> .
microinjection of the tre-myc-gata- <dig> fragment into fertilized eggs resulted in  <dig> live born pups, of which  <dig> carried the transgene as deduced by southern blot analysis .
three lines were chosen for further analysis based on the intensity of the signal on southern blots.
all heterozygous and homozygous tre-myc-gata- <dig> mice were viable and showed no defects.
timed pregnancies were set up between hnrpa2b1-rtta2s-m <dig> homozygous mice and tre-myc-gata- <dig> heterozygous mice.
dox was given to pregnant dams for one day before isolating embryos .
since it has been previously shown that overexpression of the myc-gata- <dig> transgene results in defects in the differentiation of the airway epithelium of the lung  <cit> , we examined the lungs of double transgenic pups that were treated with dox.
we found striking similarities with the published lung phenotype, in that the airways appear dilated with some bleeding also evident .
the lung phenotype was observed in all three independent tre-myc-gata- <dig> lines and appeared only in double transgenics treated with dox.
there was no obvious lung phenotype in double transgenics not treated with dox .
furthermore, dox treatment of pregnant dams for only one day also resulted in gross morphology defects in that the embryos showed loosening of the skin and signs of oedema .
these observations could be due to interference of normal gata factor functions in the skin as a result of induced myc-gata- <dig> overexpression  <cit> .
induction of the transgene for more than one day resulted in a general growth arrest and death in utero.
thus, these observations reveal defects that are specific for the induction of the myc-gata- <dig> transgene and demonstrate the utility of hnrnpa2b1-rtta2s-m <dig> line  <dig> in studying the effects of the widespread inducible overexpression of specific factors in embryonic development.
alternatively, this system could be used for timed rescue experiments of mice that have essential genes knocked out.
CONCLUSIONS
in conclusion, we showed that the methylation free cpg island of the human housekeeping hnrnpa2b1-cbx <dig> gene locus is capable of driving expression of a linked rtta2s-m <dig> transgene in mice in all tissues that were examined.
transgene expression was not found to be copy number related, suggesting that the cpg island does not completely escape chromosomal position effects in vivo.
however, in line  <dig> where the transgene is most active, it is expressed in all tissues tested.
furthermore, using this system we demonstrated that the highly expressing line  <dig> is a very useful tool for the inducible, sensitive expression of transgenes in a variety of tissues in response to dox treatment.
importantly, the inducible transgenic system reported here can be used to examine the effects of inducing widespread overexpression of transcription factors during mouse development.

 controlling gene expression in a temporally and spatially inducible manner is an important aspect of transgenic approaches. a key development has been the application of gene expression systems based on the tetracycline-resistance operon of the tn transposon of e. coli. in the absence of tetracycline, the tet repressor dna binding protein binds to a defined dna operator sequence and suppresses transcription. addition of tetracycline causes a conformational change in tetr preventing it from binding to teto. in the tet transactivator system, tetr was fused to vp a strong transcriptional activator, so that it now activates expression upon binding to teto in the absence of tc. it was further improved by developing a reverse tet transactivator which requires tc for binding teto for transactivation, thus eliminating long-term exposure to tc. also, doxycycline was introduced as an inducer due to its superior qualities of lower toxicity, longer half-life and high bioavailabilty. finally, the rtta2s-m variant generated by mutagenesis constitutes a significant improvement over the original rtta since it is induced at a 10-fold lower dox concentration compared to rtta, it shows no background dna binding and it is also more stable in mammalian cells. application of the rtta system in mice requires two transgenic lines: one line expressing the rtta under an appropriate promoter and a second line carrying the target transgene of interest linked to a tet-responsive element containing teto sequences. however, the efficiency of the tet system in transgenic animals is often negatively impacted by the chromosomal site of integration of the transgenes. this becomes particularly important when ubiquitous induction in several tissues is desirable. whereas chromosomal position effects can be partly alleviated by the use of gene domain regulatory elements such as locus control regions or insulators, these elements are often tissue specific. by contrast, regulatory elements associated with ubiquitously expressed housekeeping genes are active in all tissues. for example, methylation-free cpg islands associated with the 5' ends of housekeeping genes are known to be localized in regions of active chromatin. previous evidence from cell transfection experiments has suggested that transgenes containing cpg islands have the potential to protect from position effects, even when integrated in heterochromatin. for example, large fragments spanning cpg islands from the ubiquitously expressed human tbp-psmb and hnrnpa2b1-cbx loci were able to protect from heterochromatic silencing. these regions are structurally similar in that they contain divergently transcribed promoters embedded within an extended cpg island. in the hnrnpa2b1-cbx locus the two divergently expressed genes code for heterogeneous ribonucleoprotein a2/b and for chromobox homolog. assays in mammalian cells showed that the hnrnpa2b1-cbx cpg island gave rise to reproducible, stable and non-variegated expression from the endogenous hnrnpa2b promoter, even when integrated in centromeric heterochromatin. furthermore, the coupling of the hnrnpa2b1-cbx cpg island to the cmv promoter gave substantial improvements in the level and stability of expression, resulting in improved production of recombinant proteins in cho cells. these observations suggest that methylation-free cpg islands may harbor dominant chromatin remodeling functions. in this report, we show that the hnrnpa2b1-cbx cpg island is sufficient to generate an efficient, ubiquitously expressed rtta inducible system in transgenic mice.  we used an ~ kb dna fragment encompassing the human hnrnpa2/b1-cbx gene locus cpg island, which includes the two divergently transcribed promoters. the rtta2s-m variant was cloned under the control of the hnrnpa2/b promoter as shown and the entire kb fragment was purified and microinjected into mouse fertilized eggs. a total of founders transgenic for the hnrnpa2b1-cbx3/rtta2s-m construct were generated and seven transgenic lines were established through transmission of the transgene. transgenic founder had two integration sites, which separated upon breeding to generate lines 2a and 2b. integrity of the transgenes was established by southern blotting using the kb dna microinjection fragment as probe. for the majority of transgenic lines, we also determined transgene copy numbers and chromosomal integration sites using fish. we next carried out northern blot analysis to determine the tissue distribution and relative expression levels of the rtta2s-m transgene in the different lines. line is clearly the highest expressing line in all tissues tested, lines 2a and 2b expressed weakly in some tissues, whereas lines and barely expressed. these data show that transgene expression levels are not copy number dependent and are prone to position effects. this was confirmed by quantitative rt-pcr for lines 2a, 2b, and in heart and liver rna. plotting normalized levels of expression against transgene copy numbers shows no direct correlation between expression levels and copy numbers, for example, the single copy line expresses at much higher levels than the multi copy line 2a. the chromosomal sites of integration of the transgenes cannot readily account for these results. for example, line carrying copies of the transgene in a euchromatic site does not express, whereas single copy line also integrated in euchromatin, expresses at high levels. these observations contrast with previous work in transfected cells showing that a broader kb genomic fragment spanning the hnrnpa2b1-cbx locus expressed consistently even when integrated in centromeric heterochromatin, or that the kb hnrnpa2b1-cbx fragment when linked to a cmv promoter showed consistently detectable, though variable, transgene expression for all integration events. reasons that could account for these differences include the requirement of additional elements for position independent transgene expression which are lacking from the kb fragment we used, or that the use of antibiotic resistance in cell transfections introduces a bias for open chromatin integration, particularly when using the cmv promoter. we next focused on the utility of the highly expressing line as a tool for widespread inducible transgene expression upon dox treatment. we first obtained further evidence that the rtta2s-m transgene is indeed widely expressed in this line. by employing rt-pcr, we detected expression of the transgene in all tissues assayed, thus confirming the ubiquitous expression of the rtta2s-m transgene driven by the hnrnpa2b1-cbx cpg island. we next generated three transgenic lines carrying a tre-gfp reporter construct and crossed them with line analysis of gfp expression in the liver of adult mice treated with dox showed that tre-gfp line was induced more efficiently than tre-gfp lines and. thus, tre-gfp line was used in all subsequent experiments. because of the high levels of autofluorescence observed in the liver and other tissues, we assayed induction of gfp expression in different tissues by immunohistochemistry with an anti-gfp antibody. we observed clear induction of gfp expression in the kidney, lung, heart, spleen and liver specifically upon dox treatment of double transgenic mice. thus, taken together these results show that the rtta2s-m transgene is expressed in the liver, heart, brain, ovary, kidney, lung, spleen, muscle, thymus and blood with dox inducibility demonstrated in the liver, kidney, heart, lung, spleen and blood. to-date there have been few other studies reporting the use of the improved rtta2s-m variant in transgenic mice, for example, for the tissue specific inducible expression of genes in the retinal ganglia cells or the brain. transgenic mouse line represents an important addition to the transgenic tools that are available exploiting the superior properties of the rtta2s-m system. despite robust expression of the rtta2s-m transgene in the brain of line, we were unable to detect gfp induction in the brain, even after treatment with mg/ml dox for up to days. this is most likely due to the well-known limited penetration of the blood-brain barrier by dox. the study by michalon et al. has suggested that induction in the brain may require treating mice for longer periods with higher doses of dox. however, in the case of line where the rtta2s-m transgene is expressed in many tissues, one needs to keep in mind the potential side effects that prolonged treatment with high amounts of dox may have in tissues other than the brain. in order to test the dose response of line to dox, we crossed it with a transgenic mouse line expressing the human phospholipid transfer protein under the control of a tet responsive element, which was created for the purposes of another study. pltp is a plasma protein with several key roles in lipoprotein metabolism and, potentially, atherogenesis. we used a sensitive assay to measure pltp activity in the serum of double line 9/tre-pltp transgenic mice as a means of quantifying induction with increasing dox concentrations added to the drinking water. we found a 6- to 7-fold induction in pltp activity for dox concentrations ranging from to mg/ml. pltp activity appeared to decline with increasing dox dosage, however, these concentrations of dox are much higher than those normally used in mice for induction. thus, taken together, the data presented in figure establish a useful range of dox concentrations for inducing pltp activity and, in addition, demonstrate the superior sensitivity of induction in the serum of the rtta2s-m variant in transgenic mice with as little as mg/ml of dox. in order to demonstrate the utility of line for dox inducible expression in mouse embryos, we established a sensitive genetic system based on a myc-tagged murine gata- transgene under the control of a tre. gata- is an essential transcription factor with important functions in endoderm differentiation. importantly, gata- overexpression in transgenic embryos results in branching defects in the lung, showing that lung development is sensitive to gata- dosage. microinjection of the tre-myc-gata- fragment into fertilized eggs resulted in live born pups, of which carried the transgene as deduced by southern blot analysis. three lines were chosen for further analysis based on the intensity of the signal on southern blots. all heterozygous and homozygous tre-myc-gata- mice were viable and showed no defects. timed pregnancies were set up between hnrpa2b1-rtta2s-m homozygous mice and tre-myc-gata- heterozygous mice. dox was given to pregnant dams for one day before isolating embryos. since it has been previously shown that overexpression of the myc-gata- transgene results in defects in the differentiation of the airway epithelium of the lung, we examined the lungs of double transgenic pups that were treated with dox. we found striking similarities with the published lung phenotype, in that the airways appear dilated with some bleeding also evident. the lung phenotype was observed in all three independent tre-myc-gata- lines and appeared only in double transgenics treated with dox. there was no obvious lung phenotype in double transgenics not treated with dox. furthermore, dox treatment of pregnant dams for only one day also resulted in gross morphology defects in that the embryos showed loosening of the skin and signs of oedema. these observations could be due to interference of normal gata factor functions in the skin as a result of induced myc-gata- overexpression. induction of the transgene for more than one day resulted in a general growth arrest and death in utero. thus, these observations reveal defects that are specific for the induction of the myc-gata- transgene and demonstrate the utility of hnrnpa2b1-rtta2s-m line in studying the effects of the widespread inducible overexpression of specific factors in embryonic development. alternatively, this system could be used for timed rescue experiments of mice that have essential genes knocked out.  in conclusion, we showed that the methylation free cpg island of the human housekeeping hnrnpa2b1-cbx gene locus is capable of driving expression of a linked rtta2s-m transgene in mice in all tissues that were examined. transgene expression was not found to be copy number related, suggesting that the cpg island does not completely escape chromosomal position effects in vivo. however, in line where the transgene is most active, it is expressed in all tissues tested. furthermore, using this system we demonstrated that the highly expressing line is a very useful tool for the inducible, sensitive expression of transgenes in a variety of tissues in response to dox treatment. importantly, the inducible transgenic system reported here can be used to examine the effects of inducing widespread overexpression of transcription factors during mouse development. 
the aim of this study was to test the utility of a mammalian methylation-free cpg island to drive ubiquitous expression of the sensitive doxycycline inducible rtta2s-m tet-transactivator in transgenic mice. the ubiquitously expressing rtta2s-m transgenic mouse line described here provides a very useful tool for studying the effects of the widespread, inducible overexpression of genes during embryonic development and in adult mice. the utility of this rtta2s-m line for inducible expression in mouse embryos was also demonstrated using a gata- tet-inducible transgene to show specific phenotypes in the embryonic lung, as well as broader effects resulting from the inducible widespread overexpression of the transgene. using this line we also show highly sensitive quantitative induction with low doses of dox of an assayable plasma protein transgene under the control of a tet responsive element.
 a sensitive, ubiquitously expressed tetracycline inducible system would be a valuable tool in mouse transgenesis. however, this has been difficult to obtain due to position effects observed at different chromosomal sites of transgene integration, which negatively affect expression in many tissues. the aim of this study was to test the utility of a mammalian methylation-free cpg island to drive ubiquitous expression of the sensitive doxycycline inducible rtta2s-m tet-transactivator in transgenic mice.  an kb genomic fragment from the methylation-free cpg island of the human hnrnpa2b1-cbx housekeeping gene locus was tested. in a number of transgenic mouse lines obtained, rtta2s-m expression was detected in many tissues examined. characterisation of the highest expressing rtta2s-m transgenic mouse line demonstrated dox-inducible gfp transgene expression in many tissues. using this line we also show highly sensitive quantitative induction with low doses of dox of an assayable plasma protein transgene under the control of a tet responsive element. the utility of this rtta2s-m line for inducible expression in mouse embryos was also demonstrated using a gata- tet-inducible transgene to show specific phenotypes in the embryonic lung, as well as broader effects resulting from the inducible widespread overexpression of the transgene.  the ubiquitously expressing rtta2s-m transgenic mouse line described here provides a very useful tool for studying the effects of the widespread, inducible overexpression of genes during embryonic development and in adult mice. 