Ubiquitous expression of the rtTA2S-M2 inducible system in transgenic mice driven by the human hnRNPA2B1/CBX3 CpG island
© Katsantoni et al; licensee BioMed Central Ltd. 2007
Received: 27 March 2007
Accepted: 27 September 2007
Published: 27 September 2007
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 (Dox) inducible rtTA2S-M2 Tet-transactivator in transgenic mice.
An 8 kb genomic fragment from the methylation-free CpG island of the human hnRNPA2B1-CBX3 housekeeping gene locus was tested. In a number of transgenic mouse lines obtained, rtTA2S-M2 expression was detected in many tissues examined. Characterisation of the highest expressing rtTA2S-M2 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 (TRE). The utility of this rtTA2S-M2 line for inducible expression in mouse embryos was also demonstrated using a GATA-6 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-M2 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.
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 (tet) operon of the Tn10 transposon of E. coli. In the absence of tetracycline, the tet repressor (tetR) DNA binding protein binds to a defined DNA operator sequence (tetO) and suppresses transcription. Addition of tetracycline (Tc) causes a conformational change in TetR preventing it from binding to tetO . In the tet transactivator (tTA) system, tetR was fused to VP16, 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 (rtTA) which requires Tc for binding tetO for transactivation, thus eliminating long-term exposure to Tc . Also, doxycycline (Dox) was introduced as an inducer due to its superior qualities of lower toxicity, longer half-life and high bioavailabilty. Finally, the rtTA2S-M2 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 (TRE) 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-PSMB1 and hnRNPA2B1-CBX3 loci were able to protect from heterochromatic silencing [6, 7]. These regions are structurally similar in that they contain divergently transcribed promoters embedded within an extended CpG island. In the hnRNPA2B1-CBX3 locus the two divergently expressed genes code for heterogeneous ribonucleoprotein A2/B1 and for chromobox homolog 3 . Assays in mammalian cells showed that the hnRNPA2B1-CBX3 CpG island gave rise to reproducible, stable and non-variegated expression from the endogenous hnRNPA2B1 promoter, even when integrated in centromeric heterochromatin . Furthermore, the coupling of the hnRNPA2B1-CBX3 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-CBX3 CpG island is sufficient to generate an efficient, ubiquitously expressed rtTA inducible system in transgenic mice.
Results and discussion
Despite robust expression of the rtTA2S-M2 transgene in the brain of line 9 (Fig. 2A and 2B), we were unable to detect GFP induction in the brain (Fig. 3), even after treatment with 2 mg/ml Dox for up to 18 days (not shown). 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 9 where the rtTA2S-M2 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 conclusion, we showed that the methylation free CpG island of the human housekeeping hnRNPA2B1-CBX3 gene locus is capable of driving expression of a linked rtTA2S-M2 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 9 where the transgene is most active, it is expressed in all tissues tested. Furthermore, using this system we demonstrated that the highly expressing line 9 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.
An 8 kb genomic fragment spanning the CpG island of the HNRPA2B1-CBX3 locus was used to express the rtTA derivative rtTA2S-M2. The latter was obtained as an EcoRI-HindIII fragment, including the SV40 polyA sequence, from plasmid pUHrT62-1  and was blunt-end cloned into a Bgl II site in the first exon of the HNRPA2B1 gene. The cDNAs for GFP and human PLTP  and murine N-terminally myc-tagged GATA-6  were cloned under the control of a TetO-CMV promoter in plasmids pTRE (GFP and PLTP) or pTRE-Tight (GATA-6) (Clontech).
HNRPA2B1/rtTA2S-M2 was released from vector sequences as an 8 kb HindIII fragment (Fig. 1A). TRE-GFP-SV40polyA and TRE-hPLTP-SV40polyA were released from vector sequences as XhoI/PvuII fragments. TRE-myc tag-GATA-6-SV40polyA was released as an XhoI fragment. All fragments were purified by salt gradient centrifugation, as previously described , microinjected at approximately 0.5 ng/μL into the pronucleus of fertilized eggs of FVB/N mice and transplanted into the oviducts of pseudopregnant B10xCBA mice . Transgenic founders were identified by Southern blotting using the rtTA2S-M2, GFP, hPLTP and GATA-6 fragments as probes. The integrity of the HNRPA2B1/rtTA2S-M2 transgene was checked by hybridization using the entire HNRPA2B1/rtTA2S-M2 construct as probe. Transgene copy numbers for the HNRPA2B1/rtTA2S-M2 lines were determined from the intensity of fragments using an EcoRI/BamHI rtTA2S-M2 probe, together with a 0.9-kb Pvu I probe detecting the endogenous mouse carbonic anhydrase II (CA-II) gene. PhosphorImager analysis was performed using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). All animal experiments described in this work conformed to national and institutional guidelines.
DNA fluorescence in situ hybridization analysis
Mice (2–4 months old) were kept on drinking water containing 5% sucrose for three days, followed by drinking water containing 2 mg/ml doxycycline hydrochloride (Sigma) plus 5% sucrose for 3–4 days. Doxycycline-containing drinking water was protected from light. Negative control mice were kept on drinking water containing 5% sucrose for the same time period. The drinking water was replaced every 2–3 days. Doxycycline induction of the myc-GATA-6 transgene expression in embryos: after the identification of a vaginal plug in the morning, dams bearing single and double (HNRPA2B1/rtTA2S-M2 and myc-GATA-6) transgenic pups were given doxycycline in the drinking water (2 mg/ml supplemented with 5% sucrose) for 1–4 days, after which embryos were isolated at different gestational ages and processed for further analysis.
Northern blot analysis
RNA was isolated using the Trizol reagent and according to the manufacturer's instructions (Invitrogen). Northern blot analysis was carried out with 5 μg of total RNA as previously described . The rtTA2S-M2 fragment was used as probe for hybridization.
RT-PCR and Quantitative RT-PCR
RNA was treated with RQ1 RNase-Free DNase I (Promega) for 30 minutes at 37°C according to the manufacturer's instructions. DNase-treated RNA samples were reverse transcribed with Superscript (Invitrogen) using oligo dT. Control reactions without reverse transcriptase (RT) were also performed. Real-time PCR reactions were performed in triplicate with a Chromo 4, MJ Research RealTime PCR Cycler, as previously described . Normalization for the amount of template was done using primers specific for exon 8 of the mouse HPRT gene. Primer sequences for rtTA2S-M2 and HPRT are available upon request. Data were analyzed using the Chromo 4, MJ Research RealTime PCR software and statistical analysis was done using the comparative CT method for the relative quantitation of results . Post amplification denaturation curves showed that the primer pairs generated single products.
PLTP activity assay
Blood samples were collected from the orbital plexus of mice before the sucrose run-in period, after three days of plain sucrose administration and after three days of doxycycline administration. Blood was centrifuged at 2800 rpm for 15 minutes at 4°C and plasma samples stored at -80°C. PLTP activity was measured using a phospholipid vesicle-HDL system . Radiolabelled phospholipid vesicles were prepared by mixing egg phosphatidylcholine (Sigma) with [14C]dipalmitoylphosphatidylcholine (Amersham) and butylated hydroxytoluene (Sigma), followed by drying under N2 and sonification. EDTA-plasma samples (5 μl of plasma diluted 1:150) and phospholipid vesicles were incubated in the presence of isolated human HDL for 45 minutes at 37°C. After incubation, the vesicles were precipitated and the radioactivity transferred to HDL was counted in the supernatant by liquid scintillation. PLTP activity was expressed as a percentage of human reference plasma (100% is equivalent to 14 μmol/ml/h).
Perfusion of mice
Mice were sacrificed using an overdose of isoflurane (1-chloro-2,2,2-trifluoroethyl-difluoromethyl-ether). Subsequently, in situ perfusion fixation was performed by flushing 20 ml PBS through a cardiac puncture followed by 20 ml 4% (v/v) paraformaldehyde in PBS.
Immunohistochemistry was performed on cryosections as previously described , with the following modifications: endogenous peroxidase activity was inhibited by a 30 min incubation in 1% H2O2 in methanol. A rabbit polyclonal anti-GFP antibody (Abcam ab290) was used in a dilution of 1:200. Rabbit IgG (Santa Cruz) was used as a negative control in dilution of 1:500. Anti rabbit HRP-conjugated IgG was used as secondary antibody at a 1:500 dilution. Antigen-antibody complexes were visualized by incubation in substrate solution, containing hydrogen peroxide and 3,3'-diaminobenzidine HCl (DAB substrate kit for peroxidase, Vector Laboratories). Photographs were recorded with a digital camera (Olympus DP 50) on a light Olympus Microscope (BX 40 with U-DO Dual View).
This work was supported by the Dutch NWO and the EU (FG and JS) and by ZonMW (RdC). EK was supported by a Marie Curie Post Doctoral Fellowship (QLK1-CT-2002-51556) and an EU ERG grant. The authors are grateful to An Langeveld for assistance with the FISH experiments, to Marianne van Tienhoven for genotyping, to the EDC for animal care and to Rien van Haperen, Sanja Krpic, Paschalis Sideras and Alexandros Sountoulidis for helpful discussions.
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