Conditional Liver-specific Expression of Simian Virus 40 T Antigen Leads to Regulatable Development of Hepatic Neoplasm in Transgenic Mice*

Elanchezhiyan ManickanDagger , Jujin SatoiDagger , Timothy C. Wang§, and T. Jake LiangDagger

From the Dagger  Liver Diseases Section, NIDDK, National Institutes of Health, Bethesda, Maryland 20892 and the § Gastrointestinal Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02115

Received for publication, October 26, 2000, and in revised form, January 12, 2001




    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Adaptive epigenetic changes and toxicity often accompany constitutive expression of a transgene or knockout of an endogenous gene in mice. These considerations potentially limit the usefulness of transgenic technology in studying the in vivo functions of a gene. Using conditional gene expression technology, it is possible to override such restrictions to achieve temporal and tissue-specific manipulation of gene expression in vivo. Based on the tetracycline regulatory system, we established a binary transgenic model in which the conditional expression of two transgenes, SV40 T antigen (TAg) and lacZ, can be tightly regulated in the liver by administration of tetracycline. The mouse albumin or mouse major urinary protein promoter was used to achieve liver-specific expression of the tetracycline-responsive transcriptional activator (tTA) in one set of transgenic mice. These mice were crossed with transgenic mice carrying either TAg or lacZ under the control of the tTA-regulated promoter. Analyses of mice transgenic for both tTA and TAg (or lacZ) revealed that the liver-specific expression of the transgenes could be suppressed to undetectable levels and regulated in a reversible fashion by tetracycline administration and withdrawal. Mice with tTA and TAg transgenes developed hepatocellular adenomas and hyperplasia that could be prevented by continuous tetracycline administration. Our report demonstrates the value of this binary transgenic model in studying the physiological functions of any potential genes of interest in a liver-specific manner.




    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Transgenic mouse models have been used widely to study gene expression under in vivo physiological conditions. With tissue-specific promoters, it is possible to target gene expression exclusively in a specific tissue such as liver. However, constitutive expression of a transgene, especially if it deleteriously affects the tissues to which it is targeted, often results in prenatal or postnatal death or causes a variety of compensatory changes (1) in the overall gene expression pattern of the tissue. These changes could result in unexpected phenotypes that may not reflect the true biological functions of the transgene (2-5). In certain situations, the transgene is silenced by epigenetic events such as methylation because of its adverse effects on the cells (2, 6). Furthermore, the transgene behaves as a self-antigen, inducing negative selection of reactive T cells in the thymus and causing the animals to become immunologically tolerant to the transgene. Therefore, the immunologic response to the transgene cannot be easily studied without special manipulation. This aspect is particularly relevant in models of autoimmune or viral disease.

To circumvent these problems, it is necessary to develop a system by which the expression of a transgene can be induced at desired time points and otherwise be kept completely silent for an extended period of time. Such a model may also allow a viral or self-antigen to escape the thymic selection process so the immunologic response can be studied. The use of inducible promoters, such as heat shock, metallothionein, and murine mammary tumor virus promoters, that can be regulated by temperature, zinc, or dexamethasone is frequently associated with a high basal level of expression, a less than impressive induction of the transgene, a relative lack of specificity, and a possible toxicity of the induction method (3, 4, 7). Conditional gene expression in vivo has been achieved using a variety of model systems. One of them takes advantage of the cre-lox recombination system by which a transgene can be activated and an endogenous gene deleted in a tissue-specific and time-dependent manner (8). However, this system requires the exogenous delivery of the cre gene (usually by an adeno- or retrovirus), and the induction is irreversible. Recently several drug- or ligand-inducible systems have been developed in vitro and, to some extent, in vivo (7). These systems involve the use of a chimeric transcriptional activator that reversibly activates a target gene in response to the administration of the inducing agent. One of the systems that uses the intrinsic properties of the Escherichia coli tetracycline resistance operon has been applied widely to the generation of cell lines with tightly regulated gene expression in response to tetracycline (4). This system has also been applied to the transgenic models of lymphoid and salivary development (5), but no such model has been reported for liver-specific expression. In this study, we developed a binary transgenic mouse model that permits the tight control of transgene expression in a temporal and liver-specific fashion by tetracycline (the tetracycline-repressed regulatable system). We evaluated the efficiency of this system in transgenic mouse strains carrying one of two reporter genes, one coding for SV40 T antigen (TAg)1 and the other for beta -galactosidase (lacZ), and the tetracycline-responsive transcriptional activator (tTA) under the control of liver-specific promoter of mouse albumin (Alb) (9, 10) or mouse major urinary protein (MUP) (11, 12). tTA, in the absence of tetracycline, activates the reporter genes through binding to the reporter promoter sequence.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Generation of Transgenic Mouse Lines-- The transgenes containing tTA under the control of the mouse albumin (Alb-tTA) or mouse major urinary protein (MUP-tTA) promoter were constructed (see Fig. 1A). For construction of the Alb-tTA transgene, the EcoRI-BamHI fragment containing the tTA sequence from pUHD15.1 (22) was cloned into the EcoRI and BamHI sites of the pBSIIKS+ vector (Stratagene). The tTA fragment released by digestion of the resulting plasmid with NotI and XhoI was inserted into the pGEMAlbSVtpA vector (33). The resulting construct was digested with NheI and NsiI sites to generate the Alb-tTA transgene (~4.5 kilobase pairs). The MUP-tTA transgenic construct was generated the following way. The PstI-KpnI fragment containing the MUP promoter from MUPII (11) was inserted into the NsiI and KpnI sites of pSVSPORT1 (Life Technologies, Inc.), replacing the SV40 promoter. The EcoRI-HpaI fragment containing tTA and SV40tIVSpA from pUHD15.1 was inserted into the SalI (treated with Klenow for blunt end) and EcoRI sites of the previous construct. The resulting plasmid was then digested with ClaI and MluI to generate the MUP-tTA transgenic fragment (~4.0 kilobase pairs). These transgenes were introduced into fertilized eggs of FVB/n mice using standard transgenic techniques (13). Mice transgenic for SV40 T antigen (tetO-TAg; from Shimon Efrat, Albert Einstein College of Medicine, Bronx, NY) (14) or beta -galactosidase (lacZ; from the Jackson Laboratory, Bar Harbor, ME) (15) linked to the cytomegalovirus minimal promoter fused to the tTA-binding sequences (tetO) have been described previously. Genomic DNA isolated from tail clips of the offspring was used for genotyping. Genotyping of the Alb-tTA and MUP-tTA transgenic mice was performed using standard Southern blotting, and genotyping of the TAg mice was performed using the polymerase chain reaction (PCR) with TAg-specific primers (sense, 5'-GGA ATA GTC ACC ATG AAT GAG TAC AG-3'; and antisense, 5'-GGA CAA ACC ACA ACT AGA ATG CAG TG-3').

Administration of Tetracycline-- Slow-release tetracycline pellets (Innovative Research of America, Sarasota, FL) were implanted subcutaneously in the shoulder regions of mice using a trochar as described by the manufacturer. These pellets release 0.7 mg of tetracycline hydrochloride/day for 21 days. As negative controls, placebo tablets obtained from the same manufacturer were similarly implanted. Expression of the transgene was measured on day 20. A group of mice were killed and analyzed for their transgene expression on day 28 (7 days after the dissolution of the tetracycline pellets) to evaluate the reversibility of the transgene expression.

Protein and RNA Extraction-- 100 mg of liver or spleen tissue from the transgenic animals was homogenized in buffer containing 1% Nonidet P-40, 1% sodium deoxycholate, 1% SDS, 150 mM sodium chloride, 10 mM sodium phosphate, 2 mM EDTA, 50 mM sodium fluoride, and protease inhibitor mixture (Pefabloc SC, Roche Molecular Biochemicals) at a concentration of 1 mg/ml. Homogenates were centrifuged at 15,000 × g for 20 min at 4 °C, and the supernatants were collected. Total protein concentrations of the lysates were measured using a Coomassie Plus protein assay reagent kit (Pierce). Total RNAs from liver, spleen, brain, tongue, kidney, lung, heart, ovary, skin, and skeletal muscle were isolated using the Ultraspec-II RNA isolation reagent (Biotecx Laboratories, Houston, TX). Briefly, 100 mg of tissue was homogenized using a glass homogenizer in 1 ml of Ultraspec-II RNA isolation reagent. The RNA was precipitated with isopropyl alcohol and dissolved in diethyl pyrocarbonate-treated water.

Analysis of T Antigen and lacZ Expression-- T antigen was detected by Western blotting using mouse monoclonal anti-TAg antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and peroxidase-labeled goat anti-mouse IgG as the secondary antibody (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD) with a chemiluminescence detection kit (Santa Cruz Biotechnology). The actin level of each sample was tested as an internal protein control. Reverse transcription was performed using the Thermoscript RT-PCR system kit (Life Technologies, Inc.). Briefly, 5 µg of total RNA was mixed with TAg-specific antisense primer (10 µM), and the volume was adjusted to 10 µl using diethyl pyrocarbonate-treated water. The RNA/primer mixture was incubated at 65 °C for 5 min. 10 µl of the reverse transcription mixture was added to the RNA/primer mixture, and the reaction was continued at 50 °C for 1 h and terminated by heating at 85 °C for 5 min. 1 µl of RNase H was added for an additional 20 min at 37 °C. Synthesized cDNA was amplified by a standard PCR protocol: one cycle at 94 °C for 4 min; 35 cycles at 94 °C for 1 min, 56 °C for 1 min, and 72 °C for 1.5 min; and a final cycle at 72 °C for 5 min. PCR-amplified fragments were electrophoresed on 1% agarose gel and analyzed by Southern blotting using 32P-labeled TAg cDNA probe.

beta -Galactosidase activity in the liver or spleen lysate was measured using the Galacto-light beta -galactosidase detection kit (Tropix Inc., Bedford, MA). Briefly, 100 mg of liver or spleen tissue was homogenized in 1 ml of lysis solution containing 10 µl of a 0.1 M solution of dithiothreitol, 1 µl of 30% hydrogen peroxide, and 100 µl of protease inhibitor mixture (Complete, Roche Molecular Biochemicals; from a stock solution containing one tablet in 1 ml of water). The protein concentration of the lysate was determined, and 100 µg of lysate was used for quantitation of beta -galactosidase activity. Recombinant beta -galactosidase (Sigma) was used as a positive control, and liver lysate from nontransgenic mice was used as a negative control. The final reaction mixture containing light-emitting substrates was measured using a plate luminometer (Packard Instrument Co.), and the results of the beta -galactosidase activity were calculated as counts/min.

Analysis of Tumor Development in TAg Mice-- Offspring of the cross between Alb-tTA line 3123 and tetO-TAg were subjected to tetracycline or placebo treatment continuously for a period of 12 months. These mice were killed at the end of the follow-up period. Livers and other organs were examined for any gross morphological changes. A piece of each tissue was fixed in buffered Formalin, and 8-µm-thick sections were cut. Sections were stained with standard hematoxylin/eosin and evaluated for any histological changes.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Development of a Binary Transgenic System-- Two Alb-tTA (lines 3140 and 3123) and three MUP-tTA (lines 7552, 7545, and 7016) transgenic lines were generated. Expression of tTA could be detected by RT-PCR in the livers from all lines (data not shown). The heterozygous animals of each tTA line were crossed with the heterozygous tetO-TAg or tetO-lacZ mice to generate double-positive, single-positive, or double-negative transgenic mice for the experiment. Alb-TAg line 3140 was generated by crossing Alb-tTA 3140 mice with tetO-TAg mice, and Alb-TAg line 3123 was generated by crossing Alb-tTA 3123 mice with tetO-TAg mice. The Alb-lacZ mice were generated the same way. MUP-TAg and MUP-lacZ lines 7552, 7545, and 7016 were generated by crossing each MUP-tTA line with tetO-TAg and tetO-lacZ, respectively.

Animals obtained from each cross were divided into three groups based on the genotype, viz. double-positive animals (which contain both tTA and TAg transgenes), tTA single-positive, and TAg single-positive (Fig. 1B). The latter two groups were used as controls for the binary transgenic system in response to tetracycline administration. In each group, one animal received tetracycline treatment, the second received a placebo, and the third received tetracycline first, followed by 7 days of tetracycline withdrawal. The aim for the third group was to evaluate the reversibility of transgene expression by tetracycline regulation. To assess the tissue-specific regulation of the transgene by tetracycline, we analyzed the livers and various other organs, including the spleen. An identical scheme was followed for the generation of tTA/lacZ mice. The expression of the lacZ transgene was determined by beta -galactosidase activity.



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Fig. 1.   Construction of tTA transgenes and mouse genotyping. A, schematic diagram of tTA constructs. Two constructs were generated for the liver-specific tTA expression. The first was constructed with the mouse albumin promoter upstream of tTA, and the second was constructed with the mouse MUP promoter (see "Materials and Methods"). SV40tIVSpA represents SV40 small t intron and polyadenylation sequences. B, mouse genotyping. The genotypic pattern of Alb-TAg hybrid animals is represented. The upper panel shows the results of Southern blot analysis of genomic DNA digested with HindIII and probed with the tTA cDNA; the isolated transgenic fragment was used as a positive control. The same genomic DNA was subjected to PCR analysis using TAg-specific primers, generating a 630-base pair product, and the results are represented in the lower panel. DNA isolated from COS-7 cells was used as a positive control. Based on these findings, animals were classified as double-positive (positive for both tTA and TAg), tTA single-positive, or TAg single-positive. Not shown are the nontransgenic animals, which were negative in both assays. kbp, kilobase pairs.

Regulation of TAg Expression by Tetracycline-- After treatment, the TAg mice described above were analyzed for TAg expression in the liver, spleen, brain, tongue, kidney, lung, heart, ovary, skin, and skeletal muscle. Fig. 2 shows the levels of TAg expression in the livers and spleens of the Alb-TAg 3140 and 3123 mice and MUP-TAg 7552 mice. TAg expression was detected only in the liver and not in the spleen. RT-PCR analysis for TAg mRNA revealed a complete absence of TAg expression in all the organs listed above, except liver (data not shown). In all these lines, TAg expression was completely abrogated in the liver by tetracycline treatment of the double-positive mice. In animals after 7 days of tetracycline withdrawal (Fig. 2, third lane), TAg expression resumed, although to a lower level compared with the placebo-treated animals. This lower level may be attributed to a gradual disappearance of the tetracycline from the body and/or a less-than-precise time course of tetracycline release from the implanted pellet. The single-positive transgenic mice (TAg or tTA) were completely negative for TAg expression under all conditions, supporting the concept that the binary components of this transgenic model are essential for tetracycline-regulated gene expression. Finally, TAg expression could be detected only in the liver and not in the other tissues, indicating that the expression is tightly regulated only in the targeted tissue. All these results suggest that a tightly regulated, liver-specific gene expression by tetracycline administration can be achieved in this transgenic model. However, it is important to mention that there were considerable variations in the levels of TAg expression between the different mouse lines. In MUP-TAg lines 7545 and 7016, none of the double-positive animals expressed any detectable level of TAg (data not shown).



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Fig. 2.   Regulation of TAg expression by tetracycline in transgenic mice. Alb-TAg lines 3140 and 3123 and MUP-TAg lines 7552, 7545, and 7016 were generated as described under "Results." The offspring were genotyped at 3 weeks of age, and those harboring both tTA and TAg genes (Double) and positive for only one of the transgenes (tTA or TAg) were used in the study. Each group consisted of subgroups that were treated with either tetracycline (Tetracycline +) or placebo pellets (Tetracycline -). Some double-positive animals were first treated with tetracycline, followed by 1 week of tetracycline withdrawal (Tetracycline +(w)). Liver and spleen lysates (100 µg of protein) were analyzed for TAg and actin protein content by Western blot analysis using specific antibodies. The results are representative of at least three independent experiments. Positive and negative control lysates from cell lines (COS-7 and Huh-7 cells, respectively) showed a barely detectable actin signal because the gels were loaded with much lower protein content (5 µg). T antigen is a 85-kDa protein; actin is a 42-kDa protein.

Regulation of lacZ Expression by Tetracycline-- To address whether this tetracycline-regulated transgenic system can be used to direct the conditional expression of any transgene, we evaluated the expression of the lacZ reporter gene using the same approach. Fig. 3 shows the levels of lacZ expression in livers from these various tTA/lacZ lines. The double-positive animals from Alb-lacZ line 3140 and MUP-lacZ line 7552 demonstrated a tight regulation of lacZ expression in the liver by tetracycline, similar to the results obtained with the TAg lines above. Surprisingly, MUP-lacZ line 3123 did not show any LacZ protein activity in the absence of tetracycline, which is contrary to the results obtained with the corresponding TAg line. This could be due to a relatively lower sensitivity of lacZ detection in the livers of the animals. Similar to the tTA/TAg system, gene expression was confined to the liver and was not detected in the spleen. Discontinuation of tetracycline resulted in the resumption of lacZ gene expression in the liver. These findings support the notion that tetracycline-regulated gene expression can be accomplished with any gene of interest. Table I summarizes the findings of all TAg and lacZ lines.



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Fig. 3.   Tetracycline-mediated regulation of lacZ expression in the liver. The Alb-lacZ and MUP-lacZ lines were generated by breeding mice from the tetO-lacZ line with different tTA lines and treated with tetracycline or a placebo, and the liver lysates (100 µg of protein) were analyzed for beta -galactosidase activity as described under "Materials and Methods." The results are expressed in counts/min. Not shown are the LacZ protein activities in the spleens of these mice and in the livers of nontransgenic mice (negative controls), which were all within the background level (<15,000 cpm). Tetracycline +(w), tetracycline treatment, followed by 1 week of tetracycline withdrawal.


                              
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Table I
Regulated expression of TAg or lacZ in transgenic mouse livers
Alb-TAg or Alb-lacZ line 3140 was generated by crossing Alb-tTA 3140 mice with tetO-TAg or tetO-lacZ mice. Alb-TAg or Alb-lacZ line 3123 was generated by crossing Alb-tTA 3123 mice with tetop-TAg or tetop-lacZ mice. MUP-TAg lines 7552, 7545, and 7016 were generated similarly by crossing the respective MUP-tTA mice with tetO-TAg or tetO-lacZ mice. Genotypes of the offspring were double-positive (bigenic for both tTA and TAg or lacZ; Double), TAg single-positive (TAg or lacZ), or tTA single-positive (tTA). Mice with each genotype were implanted with either tetracycline (Tc) or placebo (P) pellets. A group of double-positive animals was treated with tetracycline, killed, and analyzed 1 week after dissipation of the tetracycline pellet (Tc/W). Each group included at least three animals, and they all behaved similarly. +, animals with TAg or lacZ expression; -, animals that failed to express TAg or lacZ.

Complete Suppression of Transgene Expression by Tetracycline-- To determine whether there is a low level expression of the transgene under suppressed conditions (by tetracycline) that is below the limit of the protein detection method above, total RNA was isolated from all groups (double-positive and tTA and TAg single-positive mice) with or without tetracycline treatment and subjected to RT-PCR analysis. RT-PCR can detect as few as 1000 copies of TAg transcripts, which translates to less than one copy/cell (10 µg of total RNA for RT-PCR, equivalent to ~104 cells), and thereby is much more sensitive than Western blotting. The results of the RT-PCR analysis of these RNA samples are shown in Fig. 4. Tetracycline treatment resulted in the complete absence of TAg transcripts compared with the placebo group (Fig. 4, lane 1). The tetracycline withdrawal group also showed detectable levels of TAg RNA, supportive of the reversal of the tetracycline regulation. Furthermore, the absence of TAg transcripts in the TAg single-positive mice is consistent with the notion that this transgene, in the absence of tTA, is completely silent.



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Fig. 4.   Complete suppression of TAg expression by tetracycline in the livers of transgenic mice. Total cellular RNA was isolated from liver and spleen tissues of various groups of mice described under "Results" and from the positive control cell line COS-7 (Positive RNA control). RNA was reverse-transcribed (RT +) and PCR-amplified using specific primers and analyzed on agarose gel. A positive control DNA was included in the assay (Positive DNA control). As a control, reverse transcription was also performed without reverse transcriptase (RT -). Specificity of the amplified fragments was confirmed by Southern blotting and probing with 32P-labeled TAg cDNA. Tc Treatment +(w), tetracycline treatment, followed by 1 week of tetracycline withdrawal.

Suppression of Hepatocellular Neoplasia by Tetracycline-- It is well known that constitutive expression of TAg in transgenic mouse models is oncogenic. At 4 weeks of age, double- and single-positive animals from Alb-TAg line 3123 were subjected continuously to tetracycline or placebo treatment for a period of 12 months and killed. Gross morphological examination of the livers, spleens, kidneys, lungs, hearts, brains, and skeletal muscles of all the animals was performed. Evidence of hepatic neoplasia was present in three out of five animals that were double-positive (Table II). One of them had a large liver tumor. Histological evaluation revealed hepatocellular adenoma with loss of lobular architecture and compression of tissues adjacent to the adenoma (Fig. 5, F and G). The hepatocytes within the adenoma were irregularly arranged with considerable pleomorphism, large heterochromatic nuclei, many mitotic figures, and intracellular fat vacuoles (lipidosis and steatosis). No well defined portal triads or central veins or sinusoids were noticed. The other two mice had multiple microscopic foci of nodular hyperplasia, and one of them had a microadenoma. These hyperplastic foci stood out as pale areas among the pink hepatic parenchyma and were distributed in all lobes of the liver (Fig. 5, B and C). Histological examination showed similar pathological changes to those of the adenoma described above. In contrast, none of the tetracycline-treated animals developed any evidence of tumor in the liver as revealed by histological examination (Fig. 5, A, D, and E). In all cases, tumors were confined to the liver only, which again supports the notion that this regulatable system can be precisely operated in a time-dependent and tissue-specific manner. All other organs appeared grossly normal without evidence of tumors. It is possible that they may have microscopic pathology, but in the complete absence of TAg expression, it is unlikely.


                              
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Table II
Regulation of tumor induction by tetracycline treatment
Double- and single-positive animals derived from Alb-TAg line 3123 were treated continuously with tetracycline or placebo for a period of 12 months and killed. Livers and other organs were examined for both gross morphological changes and histological changes. All organs other than the liver appeared normal in all mice. +/w, tetracycline treatment, followed by 1 week of tetracycline withdrawal.



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Fig. 5.   Regulatable development of hepatic neoplasia by tetracycline. Animals from Alb-TAg line 3123, which were double- or single-positive for the transgenes, were treated with or without tetracycline continuously for a period of 1 year. At the end of the treatment, the animals were killed and examined for tumor development in the liver and other organs as described under "Results." Hematoxylin/eosin-stained liver sections were examined for any histological changes. A shows the liver of a tetracycline-treated double transgenic animal, which appeared histologically normal and had no evidence of malignancy (magnification × 2.5; the same view is shown in D (magnification × 10) and E (magnification × 40)). B and C show a low power view (magnification × 2.5) of the livers from two placebo-treated animals, in which a microadenoma (B, arrow) and multiple hyperplastic foci (C, arrowheads) can be observed. F and G show the pathological features of the large hepatic adenoma from the third animal (magnification × 10 and × 40, respectively).



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we developed a binary transgenic system in which the E. coli tetracycline regulon was genetically engineered to achieve a tightly regulated, liver-specific gene expression in vivo. The liver-specific expression was accomplished using a liver-specific promoter (the mouse albumin or mouse major urinary protein promoter) to drive the tetracycline-sensitive tTA in one transgenic lineage; the other transgenic lineage contains the reporter gene (SV40 TAg or lacZ) under the control of the tetO promoter, to which tTA, in the absence of tetracycline, binds and activates. Analyses of the double transgenic mice showed that two of the three Alb-tTA lines and one of the two MUP-tTA lines exhibited the desirable phenotype of conditional expression in the liver. The expression of both reporter transgenes could be completely suppressed by tetracycline administration; such a suppression could be reversed upon discontinuation of tetracycline.

The biological significance of this system was confirmed by the phenotypic study of the TAg mice, in which the double transgenic animals developed liver neoplasia in the absence of tetracycline, and the tetracycline-treated group had normal liver histology. SV40 T antigen has been shown to be tumorigenic in transgenic mice (16-19). Constitutive expression of TAg in the liver leads to massive hepatomegaly and development of liver tumors by 5 months of age (20). Although we have not performed a detailed kinetic study of tumor development, three of five mice in the absence of continuous tetracycline developed gross and histological hyperplasia and/or neoplasia at 12 months of age. The less oncogenic potential of TAg in our model probably reflects a lower level of TAg expression than in other TAg transgenic mouse models. In a similar study by Ewald et al. (21), the tetracycline-regulated expression of TAg was targeted to the salivary glands of mice. These mice developed ductal hyperplasia at 4 months of age. Importantly, tumor development could be abolished by tetracycline administration at 4 months, but not at 7 months, of age. However, it is not clear whether the time or the stage of the cancer is crucial for tumor suppression. In our model, we do not know what the threshold time point is for tumor regression by tetracycline. Further experiments are underway to address these issues.

Several transgenic mouse models have been developed using the tetracycline-regulated system in various target tissues or organs. Gossen and Bujard (22) were able to achieve tetracycline-regulated gene expression in lymphoid cells, and Passman and Fishman (23) in cardiac muscles. In another study, the rat liver enriched activator protein promoter was used for liver-specific expression of tTA; however, reporter gene (luciferase) expression could be detected in several organs such as spleen, heart, brain, and liver under the induced conditions (24). In all these studies, it is not clear whether one can achieve near-complete suppression of the transgene by tetracycline administration with more sensitive techniques such as PCR.

Other conditional expression systems have been applied to the transgenic and knockout technology. The most notable one is the cre-lox system (8, 25). cre is often delivered by an adenoviral or retroviral vector to mice with the lox-controlled transgene (26, 27). Although one can achieve tissue-specific targeting using a tissue-specific promoter to express cre, the recombination mediated by lox is often incomplete, and the effect of viral vectors may have untoward functional effects on the targeted tissues (28). Development of cre transgenic mice under the control of tissue-specific promoters can overcome the viral effects, but the advantage of specific timing of conditional expression is lost. In addition, the cre-lox-mediated recombination is not reversible, thereby mitigating the potential of an on-off regulation. Other conditional expression systems regulatable by pharmacologic agents such as mifepristone (RU-486) (29), ecdysone (30), and rapamycin (FK506) (31) have been developed recently. A system using the antiprogestin mifepristone and progesterone receptor ligand-binding domain fusion protein to regulate gene expression has been developed in transgenic mice (32). Although several orders of magnitude of induction of the transgene can be achieved in vivo, it is not clear whether the dose of mifepristone used for induction exerts any unforeseen biological effects on the animals or what the basal level expression of the transgene is. Furthermore, the slow kinetics of regulation by mifepristone in vivo limited the usefulness of this system (32).

Our study has several biological implications. First, it is possible keep the expression of a transgene completely silent in the liver and to induce its expression at any desirable time point in a reversible manner. This would allow a precise evaluation of the true biological functions of any gene in the liver in the context of the defined physiological or pathophysiological state of the animal without other compensatory or epigenetic events. Second, this model system can be applied to the silencing of an endogenous gene in a reversible, time-dependent manner. For example, cre could be placed under the control of the tetracycline-regulated system for specific knockout of a gene, or an antisense transgene could be designed to suppress the expression of an endogenous gene in the liver. Third, by completely silencing the transgene during the fetal stage, it is possible to avert the thymic selection process of a transgene as a self-antigen and therefore to characterize the immunologic responses to the transgene later in the animal's life. This can be applied to the study of autoantigens or viral infections. The latter approach is particularly valuable for hepatitis C, in which there is no convenient small animal model. A previous study has reported the induction of the hepatitis C virus core-specific immune response using the cre-lox system to induce the expression of the core transgene in liver (28). However, the presence of a strong anti-adenoviral immunity (delivery of cre by adenovirus to the liver) precluded detailed characterization of the effects of an anti-hepatitis C virus response in vivo. We are hopeful that our model system will pave the way for the more precise study of any endogenous or exogenous genes and their interactions with the immune system in the context of viral immunopathology, hepatocarcinogenesis, autoimmunity, or other forms of liver disease.


    ACKNOWLEDGEMENTS

We thank John Vergalla for excellent technical assistance, Mark St. Clair for superb veterinary support, and Shimon Efrat for providing the tetO-TAg mice. We thank Donald J. Gardner for interpretation of the histology slides. We also thank Andrew Leiter, the New England Medical Center/Tufts Center for Gastroenterology Research on Absorptive and Secretory Processes Digestive Disease Center, and the Massachusetts General Hospital Center for Study of Inflammatory Bowel Disease for generating some of the transgenic mice.


    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Liver Diseases Section, DDB/NIDDK/NIH, Bldg. 10, Rm. 9B16, 10 Center Drive MSC 1800, Bethesda, MD 20892. Tel.: 301-402-1721; Fax: 301-402-0491; E-mail: JakeL@bdg10.niddk.nih.gov.

Published, JBC Papers in Press, January 18, 2001, DOI 10.1074/jbc.M009770200


    ABBREVIATIONS

The abbreviations used are: TAg, SV40 T antigen; tTA, tetracycline-responsive transcriptional activator; Alb, albumin; MUP, major urinary protein; PCR, polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


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