Use of the Tetracycline-controlled Transcriptional Silencer (tTS) to Eliminate Transgene Leak in Inducible Overexpression Transgenic Mice
2001; Elsevier BV; Volume: 276; Issue: 27 Linguagem: Inglês
10.1074/jbc.m101512200
ISSN1083-351X
AutoresZhou Zhu, Bing Ma, Robert Homer, Tao Zheng, Jack A. Elias,
Tópico(s)Neurogenetic and Muscular Disorders Research
ResumoThe doxycycline-inducible reverse tetracycline transactivator (rtTA) is frequently used to overexpress transgenes in a temporally regulated fashion in vivo. These systems are, however, often limited by the levels of transgene expression in the absence of dox administration. The tetracycline-controlled transcriptional silencer (tTS), a fusion protein containing thetet repressor and the KRAB-AB domain of the kid-1 transcriptional repressor, is inhibited by doxycycline. We hypothesized that tTS would tighten control of transgene expression in rtTA-based systems. To test this hypothesis we generated mice in which the CC10 promoter targeted tTS to the lung, bred these mice with CC10-rtTA-interleukin 13 (IL-13) mice in which IL-13 was overexpressed in an inducible lung-specific fashion, and compared the IL-13 production and phenotypes of parental mice and the triple transgenic CC10-rtTA/tTS-IL-13 progeny of these crosses. In the CC10-rtTA-IL-13 mice, IL-13, mucus metaplasia, inflammation, alveolar enlargement, and enhanced lung volumes were noted at base line and increased greatly after doxycycline administration. In the triple transgenic tTS animals, IL-13 and the IL-13-induced phenotype could not be appreciated without doxycycline. In contrast, tTS did not alter the induction of IL-13 or the generation of the IL-13 phenotype by doxycycline. Thus, tTS effectively eliminated the baseline leak without altering the inducibility of rtTA-regulated transgenes in vivo. Optimal "off/on" regulation of transgene expression can be accomplished with the combined use of tTS and rtTA. The doxycycline-inducible reverse tetracycline transactivator (rtTA) is frequently used to overexpress transgenes in a temporally regulated fashion in vivo. These systems are, however, often limited by the levels of transgene expression in the absence of dox administration. The tetracycline-controlled transcriptional silencer (tTS), a fusion protein containing thetet repressor and the KRAB-AB domain of the kid-1 transcriptional repressor, is inhibited by doxycycline. We hypothesized that tTS would tighten control of transgene expression in rtTA-based systems. To test this hypothesis we generated mice in which the CC10 promoter targeted tTS to the lung, bred these mice with CC10-rtTA-interleukin 13 (IL-13) mice in which IL-13 was overexpressed in an inducible lung-specific fashion, and compared the IL-13 production and phenotypes of parental mice and the triple transgenic CC10-rtTA/tTS-IL-13 progeny of these crosses. In the CC10-rtTA-IL-13 mice, IL-13, mucus metaplasia, inflammation, alveolar enlargement, and enhanced lung volumes were noted at base line and increased greatly after doxycycline administration. In the triple transgenic tTS animals, IL-13 and the IL-13-induced phenotype could not be appreciated without doxycycline. In contrast, tTS did not alter the induction of IL-13 or the generation of the IL-13 phenotype by doxycycline. Thus, tTS effectively eliminated the baseline leak without altering the inducibility of rtTA-regulated transgenes in vivo. Optimal "off/on" regulation of transgene expression can be accomplished with the combined use of tTS and rtTA. Clara cell 10-kDa protein tetracycline-controlled transcriptional silencer reverse tetracycline transactivator interleukin 13 tetracycline operator human growth hormone doxycycline matrix metalloproteinase monocyte chemoattractant protein bronchoalveolar lavage periodic acid-Schiff with diastase reverse transcription polymerase chain reaction Overexpression transgenic modeling is being used with increasing frequency to investigate the processes involved in tissue homeostasis and disease pathogenesis. This is nicely illustrated in the lung, where the Clara cell 10-kDa protein (CC10)1 or surfactant apoprotein-C promoters can be used to selectively target genes of interest to the lung parenchyma and/or airway (1Schilz R. Elias J.A. Raeburn D. Giembycz M. Airways Smooth Muscle: Modeling the Asthmatic Response in Vivo. Birkhaeuser Verlag, Basel, Switzerland1996: 241-274Google Scholar). In early studies, these promoters were used to directly drive the expression of transgenes in the lung. These studies provided impressive insights into the chronic respiratory effector functions of inflammatory mediators and the pathogenesis of asthma, adult respiratory distress syndrome, lung development, and pulmonary fibrosis (2Zhu Z. Homer R.J. Wang Z. Chen Q. Geba G.P. Wang J. Zhang Y. Elias J.A. J. Clin. Invest. 1999; 103: 779-788Crossref PubMed Scopus (1498) Google Scholar, 3Zhou L. Dey C.R. Wert S.E. Whitsett J.A. Dev. 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This system is based on the generation of transgenic mice with two molecular constructs. In the first, the promoter of choice (CC10) drives the expression of the reverse tetracycline transactivator (rtTA), a fusion protein made up of the herpesvirus VP-16 transactivator and a mutantTet repressor from Escherichia coli. This transactivating fusion protein requires doxycycline (dox) (a tetracycyline derivative) for specific DNA binding. The second construct contains multimers of the tetracycline operator (tet-O), a minimal cytomegalovirus promoter, and the gene of interest. In the absence of dox, the transactivator does not recognize or weakly recognizes its specific target sequence (tet-O), and target gene transcription occurs at low levels or not at all. The addition of dox allows the transactivator to bind in trans to thetet-O, activating the transgene of interest. This system has been used successfully in our laboratory and others to define the development-dependent and -independent processes that cause alveolar enlargement in the lung, inflammatory events that may contribute to the pathogenesis of pulmonary emphysema, cytokine-mediated protective events in oxygen toxicity, and crucial windows in lung development that define cytokine effector profiles (4Waxman A.B. Einarsson O. Seres T. Knickelbein R.G. Warshaw J.B. Johnston R. Homer R.J. Elias J.A. J. Clin. Invest. 1998; 101: 1970-1982Crossref PubMed Scopus (156) Google Scholar,10Ray P. Tang W. Wang P. Homer R. Kuhn C.I. Flavell R.A. Elias J.A. J. Clin. Invest. 1997; 100: 2501-2511Crossref PubMed Scopus (129) Google Scholar, 18Zheng T. Zhu Z. Wang Z. Homer R.J. Ma B. Riese R. Chapman H. Shapiro S.D. Elias J.A. J. Clin. Invest. 2000; 106: 1081-1093Crossref PubMed Scopus (546) Google Scholar, 19Tichelaar J.W. Lu W. Whitsett J.A. J. Biol. Chem. 2000; 275: 11858-11864Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 20Corne J. Chupp G. Lee C.G. Homer R.J. Zhu Z. Chen Q. Ma B. Du Y. Roux F. McArdle J. Waxman A.B. Elias J.A. J. Clin. Invest. 2000; 106: 783-791Crossref PubMed Scopus (138) Google Scholar). Detailed analyses of these and other rtTA-based systems have, however, revealed some properties that restrict its application. One limitation stems from the fact that rtTA can exhibit a degree of residual affinity to tet-O in the absence of dox. This manifests as definable levels of transgene activation and phenotype induction in animals (or cells) that are not receiving dox (11Baron U. Bujard H. Methods Enzymol. 2000; 327: 401-421Crossref PubMed Scopus (272) Google Scholar, 16Furth P.A. St. Onge L. Boger H. Gruss P. Gossen M. Kistner A. Bujard H. Hennighausen L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9302-9306Crossref PubMed Scopus (677) Google Scholar, 18Zheng T. Zhu Z. Wang Z. Homer R.J. Ma B. Riese R. Chapman H. Shapiro S.D. Elias J.A. J. Clin. Invest. 2000; 106: 1081-1093Crossref PubMed Scopus (546) Google Scholar,21Wang Z. Zheng T. Zhu T. Homer R.J. Riese R.J. Chapman H.A. Shapiro S.D. Elias J.A. J. Exp. Med. 2000; 192: 1587-1600Crossref PubMed Scopus (367) Google Scholar, 22Urlinger S. Baron U. Thellmann M. Hasan M.T. Bujard H. Hillen W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7963-7968Crossref PubMed Scopus (755) Google Scholar, 23Kistner A. Gossen M. Zimmermann F. Jerecic J. Ullmer C. Lubbert H. Bujard H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10933-10938Crossref PubMed Scopus (663) Google Scholar, 24Freundlieb S. Schirra-Muller C. Bujard H. J. Gene Med. 1999; 1: 4-12Crossref PubMed Scopus (226) Google Scholar, 25Forster K. Helbl V. Lederer T. Urlinger S. Wittenburg N. Hillen W. Nucleic Acids Res. 1999; 27: 708-710Crossref PubMed Scopus (97) Google Scholar). Approaches that can be used to eliminate basal transgene leakin vivo have not been well characterized. The tet-controlled transcriptional silencer (tTS) is a fusion protein made up of a mutant Tet repressor and the KRAB-AB domain of the Kid-1 protein, a powerful transcriptional repressor (24Freundlieb S. Schirra-Muller C. Bujard H. J. Gene Med. 1999; 1: 4-12Crossref PubMed Scopus (226) Google Scholar, 26Witzgall R. O'Leary E. Leaf A. Onaldi D. Bonventre J.V. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4514-4518Crossref PubMed Scopus (317) Google Scholar). It binds to tet-O only in the absence of dox. Therefore, in systems containing tTS and tet-O-driven transgenes, tTS binds to the tet-O in the absence of dox and inhibits the expression of the gene of interest. As dox is added to the culture medium, the tTS dissociates from tet-O, relieving the transcriptional suppression. At sufficient concentrations, the dox also interacts with rtTA, allowing it to bind to tet-O and activate the gene of interest. The tTS system has been shown to confer exquisite regulating ability on transiently transfected and stably transfected rtTA-regulated reporter genes in cells in culture (24Freundlieb S. Schirra-Muller C. Bujard H. J. Gene Med. 1999; 1: 4-12Crossref PubMed Scopus (226) Google Scholar, 25Forster K. Helbl V. Lederer T. Urlinger S. Wittenburg N. Hillen W. Nucleic Acids Res. 1999; 27: 708-710Crossref PubMed Scopus (97) Google Scholar,27Deuschle U. Meyer W.K.-H. Thiesen H.-J. Mol. Cell. Biol. 1995; 15: 1907-1914Crossref PubMed Scopus (228) Google Scholar). Surprisingly, the feasibility and efficacy of tTS in regulating basal gene expression in vivo in transgenic animals has not been investigated. We postulated that the principles involved in tTS inhibition of basal gene expression in vitro are also applicable to the in vivo state. To test this hypothesis, we generated transgenic mice in which the CC10 promoter targeted tTS to the lung and bred these mice with CC10-rtTA-IL-13 mice in which CC10 and rtTA were used to express IL-13 in the lung/airway in an externally regulatable fashion. These mice had been previously generated in our laboratory and shown to have a quantifiable level of transgene leak in the absence of dox administration (18Zheng T. Zhu Z. Wang Z. Homer R.J. Ma B. Riese R. Chapman H. Shapiro S.D. Elias J.A. J. Clin. Invest. 2000; 106: 1081-1093Crossref PubMed Scopus (546) Google Scholar). We then compared the IL-13 production and phenotypes of the dual transgenic CC10-rtTA-IL-13 and the triple transgenic CC10-rtTA/tTS-IL-13 mice. These studies demonstrate that tTS totally abrogates basal transgene leak and phenotype induction in our transgenic system. They also demonstrate that tTS mediates this inhibition without decreasing the ability of dox to stimulate the rtTA-regulated transgene or the ability of the transgene to induce a tissue phenotype. In order to express tTS in a lung specific fashion, the construct CC10-tTS-hGH was generated (Fig.1 A). The plasmid containing tTS, pTet-tTS, was obtained from Dr. Andrew Farmer (CLONTECH Inc., Palo Alto, CA). Oligonucleotide primers were synthesized that would introduceHindIII and BamHI restriction enzyme sites 5′ and 3′ of the tTS coding region respectively. Furthermore a silent mutation was introduced to disrupt the original BamHI site right before the stop codon. The primers were, tTSUP1: 5′-GTG AAC AAG CTT ATC GCC TGG AGA CG-3′ and tTSLO1: 5′-CTT AGT GGA TCC ATT TAC CAG GGG TCC TCT CT TGC-3′. The tTS fragment was amplified by PCR and then inserted between CC10 promoter and hGH polyadenylation and intronic sequence in place of the rtTA sequences in construct CC10-rtTA-hGH that was previously described by our laboratory (10Ray P. Tang W. Wang P. Homer R. Kuhn C.I. Flavell R.A. Elias J.A. J. Clin. Invest. 1997; 100: 2501-2511Crossref PubMed Scopus (129) Google Scholar). After verifying the junction areas and tTS DNA by sequencing, the construct was isolated by electrophoresis. The DNA was then purified through an Elutip-D column following the manufacturer's instructions (Schleicher and Schuell, Inc., Keene, NH) and dialyzed against microinjection buffer (25 mm Tris-HCl/0.5 mm EDTA, pH 7.5). Transgenic mice were generated in (CBA X C57BL/6) F2 eggs using standard pronuclear injection as previously described (6Tang W. Geba G.P. Zheng T. Ray P. Homer R. Kuhn C. Favell R.A. Elias J.A. J. Clin. Invest. 1996; 98: 2845-2853Crossref PubMed Scopus (197) Google Scholar, 28Hogan B. Constantini F. Lacy E. Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1986Google Scholar). The resulting animals and their progeny were genotyped using tail DNA and Southern blot analysis with 32P-labeled tTS DNA as a probe or PCR. For PCR reactions, primers were designed to cover the area that was unique to the tTS transgene (TableI). The primers were tTSUP2: 5′-GAG TTG GCA GCA GTT TCT CC-3′ and tTSL02: 5′-GAG CAC AGC CAC ATCTTC AA-3′. The PCR protocol was 95 °C for 5 min; 30 cycles at 95 °C for 1 min; 60 °C for 1 min and 72 °C for 1 min and a final extension at 72 °C for 10 min. A product of 472 bp was expected and detected.Table IPCR primersMoietySense primerAnti-sense primerAnneal temperatureProduct size°Cbase pairsIL-13AGACCAGACTCCCCTGTGCATGGGTCCTTAGATGGCATTG60123tTSGAGTTGGCAGCAGTTTCTCCGAGCACAGCCACATCTTCAA60472EotaxinCCATCTGTCTCCCTCCACCATGATCCCACATCTCCTTTCATGCC56546MCP-1ACCAGCCAACTCTCACTGAAGCCAGAAGTGCTTGAGGTGGTTGT60463MMP-12AAGCAACTGGGCAACTGGACAACTCTGGTGACAGAAAGTTGATGGTG57631Cathepsin KGGGAGACATGACCAGTGAAGAAGTTGCTCTCTTCAGGGCTTTCTCGT57481β-ActinGTGGGCCGCTCTAGGCACCATGGCCTTAGGGTTCAGGGGG60241bp, base pairs. Open table in a new tab bp, base pairs. CC10-rtTA-IL-13 transgenic mice were generated as described by our laboratory (18Zheng T. Zhu Z. Wang Z. Homer R.J. Ma B. Riese R. Chapman H. Shapiro S.D. Elias J.A. J. Clin. Invest. 2000; 106: 1081-1093Crossref PubMed Scopus (546) Google Scholar). These dual transgenic mice express IL-13 in the lung in an externally regulatable fashion. The first construct in these mice, CC10-rtTA-hGH, contains the CC10 promoter, rtTA and human growth hormone (hGH) intronic and polyadenylation sequences (hGH) (10Ray P. Tang W. Wang P. Homer R. Kuhn C.I. Flavell R.A. Elias J.A. J. Clin. Invest. 1997; 100: 2501-2511Crossref PubMed Scopus (129) Google Scholar). The second construct,tet-O-IL-13-hGH, contains a tet-O and minimal cytomegalovirus promoter, murine IL-13 cDNA and hGH intronic and polyadenylation sequences. The constructs are illustrated in Fig. 1A. CC10-rtTA/tTS-IL-13 triple transgenic mice were generated by breeding CC10-rtTA-IL-13 mice with CC10-tTS-hGH mice. The genotypes of the progeny of these crosses were analyzed by PCR performed with tail DNA with primers designed to determine if rtTA, tTS and/or IL-13 were present (Table I). The protocols that were used to detect rtTA and IL-13 have been previously described (2Zhu Z. Homer R.J. Wang Z. Chen Q. Geba G.P. Wang J. Zhang Y. Elias J.A. J. Clin. Invest. 1999; 103: 779-788Crossref PubMed Scopus (1498) Google Scholar, 18Zheng T. Zhu Z. Wang Z. Homer R.J. Ma B. Riese R. Chapman H. Shapiro S.D. Elias J.A. J. Clin. Invest. 2000; 106: 1081-1093Crossref PubMed Scopus (546) Google Scholar). The PCR protocols for tTS were noted above. All mice were maintained on normal water until transgene activation was desired. At that time, dox (0.5 mg/ml) was added to the animal's drinking water. Sucrose (2%) was also added to mask the bitter taste of dox and the dox water was kept in dark brown bottles to prevent light-induced dox degradation. Mice were euthanized and a median sternotomy was performed. The trachea was then isolated via blunt dissection and small caliber tubing was inserted and secured in the airway. Three successive volumes of 0.75 ml of PBS with 0.1% bovine serum albumin were then instilled and gently aspirated. Total cell counts and differentials were evaluated as described previously (18Zheng T. Zhu Z. Wang Z. Homer R.J. Ma B. Riese R. Chapman H. Shapiro S.D. Elias J.A. J. Clin. Invest. 2000; 106: 1081-1093Crossref PubMed Scopus (546) Google Scholar, 21Wang Z. Zheng T. Zhu T. Homer R.J. Riese R.J. Chapman H.A. Shapiro S.D. Elias J.A. J. Exp. Med. 2000; 192: 1587-1600Crossref PubMed Scopus (367) Google Scholar). BAL fluid aliquots were centrifuged and the supernatants were stored at −70 °C until utilized. The levels of IL-13 were determined immunologically using commercial ELISA kits as per the manufacturer's instructions (R&D Systems, Minneapolis, MN). Selected samples were concentrated 10 X by volume using Microcon YM-10 following the manufacturer's protocol (Millipore Corporation, Bedford, MA). mRNA levels were assessed using Northern analyses and/or RT-PCR as previously described by our laboratory (18Zheng T. Zhu Z. Wang Z. Homer R.J. Ma B. Riese R. Chapman H. Shapiro S.D. Elias J.A. J. Clin. Invest. 2000; 106: 1081-1093Crossref PubMed Scopus (546) Google Scholar, 21Wang Z. Zheng T. Zhu T. Homer R.J. Riese R.J. Chapman H.A. Shapiro S.D. Elias J.A. J. Exp. Med. 2000; 192: 1587-1600Crossref PubMed Scopus (367) Google Scholar). In the RT-PCR experiments, total RNA from mouse lungs was prepared using Trizol reagent (Life Technologies, Inc., Grand Island, NY) after treatment with DNase I following the manufacturer′s instructions. The RNA samples were reverse transcribed and gene-specific primers were used to amplify selected regions of each target moiety. Optimal annealing temperature and cyclings were derived for each individual cytokine. Equal amounts of RNA were tested in each reaction and β-actin was used as an internal standard. Amplified PCR products were visualized using ethidium bromide gel electrophoresis and finally confirmed by nucleotide sequencing. IL-13, matrix metalloproteinase (MMP)-12, eotaxin, monocyte chemotactic protein-1 (MCP-1), cathepsin K and β-actin mRNA were assessed using the primers and conditions listed in Table I as described previously by our laboratory (18Zheng T. Zhu Z. Wang Z. Homer R.J. Ma B. Riese R. Chapman H. Shapiro S.D. Elias J.A. J. Clin. Invest. 2000; 106: 1081-1093Crossref PubMed Scopus (546) Google Scholar, 21Wang Z. Zheng T. Zhu T. Homer R.J. Riese R.J. Chapman H.A. Shapiro S.D. Elias J.A. J. Exp. Med. 2000; 192: 1587-1600Crossref PubMed Scopus (367) Google Scholar). In selected experiments the IL-13 RT-PCR product was transferred to a nitrocellulose membrane and evaluated with a labeled internal primer, 5′-TTT CCG CGG CTA CAG CTC CCT GGT TCT CTC-3′. The tTS transcripts were evaluated as described above. In these experiments, mice were euthanized, median sternotomies were performed and right heart perfusion was accomplished with calcium and magnesium-free PBS to clear the intravascular space. The heart and lungs were then removed en bloc, inflated to 25 cm pressure with Streck tissue fixative solution (Streck Laboratories, Inc. Omaha, NE), embedded in paraffin and sectioned at 5 microns. Hematoxylin and eosin, Mallory's trichrome and periodic acid-Schiff with diastase (PAS) stains were performed in the Research Pathology Laboratory at Yale University. To evaluate the size of the alveoli, lungs from transgenic and control mice were obtained, fixed to pressure and sectioned and stained as described above. Alveolar chord length was then measured as described previously by our laboratory (18Zheng T. Zhu Z. Wang Z. Homer R.J. Ma B. Riese R. Chapman H. Shapiro S.D. Elias J.A. J. Clin. Invest. 2000; 106: 1081-1093Crossref PubMed Scopus (546) Google Scholar). The histologic mucus index is a measure of the number of mucus secreting airway epithelial cells per unit basement membrane. It was calculated from PAS stained histologic sections as previously described by our laboratory (18Zheng T. Zhu Z. Wang Z. Homer R.J. Ma B. Riese R. Chapman H. Shapiro S.D. Elias J.A. J. Clin. Invest. 2000; 106: 1081-1093Crossref PubMed Scopus (546) Google Scholar,29Cohn L. Homer R.J. MacLeod H. Mohrs M. Brombacher F. Bottomly K. J. Immunol. 1999; 162: 6178-6183PubMed Google Scholar). Lung volume and compliance were assessed as described previously by our laboratory (18Zheng T. Zhu Z. Wang Z. Homer R.J. Ma B. Riese R. Chapman H. Shapiro S.D. Elias J.A. J. Clin. Invest. 2000; 106: 1081-1093Crossref PubMed Scopus (546) Google Scholar,21Wang Z. Zheng T. Zhu T. Homer R.J. Riese R.J. Chapman H.A. Shapiro S.D. Elias J.A. J. Exp. Med. 2000; 192: 1587-1600Crossref PubMed Scopus (367) Google Scholar). In these experiments mice were anesthetized, the trachea was cannulated and the lungs were ventilated with 100% O2 via a "T" piece attachment. The trachea was then clamped and oxygen absorbed in the face of ongoing pulmonary perfusion. At the end of this degassing, the lungs and heart were removed en bloc and inflated with PBS at gradually increasing pressures from 0–30 cm. The size of the lung at each 5 cm interval was evaluated via volume displacement. Values are expressed as means ± S.E. As appropriate, groups were compared by analysis of variance with Scheffe's procedure post hoc analysis, Student's t test or nonparametric assessments (Wilcoxon's rank sum, Mann-WhitneyU test) using Stat View software for the Macintosh (Abacus Concepts Inc., Berkeley, CA, USA). To characterize the effects of tTS in our transgenic system, we generated multiple lines of mice in which tTS was targeted to the lung using the CC10 promoter (Fig. 1A). The CC10-tTS-hGH construct was prepared, linearized and microinjected as previously described (6Tang W. Geba G.P. Zheng T. Ray P. Homer R. Kuhn C. Favell R.A. Elias J.A. J. Clin. Invest. 1996; 98: 2845-2853Crossref PubMed Scopus (197) Google Scholar, 10Ray P. Tang W. Wang P. Homer R. Kuhn C.I. Flavell R.A. Elias J.A. J. Clin. Invest. 1997; 100: 2501-2511Crossref PubMed Scopus (129) Google Scholar). PCR using tail biopsy-derived DNA was used for genotyping and RT-PCR of whole lung RNA was used to evaluate tTS gene expression. Four transgene (+) founder animals were obtained from the microinjection (Fig. 1B). Each was bred onto a C57BL/6 background and independent lines were generated. In all cases, the transgene was propagated in a Mendelian fashion. tTS mRNA was readily detected in total lung RNA from transgene (+) animals. tTS mRNA was not detected in lung RNA from transgene (-) littermate control animals. In addition, tTS was not detected in a variety of extra thoracic organs in transgene (+) or (-) progeny mice (Fig. 1C and data not shown). These studies demonstrate that CC10 appropriately targets tTS to the murine lung. To determine if tTS altered lung structure, we compared the hematoxylin and eosin, PAS and trichrome evaluations, alveolar morphometry and compliance of 1–3 month old CC10-tTS-hGH mice and wild type littermate controls. In all cases, differences could not be appreciated (data not shown). Thus, tTS expression did not alter the histology, morphometry or compliance of the murine lung. To determine if tTS altered the levels of IL-13 that were produced in the absence of dox administration, we compared the levels of BAL IL-13 protein and lung IL-13 mRNA in double transgenic (CC10-rtTA-IL-13) and triple transgenic (CC10-rtTA/tTS-IL-13) mice. Single transgenic (CC10-tTS-hGH) and transgene (-) littermate control animals were also evaluated. IL-13 was not detected in the BAL fluid from the transgene (-) littermate controls or the CC10-tTS-hGH animals. IL-13 (50–110 pg/ml) was, however, readily appreciated in the BAL fluids from the dual transgene (+) animals. In striking contrast to this finding, IL-13 protein was undetectable in BAL fluids from uninduced triple transgenic animals containing the tTS construct (Fig. 2). After 10-fold BAL fluid concentration, IL-13 was still unable to be detected in BAL fluids from triple transgenic mice, whereas significant levels (0.9–1.5 ng/ml) of IL-13 were detected in similarly concentrated BAL fluids from dual transgene (+) animals (Fig. 2). These studies demonstrate that the tTS construct decreased the BAL IL-13 content of uninduced mice by at least 3 orders of magnitude. In accord with these observations, mRNA encoding IL-13 was readily detected via RT-PCR analysis in dual transgene (+) animals. In contrast, IL-13 mRNA was unable to be detected in lungs from triple transgenic tTS mice even after 35 cycles of RT-PCR analysis (Fig. 2). When viewed in combination, these studies demonstrate that the inclusion of the tTS construct totally eliminated the background leak in our dual transgenic rtTA-regulated animals. Lungs from CC10-rtTA-IL-13 animals that did not receive dox were enlarged and had larger alveoli and enhanced compliance when compared with lungs from transgene (-) littermate controls (Fig. 3). They also manifest increased BAL cellularity, eosinophil, lymphocyte and macrophage rich BAL and tissue inflammation, mucus metaplasia and enhanced accumulation of eotaxin, MCP-1, MMP-12 and cathepsin K mRNA (Fig.4, Table IIand data not shown) (2Zhu Z. Homer R.J. Wang Z. Chen Q. Geba G.P. Wang J. Zhang Y. Elias J.A. J. Clin. Invest. 1999; 103: 779-788Crossref PubMed Scopus (1498) Google Scholar, 18Zheng T. Zhu Z. Wang Z. Homer R.J. Ma B. Riese R. Chapman H. Shapiro S.D. Elias J.A. J. Clin. Invest. 2000; 106: 1081-1093Crossref PubMed Scopus (546) Google Scholar). In contrast to these findings, the lung volumes, alveolar size and pulmonary compliance of triple transgenic tTS containing animals were unable to be differentiated from the same parameters in transgene (-) littermate control animals or CC10-tTS-hGH mice (p < 0.01 comparing triple and dual transgene (+) animals for each parameter) (Fig. 3). In addition, the BAL abnormalities, mucus metaplasia and the eotaxin, MCP-1, MMP-12
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