Mouse model for lung tumorigenesis through Cre/lox ... - Nature

Oncogene (2001) 20, 6551 ± 6558 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc

Mouse model for lung tumorigenesis through Cre/lox controlled sporadic activation of the K-Ras oncogene Ralph Meuwissen1,4, Sabine C Linn1,4, Martin van der Valk2, Wolter J Mooi3 and Anton Berns*,1 1

Division of Molecular Genetics and Center of Biomedical Genetics, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands; 2Department of Experimental Animal Pathology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands; 3Division of Diagnostic Oncology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands

The onset of human lung cancer occurs through sequential mutations in oncogenes and tumor suppressor genes. Mutations in K-Ras play a prominent role in human non-small cell lung cancer. We have developed a mouse lung tumor model in which K-Ras can be sporadically activated through Cre-lox mediated somatic recombination. Adenoviral mediated delivery of Cre recombinase in lung epithelial cells gave rise to rapid onset of tumorigenesis, yielding pulmonary adenocarcinomas with 100% incidence after a short latency. The lung tumor lesions shared many features with human non-small cell lung cancer. Our data show that sporadic expression of the K-Ras oncogene is sucient to elicit lung tumorigenesis. Therefore this model has many advantages over conventional transgenic models used thus far. Oncogene (2001) 20, 6551 ± 6558. Keywords: somatic activation; Cre-lox; conditional KRas allele; lung epithelial tissue; lung tumors; nonsmall cell lung cancer Introduction Lung cancer is the leading cause of cancer-related death in man (Wingo et al., 1999). Because of important dierences in clinical behavior, lung cancer is commonly subdivided into SCLC [small cell lung cancer] and NSCLC [non-small cell lung cancer], the latter consisting of three main histologic types: adenocarcinoma (25 ± 40%), squamous cell carcinoma (25 ± 40%) and large cell carcinoma (10 ± 20%) (World Health Organization, 1981; Edwards, 1987). Pulmonary adenocarcinomas are known to harbor multiple cytogenetic and molecular abnormalities (Rom et al., 2000), in full accordance to a multi-step model for tumorigenesis (Hanahan and Weinberg, 2000). One dominant genetic alteration often found in *30% of pulmonary adenocarcinomas is an activating mutation of K-Ras (Mills et al., 1995; Rodenhuis et al., 1988; Huncharek et al., 1999). Mouse models for

pulmonary adenocarcinomas have been generated by introducing K-Ras mutations in lung epithelium by means of chemical carcinogens or by the use of transgenic mouse strains harboring an activated HRas gene driven from a lung-epithelial tissue speci®c promoter (Tuveson and Jacks, 1999). However, these approaches have major limitations. Carcinogen-induced lung tumorigenesis may aect many other genes besides the K-Ras oncogene. In most of the transgenic systems supraphysiological levels of H-Ras and N-Ras rather than K-Ras were used. Most importantly, the classical transgenic tumor model does not mimic the sporadic occurrence of Ras mutations. There is a fundamental dierence between a transgenic model in which an oncoprotein is expressed in every cell of a tissue or cell lineage, and a sporadic cancer model in which normal wild-type cells surround the incipient tumor cell. These normal cells could have a prominent suppressing eect on the proliferative capacity of cancer-prone cells. Alternatively, but not mutually exclusive, a single mutant cell might fail to support its own proliferation through autocrine mechanisms as factors diuse. This inhibition might be overcome once a single mutant cell has given rise to an aggregate of descendants forming a small patch of cells. Here, we have circumvented this problem by establishing a tumorigenesis model using sporadic activation of K-Ras in mouse pulmonary epithelium. We have chosen the Cre-lox mediated somatic recombination (Sauer, 1998) to introduce activated K-Ras expression in lung epithelium of adult mice. Using adenoviral based delivery of Cre recombinase into lung epithelial cells, we achieved sporadic K-RasV12 activation, thereby mimicking the human condition of NSCLC in which K-Ras is often mutated and is linked to aggressive tumor behavior (Rodenhuis and Slebos, 1992; Slebos et al., 1990). The model shows close similarity with a recently described `hit and run' sporadic lung tumor model (Johnson et al., 2001). Results

*Correspondence: A Berns; E-mail: [email protected] 4 Both authors contributed equally to this work Received 2 April 2001; revised 17 May 2001; accepted 16 July 2001

Previous transgenic mouse models overexpressing mutant H-Ras showed a stochastic onset of pulmonary

Conditionally activated K-Ras induces murine lung tumors R Meuwissen et al

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neoplasia (Saitoh et al., 1990; Maronpot et al., 1991; Suda et al., 1987). We decided to use another approach by generating transgenic animals harboring a conditional K-RasV12 allele, which expresses K-RasV12 only after Cre-mediated recombination. K-RasV12 is a potent oncogene and similar K-Ras mutations have often been found in pulmonary, pancreatic and colorectal adenocarcinomas (Bos, 1989; Mills et al., 1995; Pellegata et al., 1994; Oudejans et al., 1991). K-Ras has an intrinsic GTPase activity and the K-RasV12 mutation locks the protein in its active, GTP-bound state, leading to constitutive signaling to downstream targets (Manne et al., 1985; McGrath et al., 1984; Shields et al., 2000). The conditional transgenic K-RasV12 transgene (Figure 1) used here consists of a broadly active bactin promoter, followed by a GFP [Green Fluorescence Protein] expression cassette ¯anked by two lox sites and preceding a K-RasV12 cDNA combined with a PLAP [human placenta-like alkaline phosphatase] expression construct. Six founder lines were obtained with this construct. Only one founder contained a single copy, full-length transgene. Analysis of transgene expression showed ubiquitous GFP expression in a wide range of tissues (results not shown). A polyadenylation signal behind the GFP cassette prevented read-through into the K-RasV12 gene. Therefore, expression of K-RasV12 was dependent on Cre-lox mediated deletion of the GFP fragment. Since NSCLC, including pulmonary adenocarcinoma, arises from bronchopulmonary epithelium (Tsuchiya et al., 1995; Cooper et al., 1997), we targeted pulmonary epithelial cells by administering recombinant AdCre [adenoCrevirus] of sucient high titer (up to 46109 pfu/ml) intratracheally. In order to suppress perioperative infections, mice were treated with cotrimoxazole 1 week before and after AdCre administration. Perioperative mortality was less than 5%. Our experiments using adenoLacZ virus showed b-galactosidase activity in both bronchial epithelial cells as well as in the more distal alveolar pneumocytes 48 h after infection (data not shown). However, as has been reported by others (Zsengeller et al., 1995; Otake et al.,

1998), we found an extensive clearance of infected cells within 2 ± 4 weeks after infection, probably due to an immune response against the infected cells. To suppress clearance of the cells (Howell et al., 1998), mice were treated with cyclosporin A for 4 ± 6 weeks following adenovirus administration. The eective cyclosporin concentration remained roughly constant at about 40 ng/ml blood plasma and led to a marked decrease of infected cell clearance. To further validate the model, we decided to use AdCre for intratracheal infection of ACZL reporter mice (Akagi et al., 1997). In these mice, a Lac Z reporter gene is transcribed after Cre-mediated recombination and the recombined Lac Z reporter locus can be detected by staining for the resulting b-galactosidase activity. Immunostaining of lung tissue sections obtained from ACZL mice 2 weeks after AdCre showed the presence of Cre recombinase mainly in bronchial epithelial cells but also in the more distal alveolar epithelium (Figure 2a). Only sporadic staining of cells was observed. Histological staining for b-galactosidase activity in these lung tissue sections con®rmed these results. Cre-mediated recombination activity is only present in sporadicly infected bronchial epithelial cells as well as alveolar cells (Figure 2b). No

Figure 2 Validation of the model. ACZL reporter mice had been injected 107 pfu AdCre virus intratracheally. Two weeks after administration mice had been sacri®ced. (a) Immunohistochemical staining shows that Cre recombinase is expressed in alveolar pneumocyte nuclei. (b) Staining for Lac Z. b-gal -positive bronchial epithelial cells as well as alveolar type II pneumocytes are observed

Figure 1 Schematic representation of the conditional K-Ras construct. Without Cre recombinase GFP mRNA is expressed and the K-RASVal 12 oncogene behind the poly-A signal remains silent. Upon Cre mediated recombination of the loxP sites (") the KRASVal 12 oncogene is placed directly under control of the b-actin promoter. The K-RASVal 12 oncogene is transcribed together with the PLAP cDNA and because of their linkage through the IRES [Internal Ribosomal Entry Site] this results in translation of two independent proteins. Position and orientation of the PCR primers used for analysis are depicted (?) Oncogene


recombination of the LacZ locus could be detected in tissues other than the lung. The use of R26R reporter mice (Soriano, 1999) gave similar results (data not shown). We subsequently administered AdCre to conditional K-RasV12 mice. Table 1 summarizes the results. Three weeks after infection we detected intrapulmonary proliferative epithelial lesions with a reactive lymphocytic in®ltrate (Figure 3b). This intraparenchymal rather than airway lesion strongly resembled the socalled AAH [alveolar atypical hyperplasia] of the human, a lesion that is thought to precede the development of some pulmonary adenomas (Carey et al., 1992). At 5 ± 6 weeks post-infection, all mice had developed multiple lesions of this type (Figure 3a, c). The lesions subsequently developed into larger, papillary-like tumors (Figure 3d), seen at 8 weeks post-infection. The last group of mice was analysed at 9 ± 13 weeks post-infection: all had multiple pulmonary tumors causing cachexia and respiratory distress due to mass eect and bronchial obstruction. The architecture of these large nodules was again mainly papillary so that the tumors resembled human papillary adenocarcinomas (Malkinson, 1998). The tumor cells were positive for TTF-1 [thyroid transcription factor-1] (Figure 3e), a speci®c immunohistochemical marker of thyroid and pulmonary epithelium and tumors thereof (Ordonez, 2000) and often used for the identi®cation of pulmonary adenocarcinomas (Kaufmann and Dietel, 2000). The proliferative activity of these solid nodules, as shown by BrdU staining (Figure 3f), was moderate but greater as compared to the surrounding normal tissue. At 12 ± 13 weeks postinfection, some mice showed sporadic pulmonary tumors with marked nuclear enlargement, prominent nucleoli and extensive mitotic activity. These histologic indicators of tumor progression coincided with occasional emergence of macroscopic metastases to lymph nodes and kidney (Table 1). In normal control FVB/N mice undergoing the same AdCre administration, no pulmonary lesions were found. The progression observed histologically, from AAH, via small papillary tumors to full-blown adenocarcinomas, is reminiscent of that of some pulmonary adenocarcinomas in man. As has been found for the latter (Ten Have-Opbroek et al., 1997; Gazdar, 1994),

the lung tumors arising in the conditional K-RasV12 mice probably also arose from alveolar type II cells. We investigated this by immunostaining for Sp-B and C [surfactant apoproteins B and C], which are both markers for alveolar type II cells (Bohinski et al., 1993; Whitsett et al., 1995; Khoor et al., 1994). As shown in Figure 3g, h, tumors were positive for both Sp-B and Sp-C, attesting to the alveolar type II cell origin of the tumors. To demonstrate that the lung tumorigenesis requires activation of the conditional K-RasV12 allele, we performed a Northern analysis on total RNA from AdCre treated, tumor prone conditional K-RasV12 mice and controls. In all lung tumors we could detect a KRasV12 mRNA transcript of 5.2 kB, corresponding to the K-RasV12-IRES-PLAP transcript (Figure 4a). No transcript was detected in control normal lung tissue. The inactive conditional K-RasV12 allele showed the GFP containing transcript in all tissues tested (Figure 4b), even in the lung tumor samples, in accordance with the presence of unrecombined wild-type, nonneoplastic cells in the tumor mass. These results indicate that lung tumors arose exclusively after AdCre mediated activation of the K-RasV12 allele. Since we already showed that AdCre infection occurred sporadically throughout the bronchopulmonary epithelium (Figure 2), the tumors must have arisen from sporadically switched cells. We then asked, whether overexpression of K-RasV12 is sucient for the formation of a patch of mutant cells or whether additional mutations were required for their development. In the latter situation the `®eld' of cells carrying the activated K-RasV12 should extend outside the con®nes of the microscopic lesions. This permitted us to determine whether `®eld cancerization' (Sozzi et al., 1995; Park et al., 2000; Garcia et al., 1999) does play a role in this sporadic model. One might argue that in regular transgenic models `®eld cancerization' is inherent to the system, since most if not all cells are predisposed by transgene expression or tumor suppressor gene inactivation. This point has often been raised as an argument against the suitability of transgenic mice as a model for sporadic cancer. A `®eld' of preneoplastic cells could obviously serve as a basis for tumor progression, as any additional mutation might eectively synergize with the lesion present. In

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Table 1 Tumor development in conditional K-Rasv12 mice after AdCre administration Time between adenoCre administration and sacrifice (weeks)

p.f.u. of virus particles administered

6 5 5

2 3±4 5±6

107 107 46107

6

8

107746107

6

9 ± 13

106 ± 107

No. of mice*

Histopathology lungs (No. of mice with described pathology/No. of mice analysed) Only reactive infiltrate (6/6) Epithelial focal neoplasia (4/5) Multifocal glandular neoplasia/adenomatous tumors (5/5) Epithelial focal neoplasia (2/5) Papillary neoplastic lesions (6/6) Epithelial focal neoplasia (0/6) Mixed multifocal papillary neoplasias and papillary carcinomas (6/6) Lymph node metastases (3/6), kidney metastasis (1/6)

*Median age of mice at moment of AdenoCre administration is 9 weeks (range 8 ± 12 weeks) Oncogene


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Figure 4 Conditional K-RasV12 transgene expression in tumor tissue. (a) Northern blot analysis of K-RasV12 expression on total RNA from control FVB, various organs from untreated conditional K-RasV12 transgenic mice and lung tumor tissues from four independent mice treated with AdCre. Lung tumor tissues were isolated 8 weeks after AdCre administration. A 5.2 kb mRNA corresponding to the predicted transgenic KRasV12 transcript was only detected in the lung tumor lanes. (b) Identical to panel A, the same samples and amount of total RNA was hybridized with a GFP probe. A 1.5 kb GFP mRNA was detected in all tissues from the conditional K-RasV12 transgenic mouse. The transgene expression level varies between each tissue and even some GFP expression was found in the tumor RNA samples. (c) Control hybridization with an actin probe shows the relative amount of total RNA loaded in each lane

Figure 3 Histopathological analysis of lung tumor development in activated conditional K-RasV12 transgenic mice. After intratracheal AdCre administration, mice were sacri®ced at various time points. (a) After 5 weeks, multiple foci of developing alveolar adenomas were found throughout normal lung parenchyma (H&E, power®eld 206). (b) Mild dysplastic lesions along the alveolar lining were found after 3 weeks (upper and lower arrowheads, respectively; H&E, power®eld 1006). No lesions were found at earlier time-points (c) Alveolar dysplastic lesion started to form papillary structures after 5 weeks (H&E, power®eld 2006). (d) More progressive, papillary adenocarcinoma-like lesions were found at 8 weeks post-infection. (H&E, power®eld 1006). (e). Immuno-staining of papillary adenocarcinoma nodule for TTF-1 (9 weeks post-infection, power®eld 1006). (f) Positive staining for BrdU (black stain) in a large adenoma nodule (6 weeks post-infection, power®eld 1006). (g) Staining for surfactant B (power®eld 4006) and (h) surfactant C (power®eld 1006) in cytoplasm of adenocarcinoma nodules (8 weeks post-infection)

addition, the `®eld' of mutant cells might also have dierent characteristics as it might create a microOncogene

environment in which cell ± cell contacts and paracrine eects promote cell proliferation or transformation, as has also been observed in vitro (Land et al., 1986). We performed laser dissection and PCR analysis on the early dysplastic lesions or AAH, and on the histologically normal, surrounding tissue (Figure 5a). We detected the 240 bp PCR fragment characteristic for the mutant K-RasV12 allele in AAH lesions and latestage adenocarcinomas (Figure 5b), but not in surrounding tissue. This surrounding tissue only showed the 480 bp GFP fragment corresponding to the unrecombined conditional allele (Figure 5a, b). No GFP fragment was seen in AAH or adenocarcinoma lesions, suggesting that all neoplastic cells harbored the activated K-RasV12 alleles. These results indicate that activating K-RasV12 itself is sucient for the formation of the early lesions such as AAH, which then subsequently develop into papillary adenocarcinomas. Discussion Conventional H-Ras transgenic lung tumor models have previously underlined the importance of Ras expression for the development of some lung tumors (Saitoh et al., 1990; Maronpot et al., 1991; Tuveson and Jacks, 1999). We report here that sporadic expression of a K-RasV12 transgene is already enough for the rapid onset of lung tumorigenesis. One might


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Figure 5 Analysis of conditional K-RasV12 allele recombination in neoplastic and surrounding tissue. PCR analysis was performed on genomic DNA derived from laser dissected neoplastic lesions or normal cells directly surrounding these lesions. (a) Laser dissected cells from four independent AAH lesions and their respective Sur.Tis. [surrounding tissue] cells were used for PCR analysis either of the Ras Rec. [recombined K-RasV12 allele] or GFP [unrecombined allele, still containing the GFP fragment]. A 240 bp PCR product representing the recombined conditional K-RasV12 allele (primers AG2 and Ras-5-ANTI, depicted in Figure 1) was only found in AAH cells, not in the surrounding cells. The unrecombined allele, however, represented by a 480 bp fragment (primers GFP-5 and GFP-3) was only present in surrounding cells and not in AAH cells. (b) Similar experiment performed as in (a) but now laser dissected cells from four independent Ad.Car. [adenocarcinomas] and cells from their respective surrounding normal tissue were used. Identical to AAH cells, the 240 bp PCR product for a recombined allele, is only present in Ad.Car. cells and the 480 bp PCR product for the unrecombined allele can only be detected in surrounding tissue

argue that in this model the K-RasV12 transgene is expressed at elevated levels and therefore the K-RasV12 activation or `switch' represents in fact two events that usually will take place sequentially in normal tumor development: mutation and upregulation or deletion of the wt allele. This could explain the absence of `®eld cancerization' as an intermediate step and the rapid development of full-¯edged adenocarcinomas (9 ± 13 weeks post-infection) in our model. Also, the susceptibility of FVB/N mice to adenocarcinoma formation could play a role in the rapid development of these tumors and serve itself as a mechanism similar to that of ®eld cancerization (Shimkin and Stoner, 1975; Mahler et al., 1996). However, a recent new model for sporadic mutations in the endogenous K-Ras allele shows lesions very reminiscent to ours (Johnson et al., 2001). Therefore, it is more likely that sporadic activation of K-Ras in alveolar type II cells in mice is sucient to initiate this process. Interestingly, the susceptibility is not restricted to any particular developmental window as used in lung tumor induction with carcinogens (Oomen et al., 1991; Fijneman et al., 1995). Activation of the K-RasV12 allele in adult mice leads to the synchronous development of multiple tumor lesions that progress in time. It is likely that additional mutations did occur in the transition to latestage adenocarcinomas. The nature of these acquired mutations will be investigated in further studies. Another interesting observation in our model is the preferential occurrence of parenchymatous, pneumo-

cyte-type adenocarcinomas, and the absence of bronchial-type adenocarcinomas, although bronchial epithelial cells were eectively infected with AdCre (Figure 2). Probably the cellular context in which the K-RasV12 expression arises is important. This suggests that alveolar type II cells are more sensitive to K-Ras mutations than broncho-epithelial cells, or are more prone to undergo mutant ras-driven neoplastic transformation. This is in line with the observation that no K-Ras mutations were found in bronchial adenocarcinomas in another study (Cooper et al., 1997). Similarly, K-Ras mutations are distinctly uncommon in human pulmonary squamous cell carcinomas (Slebos et al., 1990), and likewise, in our model we did not encounter squamous cell carcinomas, thus further enhancing the resemblance of the murine model to the human situation. Although the lung tumors described here resemble those found in the previous H-Ras transgenic lung tumor models (Saitoh et al., 1990; Maronpot et al., 1991; Tuveson and Jacks, 1999), our model has important advantages over classical transgenic or knockout models. The moment of tumor initiation can be chosen, the frequency in which de®ned lesions are induced can be altered, and multiple lesions can be induced at the same time. This not only permits establishment of clear genotype-phenotype relationships, but also enables longitudinal monitoring of the tumorigenic process. The model is also particularly suited to test prevention and intervention strategies as oncogenic activation events can be limited in number Oncogene


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and restricted to a small time interval, comparable to that of carcinogen exposure, except that now a de®ned lesion is produced with distinct oncogenic consequences. One word of caution should be added. Recently, it has been shown that Cre activity can lead to chromosomal aberrations in cells in vitro (Loonstra et al., 2001) as well as in vivo (Schmidt et al., 2000). We cannot exclude that temporal Cre expression has served as a co-factor in the onset of tumorigenesis. However, in the elegant study of a sporadic model for K-Ras induced mutations, cited above, tumors very similar to those described here were observed (Johnson et al., 2001), arguing against a major autonomous role of Cre in tumorigenesis in this setting. The Cre/lox-K-RasV12 model should be particularly suited to test intervention protocols directed against the K-Ras pathway. In this respect it will be worthwhile to re-evaluate the ecacy of previously tested farnesyl-transferase inhibitors and other drugs directed against downstream targets of KRas (Omer et al., 2000; Gibbs, 2000) in this more critical model. To further improve the utility of this model for this particular application we are currently combining this sporadic lung tumor model with sensitive in vivo imaging using (Contag et al., 2000) a ¯oxed luciferase expression cassette. This will permit non-invasive quantitative assessment of tumor growth, metastasis, and regression.

Materials and methods Generation of conditional transgenic K-RasV12 mice A 1.2 kb human cDNA fragment encoding K-RasV12 was placed under the control of the 1.8 kb chicken b-actin promoter (Akagi et al., 1997). In between the promoter and K-RasV12 a 1.3 kb GFP [Green Fluorescent Protein] cassette (Clonetech) with a poly-A linker was placed, ¯anked by two loxP sites. This GFP cassette served as a marker for b-actin promoter activity. Behind the K-RasV12 a 2.9 kb PLAP [human placenta-like alkaline phosphatase] was placed as a reporter of K-RasV12 expression. The construct, ¯anked by SalI sites, was puri®ed by agarose gel electrophoresis and electroelution, and injected into fertilized FVB/N mouse oocytes. Screening of transgenic mice Transgenic conditional K-RasV12 founder mice were identi®ed by Southern blot analysis. Genomic DNA was extracted from tails according to standard protocols, digested with EcoRV and hybridized with a 500 bp GFP probe or 1 kb PLAP probe. Genotyping of ospring was performed by PCR on tail tip DNA, that was ampli®ed with the primers RAS-5' (5'CAGTGCAATGAGGGCCAG-3') and RAS-3' (5'-CACCCTGTCTTGTCTTTGCTG-3'), yielding a 280 bp product. Thermocycling conditions consisted of 30 cycles of 30 s at 948C, 30 s at 598C, and 50 s at 728C. The PCR mix consisted of 16PCR buer (Gibco), 200 mM dNTPs, 2.5 mM MgCl2, 0.4 mM primers, 0.5 units of Taq polymerase and 1 ml of a dilution of extracted tail DNA, in a 25 ml-volume. ACZL and R26R reporter mice were screened by PCR using the same Oncogene

conditions as described above, only the annealing temperature was changed into 608C. The primers used for ampli®cation were LZ1 (5'-CGTCACACTACGTCTGAACG-3') and LZ2 (5'-CGACCAGATGATCACACTCG-3'), for both reporter mice. Analysis of transgene transcription Expression of the transgene was analysed by Northern blotting. Total RNA was prepared from snap-frozen tissue using Trizol isolation reagent (Gibco). Northern blotting was performed according to standard protocols using 10 ± 20 mg of total RNA. The probes used were a 480 bp GFP fragment and a 280 bp K-Ras fragment. PCR analysis of recombination Microdissection of tumors and adjacent normal lung tissue was performed using a laser capture microdissection system (Arcturus). Ten-micrometer thick sections from formalin ®xed paran-embedded material were used after dewaxing in xylene and drying. The dissected material was transferred to 50 ml of proteinase K buer and incubated at 378C overnight, after which the proteinase K was inactivated for 10 min at 968C. Five microliters were used for subsequent PCR ampli®cation using primers AG2 (5'-CTGCTAACCATGTTCATGCC-3') and Ras 5-anti (5'-CCTACGCCACAAGCTCCAACTAC-3') or GFP-5 (5'-GACCACATGAAGCAGCACGAC-3') and GFP-3 (5'-CGAACTCCAGCAGGACCATG-3'). The recombined Ras allele yielded a 240 bp product while the unrecombined, GFP containing, allele yielded a 480 bp product. The PCR cycling pro®le consisted of 35 cycles of 30 s at 948C, 30 s at 598C, and 50 s at 728C. The PCR mix consisted of 16PCR buer (Gibco), 100 mM dNTPs, 2.5 mM MgCl2, 0.5 mM primers, 2.5 units of Taq polymerase in a 25 ml-volume. Immunohistochemistry BrdU [bromodeoxyuridine] 100 mg/g was administered intraperitoneally to selected mice, 2 ± 3 h before they were sacri®ced. Mice were sacri®ced via CO2 inhalation. Paraformaldehyde 4% in PBS was instilled through the trachea. Lungs were excised, ®xed in formalin and embedded in paran. Four mm thick slides were stained for BrdU (monoclonal, 470 mg/ml; DAKO), Cre (polyclonal, 1 : 15 000; Novagen), pro-Sp-B (polyclonal, 1 : 1000; Chemicon International), pro-SP-C (polyclonal, 1 : 500; Chemicon International) and TTF-1 (DAKO clone 8G7G3/1, 1 : 40 000) Antigen retrieval with microwave heating in citric acid at pH=6 was performed for BrdU, Cre and TTF-1. As secondary antibodies biotinylated goat anti-mouse (1 : 600; DAKO), biotinylated goat anti-rabbit (1 : 800; DAKO) or horseradish peroxidase labeled goat anti-rabbit (1 : 50; DAKO) were used. Visualization was achieved using biotin/ avidin-peroxidase (Vector) (BrdU, pro-Sp-B and pro-Sp-C) and diaminobenzidine as chromogen. Staining for b-galactosidase activity in ACZL reporter mice was performed according to Akagi et al. (1997). Intratracheal Adeno-Cre virus administration Adeno-Cre virus was constructed and produced according to Akagi et al. (1997). Two to 4 month old mice were treated with 9.6 mg/day co-trimoxazole orally (480 mg co-trimoxazole in 250 ml drinking water, presumed intake 5 ml/day), 1 week prior to virus administration. Co-trimoxazole was


administered to reduce perioperative mortality due to infections. One day before AdCre administration mice received 15 mg/kg cyclosporin A intraperitoneally. From the day of AdCre administration until 4 ± 6 weeks later, mice were administered cyclosporin A orally, 20 mg/kg/day. Cyclosporin A from mouse plasma samples was measured using a ¯uorescence polarization immuno assay (Chan et al., 1992). Mice were anesthetized with a mixture of 200 mg tribromoethanol and 100 ml amyl alcohol in 7.8 ml normal saline, ®ltered through a 0.22 mm ®lter, and brought to a temperature of 378C. The trachea was reached through a cranio-caudal incision in the neck. Ten ml containing 106 to 107 pfu [plaque-forming units] were injected intratracheally. Subsequently the incision was sutured.

Acknowledgments We thank Loes Rijswijk and Fina van de Ahe for expert assistance with the microsurgical procedures, Nell Bosnie for supervising the daily care of the mice, Jurjen Bulthuis for preparation of the histological slides, Paul Krimpenfort for injecting fertilized oocytes and Olaf van Tellingen for determination of cyclosporin A blood levels. This work was supported by a grant of the Dutch Cancer Society. SC Linn was a recipient of a KWF fellowship.

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