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【关键词】 specific
Departments of Urology, Microbiology, Dermatology, and Pharmacology, New York University Cancer Institute, New York University School of Medicine, New York
Veterans Affairs Medical Center in Manhattan, New York, New York
Departments of Developmental and Cell Biology and Biological Chemistry, University of California, Irvine, California
ABSTRACT
Urothelium that lines almost the entire urinary tract acts as a permeability barrier and is involved in the pathogenesis of major urinary diseases, including urothelial carcinoma, urinary tract infection, and interstitial cystitis. However, investigation of urothelial biology and diseases has been hampered by the lack of tissue-specific approaches. To address this deficiency, we sought to develop a urothelium-specific knockout system using the Cre/loxP strategy. Transgenic mouse lines were generated in which a 3.6-kb mouse uroplakin II (UPII) promoter was used to drive the expression of Cre recombinase (Cre). Among the multiple tissues analyzed, Cre was found to be expressed exclusively in the urothelia of the transgenic mice. Crossing a UPII-Cre transgenic line with a ROSA26-LacZ reporter line, in which LacZ expression depends on Cre-mediated deletion of a floxed "stop" sequence, led to LacZ expression only in the urothelium. Gene recombination was also observed when the UPII-Cre line was crossed to an independent line in which a part of the p53 gene was flanked by the loxP sequences (floxed p53). Truncation of the p53 gene and mRNA was observed exclusively in the urothelia of double transgenic mice harboring both the UPII-Cre transgene and the floxed p53 allele. These results demonstrate for the first time the feasibility and potentially wide applicability of the UPII-Cre transgenic mice to inactivate any genes of interest in the urothelium.
urothelium; bladder; tissue-specific gene knockout; Cre recombinase; uroplakin; transgenic mice
MAMMALIAN UROTHELIUM COVERS the inner surfaces of bladder, ureter, renal pelvis, and proximal urethra and exhibits many interesting biological features. It acts as an extremely potent permeability barrier separating toxic urinary substances and metabolites from the blood. When subjected to transepithelial electrical resistance measurement, urothelium displays the highest value among all epithelia measured thus far (25, 50). Perhaps consistent with this functional requirement, urothelium is exceedingly stable, with a turnover rate of 6 mo (19, 43). However, this epithelium is by no means inert because it can reversibly adjust its surface area during each micturition cycle (26, 31); it synthesizes large numbers of highly specialized membrane plaques and delivers them to the apical surface (42); it has the remarkable capacity of postnatally trans-differentiating into squamous and glandular epithelia (4); it can bind and internalize type 1-fimbriated uropathogenic Escherichia coli (34, 48); it can give rise to multiple histologic types of neoplasms, including transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, and small cell carcinoma (23, 30); and it can even secrete proteins, contributing to urine composition (12). Urothelium is therefore a structurally, biologically, and physiologically unique epithelium.
Research into how urothelium performs its physiological functions and develops various diseases has been limited, due to the lack of tissue-specific approaches. Conventional gene knockout, where a given gene is inactivated in all organs, has yielded few insights into how the gene might function in urothelium. This is largely because gene inactivation in vital organs often leads to embryonic or premature animal deaths, thus precluding analysis of the potential urothelial involvement of the gene. For instance, tight junction is believed to play a critical role in maintaining urothelial impermeability, but mice lacking claudin, a key tight junction component, die paranatally because of the loss of epidermal barrier (14). As a result, it is impossible to assess the functional roles of claudin in urothelium. Similarly, because mouse embryos lacking the retinoblastoma (Rb) gene die 14 days into gestation (9, 21, 24), the critical role of this gene in urothelial growth and differentiation cannot be assessed. Another example is that mice lacking the p53 gene succumb to thymic lymphomas at 7 mo of age (13, 15, 16), making it difficult to analyze the long-term effects of the p53 loss on urothelial growth and tumorigenesis. There are clear advantages, therefore, to be able to inactivate genes of interest in a urothelium-specific manner.
Two recent advances have formed the foundation for us to explore the feasibility of urothelium-specific knockout. The first relates to the identification and characterization of a group of urothelium-specific markers called uroplakins (UPs) (28, 46, 47, 49). UPs Ia, Ib, II, and III are integral membrane protein components of asymmetric unit membrane, the hallmark of mammalian urothelia. With the exception of UPIb, which is also detected in corneal epithelium (1), all other UPs are highly urothelium specific, as evidenced by Northern blotting, RT-PCR, and immunohistochemistry (28, 33, 4547, 49). Such a striking tissue specificity prompted Lin et al. (28) to isolate the mouse uroplakin II (UPII) gene and test whether its 3.6-kb upstream sequence was able to drive the expression of bacterial LacZ reporter gene in transgenic mice. Indeed, LacZ expression was found primarily in the urothelium, indicating that the 3.6-kb sequence is sufficient to confer urothelium specificity (28). The second advance relates to the successful application of Cre/loxP strategy in transgenic mice (36). The bacteriophage P1-derived Cre recombinase (Cre) can specifically recognize a 34-bp partially palindromic target sequence (the loxP site) and excise any sequence between the two loxP sites that are arranged in the same orientation. By placing the loxP sites in separate introns, it is possible to delete loxP-flanked ("floxed") exons, thereby inactivating the target gene wherever Cre is expressed. By driving Cre expression under the control of the UPII promoter, we wish to know whether we can achieve urothelium-specific expression of Cre and, if so, whether urothelially expressed Cre is enzymatically active in being able to mediate the recombination of floxed DNA. If successful on both fronts, this knockout system can be a powerful approach to elucidate gene functions in urothelial biology and diseases.
MATERIALS AND METHODS
Construction of UPII-Cre chimeric gene. For transgenic expression of a Cre specifically in urothelium, a chemeric gene was constructed to contain, from 5' to 3', a 3.6-kb mouse UPII promoter, a 1.4-kb Cre gene, and a 238-bp SV40 poly(A) signal. Briefly, the SV40 poly(A) signal was excised from pEGFP-1 vector (Clontech) with NotI/AflII double digestion, blunt-ended, and cloned into the SmaI site of pBluescript II KS to yield pBS-poly(A). The Cre gene was excised from pHSG-Cre (American Type Tissue Culture) with PstI digestion and cloned into the PstI site, upstream of the poly(A) sequence, in pBS-poly(A). The Cre-poly(A) fragment was retrieved by EcoRI/SpeI double digestion and cloned into the same restriction sites, downstream of the mouse UPII promoter, in pBS-UPII (51, 52) to yield pBS-UPII-Cre-poly(A). The correct orientation of each fragment was verified by restriction digestions and DNA sequencing. The complete 5.2-kb chimeric gene fragment was excised en bloc by KpnI/SpeI double digestion, purified by agarose gel and column chromatography.
Generation and genotyping of UPII-Cre transgenic mice. The purified UPII-Cre-poly(A) chimeric gene was microinjected in the pronuclei of fertilized eggs from FVB/N inbred strains, as established previously (6). Microinjected eggs (201) were transferred in the uteri of 14 mouse foster mothers, of which 6 mothers gave birth to 29 pups. Genome incorporation of the transgene was assessed by Southern blot analysis, using genomic DNA extracted from tail biopsies as previously described (51). After NcoI digestion, the DNA fragments were resolved by agarose gel electrophoresis, transferred to a nylon membrane, and hybridized with a 450-bp DNA probe located at the 3'-end of the 3.6-kb mouse UPII promoter (Fig. 1). This probe allowed the detection of both the transgene fragment and the endogenous UPII gene fragment.
RT-PCR analysis of Cre expression. For assessing the urothelial expression of Cre, surgically dissected urinary bladders of nontransgenic and transgenic F1 mice were turned inside out with a pipette tip, and their urothelia were scraped off using a chemical spatula in diethyl pyrocarbonate-treated PBS. Total RNA was extracted from the urothelial cells using the TRIzol reagent (GIBCO-BRL) according to the manufacturer's instructions. Total RNA was also extracted from other mouse tissues to evaluate the tissue specificity of Cre expression. Before reverse transcription, the RNA samples were treated with DNA-free reagent (Ambion) to remove potentially contaminated genomic DNA. After treatment, the DNase I was inactivated by the addition to the reaction mixture of a DNase inactivation reagent (Ambion). Treated RNA (5 μg) was subjected to reverse transcription and synthesis of the second-strand DNA. The reverse-transcribed products () underwent PCR amplification using Cre-specific primers (forward: 5'-CGAACGCACTGATTTCGAC-3'; reverse 5'-GGCAACACCATTTTTTCTGAC-3'), under the following conditions: 94°C for 2 min, 62°C for 1 min, and 72°C for 2 min for a total of 35 cycles.
Testing of Cre enzymatic activity using ROSA26-LacZ reporter line. Cre activity in initiating gene recombination in the urothelium was first tested by intercrossing the UPII-Cre transgenic mice (line 6) with a ROSA26-LacZ reporter line. The ROSA26-LacZ line (The Jackson Laboratory) harbors a bacterial -galactosidase reporter gene, the expression of which requires Cre-mediated deletion of a floxed "stop" sequence separating the ROSA26 promoter and the -galactosidase gene (39). Thus the -galactosidase gene is only expressed where Cre is expressed and active. Offspring heterozygous for both the UPII-Cre transgene and the ROSA26-LacZ allele were identified by Southern blotting and genomic PCR, respectively. For genomic PCR, three oligonucleotide primers were used, which allowed the detection of a 500-bp endogenous product and a 250-bp ROSA26-LacZ product (R1295: 5'-GCGAAGAGTTTGTCCTCAACC-3'; R523: 5'-GGAGCGGGAGAAATGGATATG-3'; and R26F2: 5'-AAAGTCGCTCTGAGTTGTTAT-3'). PCR conditions were 94°C for 3 min for the first cycle; 94°C for 30 s, 56°C for 30 s, and 68°C for 1 min for 35 cycles; and 68°C for 5 min for the last cycle.
-Galactosidase activity was assessed on frozen sections of mouse bladders prefixed in 4% paraformaldehyde and 0.5% glutaraldehyde in PBS (pH 7.4). Sections (15 μm thick) were incubated at 37°C for 46 h with a solution containing 0.1% X-gal (5-bromo-4-chloro-3-indolyl -D-galactopyranoside), 0.2 mM potassium ferricyanide, 0.2 mM potassium ferrocyanide, 1.3 mM MgCl2, 15 mM NaCl, and 44 mM HEPES (pH 7.4). After being washed, the sections were counterstained for 1 min in 1% neutral red and mounted with an aqueous mounting medium.
Generation and genotyping of UPII-Cre/floxed p53 double-transgenic mice. For additional evaluation of the activity of urothelially expressed Cre, the UPII-Cre transgenic mice (line 6) were crossed with floxed p53 transgenic mice. Floxed p53 mice were generated by inserting the two loxP sequences in introns 4 and 6 of the p53 gene, using the homologous recombination approach (29). The neo selective gene was removed during the ES-cell stage via transient expression of Cre. Cre-mediated recombination of floxed p53 allele would delete exons 5 and 6, which together encode part of p53's DNA-binding domain (29). The first crossing involved homozygous UPII-Cre mice and homozygous floxed p53 mice, yielding heterozygotes for both genes. The second crossing among the heterozygous siblings yielded desired homozygotes for both genes, along with several other genotypes (see Fig. 4). Southern blotting was used for genotyping the UP-Cre transgene as described, whereas PCR was used to identify floxed p53 allele(s) using two oligonucleotide primers surrounding the second loxP site (forward primer: 5'-GCTGCAGGTCACCTGTAG-3'; reverse primer: 5'-CATGCAGGAGCTATTACACA-3'; see Fig. 4). PCR was carried out for 40 cycles under the following conditions: 94°C for 45 s, 53°C for 45 s, and 72°C for 45 s. The anticipated size of the PCR product for wild-type p53 allele was 119 and 158 bp for the floxed allele.
Detection of recombined p53 gene and mRNA in urothelium. For detection of the p53 gene deletion, genomic DNA was isolated from mouse urothelium and several other tissues of double-transgenic mice harboring both UPII-Cre and floxed p53 genes and was subjected to PCR using oligonucleotide primers located in exons 4 (5'-TGGGACAGCCAAGTCTGTTA-3') and 7 (5'-CATGCAGGAGCTATTACACA-3') of the p53 gene. PCR was performed under the following conditions: 94°C for 45 s, 55°C for 45 s, 72°C for 45 s for 40 cycles. Amplification of the wild-type and truncated p53 gene would give rise to a 1.5-kb and a 500-bp PCR product, respectively.
RT-PCR was also performed using total RNA extracted from mouse urothelia of various double- and single-transgenic mice to confirm the truncation of p53 mRNA. The primers were the same as the ones used for detecting p53 gene deletion, and the PCR conditions were as follows: 94°C for 20 s, 53°C for 20 s, and 72°C for 20 s for 40 cycles. The predicted sizes of the wild-type and truncated p53 products were 381 and 85 bp, respectively.
Immunohistochemical staining and in situ hybridization analysis of uroplakin. A pan-uroplakin antibody raised in rabbits (47) was used to assess uroplakin expression in human and mouse urothelia. Paraffin sections were routinely processed and subjected to antigen unmasking using microwave treatment at full power for 20 min in 0.01 M citrate buffer (pH 6.0). The sections were then incubated with the primary antibody (1:10,000 dilution) followed by a peroxidase-conjugated goat anti-rabbit secondary antibody and were routinely developed. The antibody-stained sections were counterstained with hematoxylin.
For in situ hybridization analysis, sense and antisense RNA probes for mouse UPII were prepared by in vitro transcribing of a partial cDNA clone of mouse UPII in the presence of digoxigenin-UTP (Roche). The labeled RNA probes were then incubated with paraffin sections of normal mouse bladders, and the hybridization was visualized by anti-digoxigenin immunohistochemical staining.
RESULTS AND DISCUSSION
Generation and characterization of UPII-Cre transgenic mice. To develop a Cre/loxP-based gene knockout system for urothelium, we constructed a transgene in which Cre expression was under the control of a 3.6-kb mouse UPII promoter (Fig. 1A), which we have demonstrated to be able to drive the urothelial expression of a bacterial LacZ reporter gene, a human growth hormone gene, and several oncogenes (8, 15, 22, 28, 51, 52). Microinjection of the transgene into the mouse embryos resulted in three founder mice harboring various copy numbers of the UPII-Cre transgene arranged in head-to-tail and head-to-head orientations (Fig. 1B). All founders transmitted their transgenes to the offspring in a Mendelian fashion. RT-PCR detected Cre expression in urothelia of transgenic F1 and F2 mice derived from the three founders, but not in nontransgenic siblings (Fig. 2A). Within a transgenic mouse, Cre expression was confined to the urothelium and was not detected in any other issues, as evidenced by RT-PCR analysis (Fig. 2B). These results established the urothelial specificity of Cre expression and set a stage for urothelium-specific knockout.
Functional activity of the urothelially expressed Cre. To evaluate whether urothelially expressed Cre was active, we first crossed the UPII-Cre transgenic mice (line 6) with a ROSA26-LacZ reporter line. The ROSA26-LacZ mouse line harbors a targeted gene locus that contains a bacterial LacZ reporter gene, the expression of which relies on the deletion of a loxP-flanked stop sequence that separates the ROSA promoter and the LacZ gene (39). Therefore, the LacZ gene is transcribed only where Cre is expressed and active. Bitransgenic mice were then generated that harbored both the UPII-Cre and the ROSA26-LacZ allele (Fig. 3B). -Galactosidase activity was detected in the urothelial cells (Fig. 3C, b-d), but not in nonurothelial tissues (data not shown) of the double-transgenic mice nor in the urothelial cells of the wild-type controls (Fig. 3Ca). This result indicated that the uroplakin promoter-driven Cre was capable of mediating gene recombination specifically in the urothelium. In the double-transgenic mice, -galactosidase could be detected in multiple urothelial layers (Fig. 3C, c and d), consistent with the early onset of uroplakin promoter activity in normal mouse urothelium (Fig. 3A, b and c).
To further establish the ability of urothelially expressed Cre to recombine any floxed genes of interest, we crossed the UPII-Cre transgenic mice (line 6) with floxed p53 mice in which exons 5 and 6 of the p53 gene had been flanked by two loxP sequences (Fig. 4A; see Ref. 29). After a second crossing, we generated double-transgenic mice homozygous for both UPII-Cre transgene and floxed p53 alleles (Fig. 4B) and assessed these mice for Cre-mediated DNA recombination. A truncated form of p53 PCR product (500 bp; instead of 1.5 kb for wild-type p53) was detected in urothelium, but not in any other tissues (Fig. 5, A and B), indicating that Cre is capable of excising the floxed p53 DNA sequence and that such a recombination event is urothelium specific. Consistent with the p53 gene recombination, a truncated p53 mRNA was detected by RT-PCR in the double transgenics harboring both the UPII-Cre transgene and the floxed p53, but not in wild-type mice or single transgenic mice harboring either gene (Fig. 5C). Of note, the truncation of the p53 mRNA in urothelium was incomplete, since a residual amount of full-length p53 mRNA could still be detected in the double transgenics (Fig. 5C, lanes 7 and 8). This may simply reflect the potential contamination of the urothelial preparations during bladder scraping by nonurothelial cells, the latter of which did not express Cre and therefore contained wild-type p53. Alternatively, the truncation of p53 within the urothelial cells may be heterogeneous, occurring only in some of the urothelial cells, a phenomenon that has been reported in other organ systems (35, 44). The truncation of p53 gene or other genes only in some cells may actually be advantageous because it mostly resembles gene inactivation in natural circumstances.
Unique features of UPII promoter-based gene knockout system. A key feature of the UPII-based Cre/loxP knockout system is the strict urothelium specificity. Not only is Cre expression urothelium specific, but also recombination of floxed DNA takes place exclusively in urothelium. Our system therefore confines gene inactivation to urothelium, thus avoiding potential complications resulting from gene deletion in other organs. Several recent studies reported gene expression in urothelia of transgenic mice using promoters of the K19 gene and the fatty acid-binding protein (FABP) gene (17, 35), but those promoters were not completely urothelium specific. For instance, it is well known that K19 gene is expressed in all simple epithelia, including liver, lung, and kidney (10, 32). Thus the phenomenon that a K19 promoter drove the SV40T to express mainly in urothelium (17) is likely a chromosomal insertion site-dependent event and is unlikely to be reproducible. In other words, it seems unlikely that this K19 promoter can be used again to express Cre specifically in urothelium. Similarly, FABP is normally expressed by hepatocytes and enterocytes (38). It is surprising, therefore, that transgenic mice bearing the FABP-Cre transgene express Cre not only in enterocytes but also in urothelium (35). It is unclear whether this ectopic Cre expression is because of promoter leakage or a chromosomal insertion-site effect. Supporting the second scenario is the fact that FABP-driven reverse tetracycline trans-activator expression is not found in the urothelium (35). More recently, Bex and colleagues (5) described a urothelium knockout method in which a fusion gene consisting of Cre and a mutant of human estrogen receptor is ubiquitously expressed under the control of the ROSA26 promoter. Cre expression in urothelium is achieved by intravesicular administration of tomoxifen, a ligand capable of binding to the estrogen receptor mutant. An advantage of this approach is obviously the time-controlled inducibility of Cre expression. The disadvantage is that it requires a survival surgery of each adult mouse to intravesicularly introduce tomoxifen-containing pellets (5). Moreover, because of cell renewal, gene deletion by this approach may be only temporary, since urothelial cells harboring the gene deletion will be replenished some time after the procedure by nascent cells without gene deletion, thus diminishing the chance for the long-term development of genotypic and phenotypic alterations in urothelium. Individual variation of Cre induction because of procedure variability and varied ligand accessibility is also possible. The reported, severe degree of urothelial necrosis caused by organic solvents of tomoxifen could further hasten urothelial renewal and complicate the interpretation of urothelial growth and tumorigenesis if a tumor suppressor gene is to be deleted. Although reintroduction of the ligand could somewhat alleviate the cell renewal problem, it requires additional surgeries, and the time frame of ligand reintroduction could be hard to define. Another shortcoming of this approach is that it does not allow analysis of the effects of gene deletion during early stages of mouse development. Taken together, it appears that each existing approach has pros and cons, and the specific objective of a given investigation will dictate the use of a particular approach.
Potential applications of the urothelium-specific knockout system. The establishment of a urothelium-specific knockout system affords new opportunities for a wide range of applications in studying urothelial biology, physiology, and diseases. First, the system can be used to explore the molecular basis of how urothelium reversibly adjusts its surface area during micturition. The superficial (umbrella) cells of the urothelium contain numerous fusiform vesicles that can insert into the apical membrane, thus expanding the surface area (2, 27, 31, 42). It has been hypothesized that Rab family proteins, some of which have recently been shown to be fusiform vesicle-associated, play crucial roles in vesicular transport and fusion (7, 40). It will therefore be extremely interesting to inactivate some of the Rab proteins in urothelium and to determine whether their loss will impair vesicular transport, hence surface area expansion. Second, the system will help elucidate the molecular basis of the permeability barrier function of urothelium and the pathological consequence of the loss of this function. For example, tight junctions, together with UPs, are believed to play a key role in urothelial impermeability (20, 25, 42, 50). Would urothelial inactivation of key tight junction components lead to a leaky urothelium and inflammation in the bladder wall Third, the system will be useful for studying the urothelial response to E. coli infections. Upon E. coli attachment, urothelium releases large amounts of cytokines, a process believed to be mediated by lipopolysaccharide receptor Toll-like receptors present on the urothelial cells (3, 37). Generation of knockout mice deficient in some of these toll-like receptors in urothelium will help define the detailed mechanisms underlying innate host immunity. Fourth, the system will provide invaluable tools for studying urothelial tumorigenesis. Several tumor suppressor genes, including pRb and p53, are mutated and/or dysfunctional in human bladder cancer (11, 15, 18, 41), but the causative relationship between their defects and bladder tumorigenesis and progression remains obscure. It is now possible to inactivate these genes singly or in a combination in urothelium, thus alleviating the premature animal deaths caused by the whole body knockout and allowing analysis of the long-term effects of gene inactivation. Finally, any novel genes identified through differential displays, subtraction libraries, and cDNA microarrays can be assessed for their in vivo roles in urothelial biology and diseases.
GRANTS
This work was supported, in part, by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-522206 (to Xue-Ru Wu and T.-T. Sun), DK-56903 (to X.-R. Wu), and DK-39753 (to T.-T. Sun) and by a Merit Review Award from the United States Veterans Affairs Administration (to X.-R. Wu).
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.
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