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发表于 2009-4-21 13:32 |只看该作者 |倒序浏览 |打印

作者：AndréSchneider, YahuaZhang, YoufeiGuan, Linda S.Davis,  Matthew D.Breyer,作者单位：1 Universitätsklinikum Eppendorf, 20246 Hamburg, Germany; Division of Nephrology,Department of Medicine, Veterans Affairs Medical Center, and Department of Molecular Physiology and Biophysics,Vanderbilt University, Nashville, Tennessee 37212-2372



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      【摘要】4 m2 |6 y7 X/ X! l
   The Cre/loxP transgenic system may be used to achieve temporallyand/or spatially regulated gene deletion. The Mx1Cre mouse expresses Cre recombinase under control of the IFN-inducible Mx1 promoter. Mx1Cre mice were crossed with areporter strain (ROSA26tm1Sor) in which -galactosidase activity isexpressed only after Cre -mediated recombination to determinethe cellular pattern of Cre -mediated genetic recombinationin the kidney and other tissues. Widespread recombination was observedin vascular endothelium as well as in the liver and spleen.Recombination was restricted to subsets of stromal cells in uterus,duodenum, colon, aorta, and kidney. In the cortex, -galactosidaseactivity was detected in a subset of tubules and all glomerular cells,including endothelium, mesangium, and podocytes. No -galactosidaseactivity was detected in proximal tubules. Costaining of kidneys withsegment-specific markers demonstrated induction of -galactosidaseactivity in collecting duct, with sporadic labeling of the thickascending limb but no significant labeling of distal convolutedtubules. We conclude that Mx1 -driven gene recombination isspatially as well as temporally restricted. The Mx1Cre transgene should prove a useful reagent to achieve temporally regulatedrecombination in endothelial, glomerular, and distal renal epithelia in mice.
      【关键词】 Cre recombinase liver spleen interstitium collecting duct glomerulus) o4 S) s! O" |! |, m
   INTRODUCTION5 j( e# k" o! A8 R0 P6 ^4 S; h
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GENE-TARGETING STRATEGIES have helped to elucidate the roles of several genes indevelopmental biology. However, the use of traditional knockout animalsdoes not allow examination of the physiological role of a gene when itsdeletion leads to intrauterine or perinatal death. Furthermore,identification of specific roles for a gene in particular tissues orcell types may be obscured, because widespread deletion of the gene inmultiple cell types or tissues could equally account for a particularphenotype. Conditional gene-targeting systems have been developed toaddress these deficiencies. The Cre/loxP system allowsspatially or temporally regulated gene targeting mediated by twoseparate transgenes: loxP sites and Cre recombinase( 10, 18, 19, 21 ). LoxP sites comprise aspecific 34-bp DNA sequence that is engineered to flank the gene to bedeleted (i.e., a "floxed" allele). LoxP sequencesprovide recognition sites for the second component, Cre recombinase, a 343-amino acid protein from the bacteriophage P1 that binds to the loxP sites and mediates excision of the interveningnucleotide sequence ( 15, 19 ). By placing Cre recombinaseexpression under the control of an inducible or tissue-specificpromoter, temporal or spatial control of genetic deletion can beachieved ( 10, 18, 19, 21 ).

The Mx1 Cre transgenic mouse expresses a modified Crerecombinase under control of the inducible Mx1 promoter( 10 ), part of the viral defense system that is normallysilent in healthy mice ( 1, 7 ). Mx1 geneexpression is potently induced by IFN or intraperitoneal injection ofthe IFN inducer polyinosinic-polycytidylic acid (pI-pC), a syntheticdouble-stranded RNA. Previous studies of Mx1Cre micereported tissue-dependent efficiency of Mx1Cre -mediated recombination of a floxed DNA polymerase allele as determined bySouthern blot ( 10 ). After induction with pI-pC, nearly100% recombination was found in liver, spleen DNA, and hematopoetic cells, but only 50% recombination was detected in kidney, heart, andlung DNA by using Southern blot hybridization ( 3, 10, 14 ).The basis for this difference is poorly understood, and thecell-specific pattern of recombination in kidney and other tissues wasnot determined.
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The purpose of the present study was to determine whether incomplete Cre -mediated recombination observed in whole kidney DNA results from global but inefficient recombination throughout the kidneyor from a highly efficient recombination restricted to certain celltypes of the kidney. The latter finding would allow the Mx1Cre transgenic mouse to be used as a system for inducible gene targeting in specific regions or cell types within the kidney.

MATERIALS AND METHODS
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Chemical reagents and transgenic animals. pI-pC and other reagents were purchased from Sigma (St. Louis, MO)unless stated otherwise. C57Bl/6 mice were purchased from HarlanSprague-Dawley (Indianapolis, IN). Transgenic mice carrying Cre under control of the IFN-inducible Mx1 promoter( Mx1Cre mice) were generously provided by Dr. Joachim Herz(University of Texas, Health Science Center). B6,129-GtRosa26 tm1Sor mice (no. 3309, Jackson Laboratory, BarHarbor, ME) were used as a reporter strain. The LacZ genehas been inserted into the ROSA26 locus. The Rosa26 promoter is auniversally expressed gene ( 17 ), but in this strain itstranscription is suppressed by placement of an upstreampolyA containing a "stop" sequence flanked by loxP sequences (i.e., loxP /polyA / loxP-LacZ ). When crossedwith a Cre transgenic strain, genetic recombination occurs at the two LoxP sites, excising the intervening stop sequence, therebyallowing LacZ to be expressed. LacZ expression can therefore be utilized to localize sites of Cre recombinase activity( 17, 22 ). Staining for -galactosidase tissues of Mx1Cre × B6, 129-Gtrosa26 tm1Sor transgenicmice thereby reveals the cellular distribution of the Cre -mediated recombination. Animals were housed understandard conditions and were fed a regular rodent chow containing 6鷗 (wt/wt; TEKLAD Premier Laboratory Diets, Madison, WI).

Induction of Mx1Cre expression. Mice were injected intraperitoneally with 250 µl of 1 mg/ml pI-pC RNAor 250 µl saline every other day for a total of three injections.
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Preparation of tissue and immunohistochemistry. Multiple organs, including liver, spleen, stomach, duodenum,lung, and kidneys, of the double transgenic mice were harvested atdeath 3 days after the final pI-pC injection. After fixation with 4%paraformaldehyde 0.25% glutaraldehyde in PBS for 2 h at 4°C,tissue sections were cut with a vibratome into 200-µm slices. Todetect -galactosidase activity, these slices were bathed twice inpermeabilization solution (2 mM MgCl 2, 0.01% sodiumdeoxycholate, and 0.02% Nonidet P-40 in PBS) for 30 min and thenstained with 1 mg/ml5-bromo-4-chloro-3-indolyl- D -galactopyranoside( -galactosidase; Sigma) in staining solution (2 mMMgCl 2, 5 mM potassium ferricyanide, potassium ferrocyanide,and 20 mM Tris, 7.4 pH, in PBS) at room temperature in the dark for48 h ( 2, 13 ). Tissues were washed, dehydrated througha graded ethanol series, and embedded in paraffin by using standardprocedures. Serial 5-µm sections were cut and examined by lightmicroscopy. To define the -galactosidase-positive nephronsegments, costaining was performed with a goat anti-human Tamm-Horsfall antibody (1:2,500; Organon-Technika) that specifically recognizes medullary and cortical thick ascending limb (TAL), as wellas the early portion of the distal tubule ( 8, 23 ), or thelectin Dolicus biflorus (Vector Laboratories) thatspecifically recognizes mouse collecting duct( 11 ). A commerically available anti-aquaporin-2(AQP2) antibody was used to specifically identify collecting ductprincipal cells (AQP21-A, anti-rat AQP2 IgG no. 2, Alpha DiagnosticInternational, San Antonio, TX) ( 6 ). To define distalconvoluted tubule (DCT) segments, an anti-thiazide-sensitive NaClcotransporter (TSC) antibody was used (generously provided by Dr. MarkKnepper, National Institutes of Health, Bethesda, MD) ( 9 ).Staining was localized by using biotinylated D. biflorus (1:250), or a biotinylated anti-IgG secondary antibody was applied to -galactosidase-stained sections. Biotin was identified by usingstreptavidin coupled to horseradish peroxidase and was visualized withdiaminiobenzidine (Vectastain ABC kit, Vector Laboratories). Sectionswere viewed and imaged with a Zeiss Axioskop and spot-cam digitalcamera (Diagnostic Instruments).
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Genotyping for Cre and LacZ-ROSA by PCR. Transmission of Mx1Cre and lacZ genes to theintercrossed offspring of the two transgenic strains was determined byPCR of genomic DNA isolated from mouse tail segments by using a DNAisolation kit (Qiagen). A 370-bp Cre -specific fragment wasamplified with primers ACCTGAAGATGTTCGCGATTATCT andACCGTCAGTACGTGAGATATCTT. An 822-bp LacZ -specific fragmentwas amplified with primers GCATCGAGCTGGGTAATAAGGGTTGGCAAT andGACACCAGACCAACTGGTAATGGTAGCGAC. Internal control primers to amplify a 150-bp fragment in all genomic DNA samples wereCAAATGTTGCTTGTCTGGTG and GTCAGTCGAGTGCACAGTTT. Amplification wasperformed for all fragments by using 40 cycles with 1 min at 94°C,30 s at 60°C, and 30 s at 72°C. Genotyping results weredetermined from PCR reactions run on 1% agarose gels.

RESULTS

Transgenic mice carrying both the Mx1Cre transgene andthe LoxP -stop- LoxP -regulatedROSA26 tm1Sor transgene were obtained by intercrossingC57/B6 Mx1Cre mice with B6, 129-Gt ROSA 26 tm1Sor mice. Transmission of both transgenes was confirmed by PCR analysis using Cre - and lacZ -specific primers. Mx1Cre expression was induced by interperitoneally injectingpI-pC three times into at least five individual, 8-wk-old doubletransgenic Mx1Cre ×Rosa26/ lacZ animals foreach tissue studied. Saline injections were also performed inthree double-transgenic animals to determine the level of background staining and to serve as a control for spontaneous noninduced Cre -mediated recombination. Three days after the thirdinjection of pI-pC, animals were killed and organs were harvested.

Saline injection did not induce -galactosidase activity (Figs. 1 and 2, bluestaining)in any tissue studied including liver spleen and kidneys. Incontrast, pI-pC injection induced intense -galactosidase activity,particularly in liver and spleen (Fig. 1 ), in accordance withpreviously reported results using Southern blot ( 3, 10, 14 ). -Galactosidase activity was also widely present inpulmonary endothelium, cerebral endothelium (Fig. 1 ), and theepithelium of the choroid plexus in the brain (not shown). Cre -mediated recombination was also observed insubpopulations of stroma and smooth muscle in aorta and uterus but wascompletely absent in cardiac muscle (not shown).

Fig. 1. The histological distribution of Cre -mediatedgene recombination. In all figures, the blue reaction product showsregions positive for gene recombination after induction of Crerecombinase [inducer polyinosinic-polycytidylic acid (pI-pC)injection] in -galactosidase reporter mice. Depicted are spleen(×200), liver (×200), and control liver (×50) showing regions ofhepatic -galactosidase activity in tissue from a noninduced mouse(i.e., saline injection rather than pI-pC). Also depicted are brain(×100) showing recombination restricted to endothelium and brainvessel (×400 area outlined in the left panel). Vascularendothelial labeling of vessel (shown in outlined topright corner at low magnification), lung (×100), aorta(×100), uterus (×100), and uterus (×400 from area outlined in top left corner) also show recombination primarily ininterstitium and endothelium, with little recombination in smoothmuscle, as shown in the ×400 photomicrograph in the bottomleft.
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Fig. 2. Top left : ×1 magnification photograph of amouse kidney vibratome section stained with -galactosidase. Note thepapilla is devoid of the bluish reaction product. Topmiddle : ×50 magnification photomicrograph of a microtome-cutsection of a -galactosidase stained kidney. Reaction product islimited to restricted segments in the cortex and outer medulla. Top right : kidney section from a mouse not preinjected withpI-pC shows absence of reaction product despite comparable staining. c,Cortex; om, outer medulla; p, papilla. Center left : ×50photomicrograph of a -galactosidase-stained (blue) kidney, costainedwith an antibody to the thick limb-specific Tamm-Horsfall protein(brown reaction product). Center middle : ×200photomicrograph of the same kidney showing -galactosidase stainingin the outer medulla predominates in Tamm-Horsfall-negative structures.Red arrow, blue staining vascular endothelium. Center right :kidney costained for the distal convoluted tubule marker (TSC)1.TSC-positive tubules do not exhibit recombination. Bottom :kidneys were stained for collecting duct-specific markers. Bottom left : staining with Dolicus biflourus (DB)shows variable intensity of blue reaction product in D. biflourus  tubules. Bottom middle and right :×630 photomicrographs showing that staining of cortical collectingcells was confirmed with a costain for aquaporin-2 (AQP2), which isspecific for collecting duct principal cells. Note, although someprinicipal cells exhibit gene recombination-positive (yellow arrow),other principal cells are negative (blue arrow). Prinicipal cells ofthe outer medulla exhibit a greater percentage of recombination thanthose of the cortex.

In the kidney, intense -galactosidase staining was presentthroughout the outer medulla, reflecting highly efficient Cre -mediated recombination in this region of the kidney(Fig. 2 ). Little staining was detected in the papilla. In the cortex,only sporadic -galactosidase activity was detected in the proximaltubule (Fig. 2 ). Cortical -galactosidase activity was apparent inglomeruli, microvascular endothelium, and collecting ducts (Fig. 2 ). Inthe glomerulus, -galactosidase activity was expressed in allcellular compartments, including the mesangium, and visceral epithelialcells (i.e., podocytes; Fig. 2 ) but was most intense in the endothelium.

Proximal tubules, identified by the presence of a brush-bordermembrane, showed no evidence of -galactosidase activity. Similarly, costaining with TSC antibody, a marker for DCT, showed no evidence of -galactosidase activity in the DCT. Colabeling with Tamm-Horsfall antibody, to identify TAL, revealed only sporadic -galactosidase labeling of the cortical TAL (Fig. 2, C and D )and more extensive, but still limited, labeling of the medullary thicklimb. In contrast, colabeling with collecting duct markers, includingthe lectin, D. biflorus, and AQP2, confirmed robust Cre -mediated recombination that was more efficient in theouter medulla (outer medullary collecting duct) than in the cortex(cortical collecting duct) (Fig. 2 ) and included both AQP2-positive andAQP2-negative cells.3 C9 ~7 U6 Z2 k$ @" g
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Finally, recombination in the gastrointestinal tract wascharacterized (Fig. 3 ). These studiesalso revealed segmental heterogeneity of epithelial recombination,demonstrating widespread induction of -galactosidase activity ingastric epithelium but only sporadic -galactosidase activity induodenum and colonic epithelium. In contrast, abundant -galactosidase activity was evident in the submucosal interstitiumand vascular endothelium in these regions of the aerodigestive tract.
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Fig. 3. Mx1 -driven, Cre -mediatedrecombination in the aerodigestive tract. PAS was used as acounterstain in colon and one of the duodenum sections. Topleft : widespread recombination in gastric epithelium (×100). Top middle : sporadic recombination in duodenal epithelium(×400). Top right : widespread recombination in the duodenalvasculature and intersititum (×200). Photomicrograph (×10) ofrecombination in the colon and ×200 photomicrograph of the samesection ( bottom left and right,respectively).
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DISCUSSION
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The generation of mosaic knockout mice with temporally regulatedor cell-specific promoters driving Cre recombinase expression providesa novel and direct approach to study gene function, allowing for theselective control of gene expression in kidney and other tissues. Thepresent studies defined the spatial pattern of genetic recombinationmediated by the temporally regulated Mx1Cre transgene. Induction of this transgene in adult mice (by injection with pI-pC) resulted in Cre -mediated recombination in a variety oforgans, including liver, spleen, kidney, lung, gastric epithelium, and endothelial cells in brain, uterus, and intestine. Mx1 induction of Cre -mediated recombination in the liver, asreflected by -galactosidase activity, was particularly efficient,approximating 100%. Because no -galactosidase activity was detectedin liver (or any other tissue) obtained from saline-injected controls,we conclude that baseline and endogenous activation of the Mx1 transgene is tightly regulated and normally insufficientto cause recombination in these tissues at any point in their development.5 V  l* s3 z+ J- I! Y" f, p/ k7 f

The Mx1 gene was originally discovered as a mouse influenzavirus resistance locus. Mx1 proteins are ubiquitous and abundant stablecytoplasmic proteins that can be detected for several days after IFNinduction in mice and in humans (where Mx1 gene homologs have been found) ( 1 ). Although a high level of Mx1 gene activity in cells of the immune system might beexpected, our studies additionally show that the Cre transgene, driven by a 2.3-kb fragment containing the Mx1 promoter, results in IFN-inducible gene expression in a diverse set ofcells outside of the classic immune system.& z: X- k: F8 c; o3 v; V

Mx1Cre -driven recombination was spatially restricted inseveral other organs, including spleen (where recombination wasinefficient in the follicles) and aorta (where recombination appears tobe restricted to stromal and smooth muscle cells). Interestingly, epithelial recombination appeared heterogenous in the gastrointestinal tract, where it was robust in stomach but essentially absent in duodenum and colon. Similarly, epithelial recombination in kidney washeterogenous and predominated in the collecting duct. Staining withAQP2 antibodies suggests variable efficiency of recombination in bothintercalated cells (AQP2 negative) and principal cells (AQP2 positive).No recombination was detected in DCT cells (TSC1 positive). Efficient Mx1 -driven, Cre -mediated recombination was alsoevident in glomeruli and renal endothelium.+ L- c  p. c0 K8 {9 K: J( C

Because the -galactosidase knocked into the endogenous ROSA26 allele(i.e., without the preceding floxed stop sequence) is widely andefficiently expressed in all tissues ( 22 ), we interpret the restricted recombination as reflecting differential induction ofthe Mx1 -driven Cre expression in the constituentcells. Although it is possible that these cells are exposed todifferent levels of local IFN production stimulated by pI-pC, thisseems unlikely because previous studies found similar recombinationefficiency whether pI-pC was used to induce endogenous IFN or exogenousIFN was administered ( 10 ). Rather, different levels of Cre expression, in these various cell types and tissues,appear more consistent with the available data. For instance, thepredominant recombination in the renal collecting duct corresponds witha recent report that endogenous Mx1 protein expression in humanspredominates in distal tubule and collecting ducts in human kidney( 5 ).7 C9 n1 F. y5 R7 J+ r7 {4 J

Although studies of mice with inducible Cre transgene havebeen published ( 10, 20 ), reports of the heterogeneity ofcells exhibiting inducible gene targeting within the discrete tissues has not previously been reported. Other groups have shown noninducible segment-specific gene targeting by using renal-specific promoters including -glutamyl transferase ( 16 ) or a kidneyandrogen-regulated protein (KAP) promoter, exclusively targetingproximal tubule cells ( 4 ). Use of the KAP promoter fusedto the human angiotensinogen gene confers constitutive expression inproximal tubule cells of male mice. Because the KAP promoter isandrogen regulated, Cre expression is only detected inkidneys of female mice after exogenous testosterone. Extrarenalexpression of the KAP-driven transgene appears to be limited toepidydimus in male mice ( 4 ). Conversely, the AQP2 promoterspecifically targets principal cells in the collecting duct( 12 ). No gender dependence of renal AQP2 transgeneexpression was reported; however, extrarenal expression was found inthe vas deferens and the testis in male mice ( 12 ). Genderdifferences in the production and action of the Mx1 gene have not been reported and would not be predicted because of similar response to viral infection in male and female mice. Furthermore, incontrast to these other transgenes, the Mx1Cre mouse allows for temporal control of gene disruption not only in collecting duct butalso in endothelium and glomeruli as well.# C, u' k, D+ ?( y, u& ]3 i2 C
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In summary, the present studies demonstrate that efficient temporallyregulated and spatially restricted gene targeting in the kidney can beachieved by using the Mx1Cre/loxP transgenes. This transgeneshould be particularly useful for deleting floxed alleles of geneshighly expressed in vascular endothelium, glomerulus, and collectingduct. In contrast, the Mx1Cre transgene does not appear tobe suitable for genetic targeting of cells in the proximal tubule.These transgenic mice should also be useful in achieving temporalcontrol of gene-mediated deletion in the spleen, liver, and gastric epithelium.

ACKNOWLEDGEMENTS

Support for this project was provided by the DeutscheForschungsgemeinschaft (DFG Schn 581/2-1; A. Schneider),an American Heart Association TN affiliate award (Y. Guan), NationalInstitute of Diabetes and Digestive and Kidney Diseases Grant DK-38226(M. D. Breyer), and a Veterans Administration Merit Award (M. D. Breyer). Infrastructural support was provided by National CancerInstitute award P30 CA68485.0 e# k8 z! f% {% L  I/ r/ r
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