Preparation of enzymatically active human Myc-tagged-NCre recombinase exhibiting immunoreactivity...

MOLECULAR REPRODUCTION AND DEVELOPMENT 73:1345–1352 (2006)

Preparation of Enzymatically Active HumanMyc-Tagged-NCre Recombinase ExhibitingImmunoreactivity With Anti-Myc AntibodySATOSHI WATANABE,1* DAISUKE HONMA,1 TADASHI FURUSAWA,1

TAKAYUKI SAKURAI,2 AND MASAHIRO SATO2

1Department of Developmental Biology, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan2Division of Basic Molecular Science and Molecular Medicine, School of Medicine, Tokai University,Bohseidai, Isehara, Kanagawa, Japan

ABSTRACT The Cre-loxP system has beenrecognized as a tool for conditional gene targetingin mice. However, most anti-Cre antibodies fail toreact with Cre expressed in vivo. In an attempt todirectly detect Cre by antibodies in vivo, we con-structed the tagged-NCre (NCreMH) gene by connect-ing the humanMyc andHis tag sequences to the 30 endof the NCre gene carrying a nuclear localizing signal(NLS) sequence. The production of NCre protein andthe recombinase activity were detected after co-transfection with pCMV-NCreMH and pCETZ-17carrying the loxP-flanked lacZ gene into NIH3T3 cells.This activity was also confirmed in vivo after genetransfer of pCMV-NCreMH and pCRTEIL-6 carryingloxP-flanked HcRed1 and EGFP cDNAs, into oviductalepithelium by electroporation. Immunohistochemicalstaining using anti-Myc antibody demonstrated thatthe area positive for enhanced green fluorescentprotein (EGFP) fluorescence was immunostained withthe antibody. These findings indicate that NCreMH isuseful as an alternative to NCre for gene targeting.Mol. Reprod. Dev. 73: 1345–1352, 2006.� 2006 Wiley-Liss, Inc.

Key Words: Cre-loxP; immunostaining; mamma-lian cells; Myc tag; recombination; transgene expres-sion

INTRODUCTION

The Cre-loxP system is a DNA recombination systemderived from Escherichia coli P1 phage. In this system,Cre DNA recombinase recognizes specifically the loxP,34-bp nucleotide sequence, and catalyzes the recombi-nation of a sequence flanked by the loxP sites (Hoesset al., 1984). This Cre-loxP system is active not only inprokaryotic cells, but also in eukaryotic cells such asyeast and insect and mammalian cells (Sutherland andWitke, 1999; Van Duyne, 2001). Therefore, it has beenwidely used for DNA recombination technology incultured cells and animals (including transgenic (TG)and knockout (KO) mice) (Bernstein and Breitman,1989). In conditional or inducible gene targeting experi-

ments, theCre-loxP is especially powerful because it canevade the potential embryonic lethality or postnataldeath ofmutantmice, and allowsus to examine stage- ororgan-specific gene function (Rajewsky et al., 1996;Sauer, 1998). However, it has been difficult to detect theCre protein expressed in these gene-engineered animaltissues through a direct approach (i.e., immunohisto-chemical staining using anti-Cre antibody), which maybe in part because of an inability to obtain anti-Creantibody with a high degree of specificity. Althoughproduction of anti-Cre monoclonal antibody has beenreported by Schwenk et al. (1997), it has not beenwidely used. Therefore, most researchers have had tocross Cre-expressing TG mice with TG or KO mice,which always carry loxP-flanked sequences and the lacZgene, tomonitor localized expression ofCre recombinase(Rajewsky et al., 1996).

In this study, we first constructed tagged-NCre(NCreMH) cDNA by joining the human Myc epitope(Kolodziej and Young, 1991) and 6X His tag (Schmittet al., 1993) on the 30 end ofNCre gene (Sato et al., 2000).The expression vector (pCMV-NCreMH) was thenconstructed by placing the NCreMH downstream ofthe cytomegalovirus (CMV) promoter. In vitro and invivo transfection experiments revealed that NCreprotein produced from pCMV-NCreMH possessed Crerecombinase activity with immunoreactivity againstanti-Myc antibody.

MATERIALS AND METHODS

Vector Construction

Tagged-NCre expression vectors. The nuclearlocalizing signal (NLS) attached Cre (NCre) and poly(A)

� 2006 WILEY-LISS, INC.

Takayuki Sakurai’s present address is Department of Organ Regen-eration, Graduate School of Medicine, ShinshuUniversity 3-1-1 Asahi,Matsumoto 390-8621, Japan.

*Correspondence to: Satoshi Watanabe, Department of Developmen-tal Biology, National Institute of Agrobiological Sciences, Ikenodai 2,Tsukuba, Ibaraki, 305-8602, Japan. E-mail: [email protected]

Received 12 December 2005; Accepted 22 January 2006Published online 7 August 2006 in Wiley InterScience(www.interscience.wiley.com).DOI 10.1002/mrd.20482

signal of the thymidine kinase gene were isolated bydigestion of pMC-Cre (Taniguchi et al., 1998)withEcoRIand subcloned into the EcoRI site of pBluescript KS(þ)(Stratagene, La Jola, CA) to obtain the pKS-CpAplasmid. A 549-bp portion at the 30 side of the NCrecDNA was PCR-amplified with CreEV (50-CTTATAA-CACCCTGTTACGTATAGC-30) and CreKp (50-GGGGTACCATCGCCATCTTCCAGCAGGCGCAC-30) primersusing pMC-Cre as a template. The resulting PCRproducts, lacking the ‘‘stop’’ codon of the Cre proteinitself, were next digested with EcoRV and KpnI, andsubcloned between the EcoRV and KpnI sites of pKS-CpA. The obtained plasmid was termed pNCre-Stop*.The Cre cDNA lacking the stop codon was dissected bydigestion of pNCre-Stop* with SalI and KpnI, andsubcloned between the XhoI and KpnI sites ofpcDNA3.1mycHis A (Invitrogen, Carlsbad, CA) in orderto add the human Myc and poly-histidine tag at the C-terminus of theNCre protein. The obtained plasmidwasnamed pCMV-NCreMH (Fig. 1).

The modified human progesterone receptor ligandbinding site was similarly PCR-amplified with CreHd(50-GTGGCAGATCCCACAGGAGTTTGTC-30) andCPRKp (50-GGGGTACCGCAGTACAGATGAAGTTGTTTG-AC-30) primers using pCrePR (Tsujita et al., 1999) as thetemplate. After digestion of the PCR products withHindIII and KpnI, these products were subclonedbetween theHindIII andKpnI sites of pCrePR to obtainpNCrePR. TheNCrePR fragment lacking the stop codonof NCre protein was isolated by digestion ofpNCrePR with SalI and KpnI, and then subclonedbetween XhoI and KpnI sites of pCMV-NCrePRMH(Fig. 1). All the plasmids were confirmed by sequencingwhether the inserts had been correctly introduced intothe vectors.

Reporter vectors. Biological activity of the tagged-NCre recombinase was assessed by measuring thereporter gene products. The plox-puro/Luc (Fig. 1) wasconstructed as follows. The mouse phosphoglyceratekinase (PGK)-puro-p(A) cassette was isolated by diges-tion of the pPGK-puro plasmid (Watanabe et al., 1995)with SalI, and subcloned into the EcoRI site of ploxP-2(Taniguchi et al., 1997) through blunt-end ligation toobtain pLPL plasmid. Two copies of the PGK-1 poly(A)signal obtained by digestion of pPGK-puro with EcoRVand SalI were subcloned into the EcoRV site of pLPL.The resulting plasmidwas termed ploxP-puro. TheCAGpromoter isolated frompCAGGS(Niwaetal., 1991) afterdigestionwithSalI andXbaIwas subclonedbetween theSalI and XbaI sites of pBluescript KS(þ) to obtain thepKS-CAG plasmid. Firefly luciferase (luc) cDNA andpoly(A) signal isolated from Picagene basic vector 2(Dainippon Ink Co., Tokyo, Japan) by digestion withHindIII and NotI were subcloned between the HindIIIand NotI sites of pKS-CAG to obtain the pCAG/Lucplasmid. The loxP-puro-3 X p(A) fragment isolated fromploxP-puro after digestion with SalI and XbaI wassubcloned into theEcoRVsite of pCAG/Luc by blunt-endligation to obtain the plox-puro/Luc plasmid. pCETZ-17(Fig. 1; (Sato et al., 2000)) can express enhancedgreen fluorescent protein (EGFP) in the absence of Creadministration when introduced into cells, but expres-sion of lacZwill be induced after transfection of the Cre-expression plasmid into CETZ-17-incorporated cells.plox-puro/Luc is silent without Cre-mediated recombi-nation, but luc expression will be induced when Cre-mediated excision of the PGK-puro-p(A) cassetteflanked by loxP sequences occurs. The reporter plasmidpCRTEIL-6 (Fig. 1) was constructed through twocloning steps. First, pCRT-17, an intermediate productfor pCRTEIL-6, was constructed by inserting theHcRed1 cDNAþpoly(A) sites of the SV40 gene (Clon-tech Laboratories, Palo Alto, CA) in front of the 50 end ofthe chloramphenicol acetyltransferase gene (CAT) inthe pCAG-CAT-lacZ (Bernstein and Breitman, 1989),from which lacZ had already been removed. A DNAfragment containing EGFP cDNA, internal ribosomalentry site (IRES), and firefly luc cDNA was then placedimmediately downstream of the loxP site located at the30 end ofCAT in the pCRT-17 to obtain pCRTEIL-6. The

Molecular Reproduction and Development. DOI 10.1002/mrd

Fig. 1. Schematic representation of the vectors used for the Cre-mediated recombination event.A: pCMV-NCreMHwas constructed byinserting the PCR-amplified NCre fragment into the pcDNA3.1my-cHIS-A plasmid. B: pCMV-NCrePRMH was constructed by insertingtheNCrePR fragment (containingNCre and the ligand-binding domainof the human progesterone receptor). C: plox-puro/Luc vector containsloxP-flanked PGK-puro cassette carrying three repeats of the poly(A)signals. After Cre-mediated recombination, expression of luc becomesdirectly controlled by the upstreamCAG promoter.D: pCETZ-17 vector(Sato et al., 2000) comprises the CAG promoter, loxP-flanked sequence(EGFP cDNA and CAT), and lacZ. After Cre-mediated recombination,lacZ expression commences under the control of the CAG promoter. E:pCRTEIL-6 comprises the CAG promoter, loxP-flanked sequence(HcRed1 cDNA and CAT), EGFP cDNA, IRES, and luc cDNA. AfterCre-mediated recombination, EGFP and luc expression commencesunder the control of the CAG promoter. CAG, cytomegalovirusenhancerþ chicken b-actin promoter; CAT, chloramphenicol acetyl-transferase gene; CMV, cytomegalovirus promoter; EGFP, enhancedgreen fluorescent protein gene; HcRed1, HcRed1 fluorescent proteingene; IRES, internal ribosomal entry site; luc, firefly luciferase cDNA;MH, humanMyc epitope andHis tag; P, mouse PGK-1 gene promoter;p(A), poly(A) signal of bovine growth hormone gene or mouse PGK-1gene; PR, ligand-binding domain of the progesterone receptor; Puro,puromycin-resistant gene; SVp(A), SV40 poly(A) signal.

1346 S. WATANABE ET AL.

resulting pCRTEIL-6 plasmid has a backbone of pBlue-script SK(�) (Stratagene).

Cell Culture and Transfections

NIH3T3 (Furusawa et al., 2003) was cultured inDulbecco’smodifiedEagle’smedium (DMEM;SigmaCo.Ltd., St. Louis,MO) supplementedwith10% fetal bovineserum (FBS), 50 units of penicillin, and 50 mg ofstreptomycin per ml at 378C under an atmosphere of5% CO2. To examine the recombinase activity of thetagged-NCre, stable transformants carrying testerplasmids (CETZ-17 and lox-puro/Luc) were obtained.The pCETZ-17 plasmid was digested with SpeI andpurified in agarose gels (#01157-66; Nacalai Tesque Co.,Kyoto, Japan) to remove the vector backboneprior to usefor transfection. The plox-puro/Luc was digested withSalI and similarly purified. The cells were seeded in a60-mm dish (#3002; Becton Dickinson, Franklin Lakes,NJ) at a density of 2�105 cells. The next day, either theCETZ-17 or plox-puro/Luc fragment (2 mg each) wasmixed with SalI fragments containing 0.2 mg of PGK-puro-p(A) (Watanabe et al., 1995), and immediatelyencapsulated by FuGENE6 reagent (RocheDiagnostics,Mannheim,Germany). Thesemixtureswere then addedto each dish. The cells were passaged to 3� 100-mmdishes 24 hr after transfection and 1 day laterselected in a medium containing 1 mg/ml of puromycin(#P8833; Sigma) for 7 days. The surviving cell colonieswere picked up and propagated in a fresh 100-mmdish (#3003; Becton Dickinson) until transfectionby Cre expression vectors. pCMV-NCreMH, pCMV-NCrePRMH, and pCAG/NCre (Sato et al., 2000) weretransiently expressed in the stable transformantscarrying the tester plasmid components. For this,NIH3T3 cells were seeded in a 100-mmdish at a densityof 7�105 cells. The next day, a mixture containing 6 mgof each of the circular NCre expression plasmid andFuGENE6 reagentwas added to each dish.Mifepristone(RU486) (#M8046, Sigma) at final concentration of100 nM was added to the dish containing pCMV-NCrePRMH transfectants 16 hr after transfection. Forthe luc assay, culture media were removed 48 hr aftertransfection, and then the cells were lysed in situ byadding 100 ml of lysis buffer (#E194A; Promega Co.,Madison, WI) at room temperature. For histochemicalstaining for b-galactosidase activity, cells were fixedwith 4%paraformaldehyde (PFA) containingDulbecco’smodified phosphate-buffered saline without Ca2þ andMg2þ, pH 7.2 [PBS(�)] for 5 min at room temperature,and then incubated overnight at 378C in the presence ofX-Gal, a substrate for b-galactosidase, as describedpreviously (Sato et al., 2000). In some cases, thefixed cells were subjected to immunohistochemicalstaining using anti-b-galactosidase antibody, asdescribed below.

Western Blot Analysis

NIH3T3 cells were homogenized in sample buffer(10% sucrose (w/v), 3% SDS (w/v), 60 mM Tris-HCl (pH6.8)). After the supernatants were treated with b-

mercaptoethanol, they were separated by electrophor-esis under reducing conditions on a 10% polyacryla-mide-SDS-gel and transferred to nylon membranes(Immobilon-P; Millipore Co., Bedford, MA). Blots wereblocked with 5% nonfat dry milk in Tris-buffered saline(TBS: 50mMTris-HCl (pH7.4), 150mMNaCl), and thenincubated with polyclonal anti-Cre antibody (#69050-3;Novagen Co., Madison, WI) or polyclonal anti-Myc Tagantibody (#06-549; Upstate Co., Lake Placid, NY). EachantibodywasdilutedwithTBScontaining5%nonfat drymilk to 1/1,000andused.AfterwashingwithTBST (TBScontaining 0.05% Tween 20), blots were incubated withhorseradish peroxidase (HRP)-linked anti-rabbit IgG(#NIF824; Amersham Pharmacia Biotech., Little Chal-font, UK) in TBS containing 5% nonfat dry milk. Blotswere then rewashed with TBST. Cre proteins wereanalyzed with the aid of a ChemiDoc XRS imageanalyzer (Bio-Rad Co., Hercules, CA).

Luciferase Assay

Luc activity for testing tagged-NCre-mediated exci-sion activity was measured using a Luciferase AssaySystem (Promega Co.) and a luminometer (#CT-9000D;Dia-Iatron, Tokyo, Japan), following the protocolsdescribed by the manufacturers. Relative light units(RLU) obtained with luc were measured for 5 secfollowing a 2 sec delay after the addition of the lysate(10 ml) to 100 ml of luciferase assay substrate (PromegaCo.) using a luminometer. In order to compensate forthe variety of DNA introduction efficiencies, fireflyluc activity was normalized against the protein concen-tration in each sample. Protein concentration wasdetermined by the Bedford method (#500-0116; Bio-Rad Co.).

Immunohistochemistry of Cultured Cells

NIH3T3 cells grown on poly-L-lysin-coated coverglasses were co-transfected with pCETZ-17 and NCreexpression vectors. At 48 hr after transfection, the cellswere fixedwith 4%PFA for 10min at room temperature,permeabilized with 0.2% Triton X-100, washed inPBS(�), and incubated with rabbit polyclonal anti-Myctag antibody diluted 1/300 or mouse monoclonal anti-b-galactosidase antibody (#G80211; Sigma) diluted 1/200.After three rinses with PBS(�) containing 0.1% (v/v)goat serum, the slides were incubated with Alexa Fluor488 goat anti-mouse IgG (#A-11029; Molecular ProbesCo., Eugene,OR; diluted 1/400) (for anti-b-galactosidaseantibody) or Alexa Fluor 594 goat anti-rabbit IgG (#A-11037; Molecular Probes Co.; diluted 1/400) (for anti-Myc tag antibody). After rinsing, the cover glasses weremounted on the slide glasseswithVectashieldmountingmedium (Vector Laboratories Co., Burlingame, CA).About 100 positive double-labeled cells were countedunder fluorescence microscopy (Axiovert; Zeiss Co.,Oberkochen, Germany).

Intraoviductal Introduction of Plasmid DNA

In vivo gene transfer to oviductal epithelium (GTOVE)was performed according to the method reported by Sato


ACTIVE TAGGED-NCre FOR GENETIC ENGINEERING IN MAMMALS 1347

(2005). Experiments using animals were carried out inaccordance with the Guide for the Care and Use ofLaboratory Animals at TokaiUniversity. All efforts weremade to minimize the number of animals used and theirsuffering. Female B6C3F1 mice (8–15 weeks old;purchased from CLEA Japan, Inc., Tokyo, Japan) wereanesthetized with sodium pentobarbital. For DNA injec-tion, 1 ml of a solution containing circular plasmid DNA(pCMV-NCreMH and pCRTEIL-6; 0.2 mg each) wasinjected into the ampulla of an oviduct. Immediatelyafter DNA injection, the oviductal regions were placedbetween tweezer-type electrodes and then administrated8� 50 msec square-wave pulses of 50 V from a pulsegenerator (#T820; BTX Genetronics, Inc., San Diego,CA). Similarly, pCAG-EGFP (¼pCE-29; (Sato et al.,2002)) or pCRTEIL-6alone (0.2mg each)was also injectedas controls.Thedayaftergene transfer, theoviductsweredissected and inspected for EGFP-derived green andHcRed1-derived red fluorescence. Some oviducts weresubjected to cryostat sectioning and subsequent immu-nohistochemical analyses.

Immunohistochemistry of Mouse Oviducts

For immunohistochemistry, oviducts from the micereceiving GTOVE were fixed in 4% PFA in PBS(�) for 2daysat 48C,and then immersed in30%sucrose inPBS(�)formorethan2daysat48C,embedded inTissue-TekOCTcompound (Sakura Finetek Co., Tokyo, Japan), andfinally frozen before serial cryostat sectioning (10–30mm in thickness). Sections were permeabilized in �208Cacetone for 10 min, washed in PBS(�), and blocked for 2hr at room temperature in PBS(�) containing 10% (v/v)normal goat serum and 0.1% (v/v) Triton X-100. Theywere then incubatedwithanti-Cre (diluted1:300) or anti-b-galatosidase (diluted 1:200) antibody overnight at 48C.After washing with PBS(�) containing 0.1% Triton X-100, the sections were next reacted with Alexa Fluor 488goat anti-mouse IgG (diluted 1/400; for anti-b-galatosi-dase antibody) or Alexa Fluor 594 goat anti-rabbit IgG(diluted 1/400; for anti-Cre antibody) for 2 hr at roomtemperature. Sections were mounted in Vectrashieldmounting medium.

Fluorescence of the cryostat sections was observedusing an Olympus BX60 fluorescence microscope(Olympus, Tokyo, Japan) with DM505 filters (BP460-490 and BA510IF) or DM600 filters (BP545-580 andBA6101F), which were used for EGFP and HcRed1monitoring, respectively. For detection of fluorescencein dissected oviducts, an Olympus BX40 dissectingmicroscope was used. Microphotographs were takenusing a digital camera (FUJIX HC-300/OL; Fuji Film,Tokyo, Japan) attached to the fluorescence microscopeand printed out using a Mitsubishi digital color printer(CP700DSA; Mitsubishi, Tokyo, Japan).

RESULTS

Biological Activity of the Tagged-NCre

We constructed the tagged-NCre cDNA fragmentcontaining Myc and His epitopes at the 30 end of NCre

or NCrePR gene. These fragments were inserted down-stream of the CMV promoter. The resulting expressionvectors were termed pCMV-NCreMH and pCMV-NCrePRMH. We first examined the recombinase activ-ity of the tagged-NCre by introducing these vectors intoNIH3T3 cells with the plox-puro/Luc reporter vector.Synthetic steroid RU486 (at final concentration of100 nM) was administrated to the culture medium16 hr after transfection with pCMV-NCrePRMHþplox-puro/Luc. pCAG/NCre or pMC-Cre was also intro-duced with plox-puro/Luc as positive controls. The lucactivity was measured 48 hr after transfection. Aremarkable increase in luc activity was observed in thecells that had undergone transfection with pCMV-NCreMH (Fig. 2). The level of luc activity, however,was lower than that in cells transfected with pCAG/NCre, a positive control vector carrying a strongpromoter, but still higher than that in cells transfectedwith pMC-Cre (Taniguchi et al., 1998), another positivecontrol vector carrying a weak promoter. An increase inluc activity was also noted after transfection withpCMV-NCrePRMH and subsequent treatment withRU486, but the level was about half of that achievedby transfection of pCMV-NCreMH (Fig. 2). Three otherindependent experiments yielded similar results. Theseresults indicated that (1) pCMV-NCreMHcan excise theloxP-flanked DNA fragment in plox-puro/Luc reportervector efficiently, and (2) pCMV-NCrePRMH acts in anRU486-dependent manner. In conclusion, the tagged-NCre exhibits recombinase activity.

Western Blotting Using Anti-Myc Antibody

Western blotting was performed to ascertain whetherNCre protein is detectable in the NIH3T3 cells


Fig. 2. Luc activity in NIH3T3 cells transiently co-transfected withthe NCre expression vectors and plox-puro/Luc. For transfection withpCMV-NCrePRMH and plox-puro/Luc, RU486 was added aftertransfection to activate the NCrePRMH protein. Firefly luc activitywas normalized against Renilla luc activity in order to compensate fordifferences in transfection efficiency and shown as RLU/mg of protein.The normalized activities were analyzed statistically by a two-tailedStudent’s t-test. The mean values are presented with their standarderrors for each transfection group. Negative control, cells nottransfected by the NCre expression vectors. pCAG/NCre and pMC-Cre were used as positive controls (PC).


transfected with the tagged-NCre expression vectors.Cells transfected with pCAG/NCre (Sato et al., 2000)and pMC-Cre were used as positive controls. Whencellular lysates prepared 48 hr after transfection weresubjected to Western blotting analysis using anti-Mycand anti-Cre polyclonal antibodies, the tagged-NCreprotein from pCMV-NCreMH could be detected by anti-Myc antibody (Fig. 3A). Unfortunately, we failed todetect pCMV-NCrePRMH-derived proteins (Fig. 3A).Cre proteins from cells transfected with pCAG/NCreand pCMV-NCreMH were successfully detected byanti-Cre antibody (Fig. 3B), but those from pMC-Creand pCMV-CrePRMH transfected cells were not(Fig. 3B). Our inability to detect Cre proteins in cellstransfected with pMC-Cre or pCMV-CrePRMH may berelated to production of small amounts of the protein.These results were consistent with the low degree of lucactivity in cells transfected with pMC-Cre or pCMV-CrePRMH, as shown in Figure 2.

Immunostaining of Cells Transfected With theTagged-NCre Expression Vectors

The cytological localization of the tagged-NCre intransfected cells was next examined. NIH3T3 cells were

first transfected with the tagged-NCre expressionvectors and pCETZ-17, then 2 days later, they weresubjected to histochemical staining for lacZ activity inthe presence of X-Gal. All the Cre expression vectorssuccessfully induced lacZ expression in the transfectedcells (data not shown). To examine the intracellularlocalization of tagged-NCre and lacZ gene products,NIH3T3 cells co-transfected with pCMV-NCreMH andpCETZ-17 vectors were subjected to immunostainingusing anti-Myc and anti-Cre polyclonal antibodies.Immunostaining using anti-Myc antibody demon-strated that NCreMH protein was predominantlylocalized in the nuclei of transfected cells (Fig. 4b,d).Immunostaining using anti-Cre antibody also yieldedpositive staining throughout the cytoplasm, butclear nuclear staining did not occur (Fig. 4f,h). Cellstransfected with pCAG/NCre alone exhibited immunor-eactivity with the anti-Cre antibody, but the reactionwas not limited to nuclei (data not shown). On theother hand, lacZ products were seen in section ofthe cytoplasm closely to the nucleus with which anti-Myc antibody had reacted strongly (Fig. 4c,g).Notably, staining with anti-b-galactosidase antibodyyielded clear specific signals (Fig. 4d,h). In cells co-transfected with pCMV-NCrePRMHþpCETZ-17 andsubsequently treated with RU486, as well as in cells co-transfected with pMC-CreþpCETZ-17, no positivestaining was seen with these two antibodies (data notshown). In these cells only a small amount of NCreprotein appeared to have been produced, which is ingood agreement with previous data on luc activity (seeFig. 3).

Tagged-NCre Expression in Mouse Oviducts

We next examined whether the tagged NCre pos-sesses its recombinase activity in vivo. Gene deliveryinto oviductal epithelium, based on DNA injectioninto the lumen of oviducts and subsequent in vivoelectroporation (Sato, 2005), was used for this purpose.Instillation of the pCAG-EGFP plasmid yielded brightgreen fluorescence throughout the ampulla portion(Fig. 5b), but not red fluorescence (Fig. 5c). When thepCRTEIL-6 plasmid was introduced alone, no EGFPfluorescencewas observed (Fig. 5e), but red fluorescencewas observed in some oviductal epithelial cells (Fig. 5f).Co-injection of the pCRTEIL-6 and pCMV-NCreMHplasmids resulted in both red and green fluorescence,although theHcRed1-derived redfluorescence appearedto be weaker than the EGFP-derived green fluorescence(indicated by arrows in Fig. 5h,i). These findingsindicate that the recombinase activity of pCMV-NCreMH was maintained even in vivo. To examinethe localized expression of EGFP and HcRed1 inthe oviducts after co-transfection, cryostat sectionswereprepared and inspected for EGFP and red fluorescence(see Fig. 6). Strong, but patchy EGFP fluorescence wasobserved in some oviductal epithelial cells (indicated byarrows in Fig. 6b) and obvious red fluorescence was notobserved in the EGFP-positive area (Fig. 6c). Whensections adjacent to those shown in Figure 6a–c were


Fig. 3. Immunoblot analysis of cellular lysates (corresponding to50 mg of protein in each lane) derived from NIH3T3 cells 48 hr aftertransfection. NCre protein was detected in cells transfected withpCMV-NCreMH using anti-Myc (A) and anti-Cre (B) antibodies. Theasterisks in (B) indicate the specific band forNCre protein. Transfectedvectors are shown above each lane. pCMV-NCrePRMHþRU486indicates that cells were incubated in a medium containing RU486 16hr after transfection of pCMV-NCrePRMH.Molecular weight in kDa isalso shown on the righthand side.


immunostained with anti-Myc and anti-Cre antibodies,the EGFP-positive areas were also positive for anti-Mycantibody (indicated by arrows in Fig. 6e). In particular,nuclear areas appeared to be intensely stained. EGFP-negative oviductal epithelia were unreactive to anti-

Myc antibody (indicated by arrowheads in Fig. 6e) andclear staining with anti-Cre antibody did not occur(Fig. 6f). Negative control staining using the secondantibody alone did not yield specific fluorescence (datanot shown). These findings clearly indicate that NCre


Fig. 4. Indirect double-immunofluorescence analysis of NIH3T3cells after co-transfection of pCMV-NCreMH and pCETZ-17 vectors.Cells were stainedwith anti-Myc polyclonal antibody (a–d) followed bya goat anti-rabbit IgG-Alexa Fluor 594 conjugate for detection of Creprotein (red). Similarly, cells were stained for detection of NCreMHwith anti-Cre polyclonal antibody (e–h) followed by Alexa Fluor 594(red). Each group of transfected cells was also stained with anti-b-

galactosidase monoclonal antibody followed by a goat anti-mouse IgG-AlexaFluor 488 (green). Photographswere takenunder bright (a and e)and dark field (b–d and f–h). b: Cells stained with anti-Myc antibody;(c) and (g), cells stained with anti-b-gal antibody; (d), merged image of(b) and (c); (f), cells stainedwith anti-Cre antibody; (h),merged image of(f) and (g). Bar¼ 10 mm.

Fig. 5. Cre-mediated excision in vivo. a–c: Oviducts transfectedwith pCAG-EGFP alone showing EGFP-derived green fluorescence(but not HcRed1-derived red fluorescence). d–f: Oviducts transfectedwith pCRTEIL-6 alone showing HcRed1-derived red fluorescence (butnot EGFP-derived green fluorescence). g–i: Oviducts co-transfectedwith pCRTEIL-6 and pCMV-NCreMH showing both types of fluores-

cence (indicated by arrows in (h) and (i)), although the greenfluorescence appears to be stronger than the red fluorescence. a,d,g:Photographs taken under light (Light); (b,e,h) photographs takenunder UV illumination using filters for detection of green fluorescence(EGFP); (c,f,i) photographs taken under UV illumination using filtersfor detection of red fluorescence (HcRed1).


protein expressed from pCMV-NCreMH in vivo can bedetected by anti-Myc antibody.

DISCUSSION

WegeneratedNCreMH by connecting the humanMycepitope sequence (comprising 30 nucleotides encoding10 amino acids) to the 30 end of NCre. The proteinproducts fromNCreMH are easily detected by anti-Mycantibody. Cre recombinase activity was observed inNIH3T3 cells co-transfected with an expression vector(pCMV-NCreMH) and a loxP-sequence-incorporatingreporter (Fig. 2). Western blotting and immunofluores-cence microscopic analyses revealed the presence ofNCreMH protein in cellular lysates of the transfectants(Fig. 3). In addition, we confirmed that the NCreMH

protein was functional in vivo and immunologicallydetectable by anti-Myc antibody (Fig. 6e).

In this research, we prepared NCreMH encodingthe human Myc-tagged Cre protein using theepitope tagging technology reported by Kolodziej andYoung (1991). Proteins derived from the resultingNCreMH were detectable by anti-Myc antibody inWestern blotting and immunohistochemical systemsunder conditions of decreased background (Figs. 3and 4). One may suppose that the addition of the Mycepitope at the C-terminus of the NCre protein wouldinhibit the enzymatic function of NCre itself, butour present experiments demonstrated that NCreMHgene products in fact exhibited recombinase activity(Figs. 2, 4, and 5).


Fig. 6. Cryostat sections of oviducts sampled 1 day after introduc-tion of pCRTEIL-6 and pCMV-NCreMH DNA (0.5 mg each) andsubsequent in vivo electroporation. (a) and (b–f) were photographedunder light, and light and UV illumination, respectively. Note thatstrong, but patchy EGFP fluorescence present in some oviductalepithelial cells (indicated by arrows) in (b).When the same section in (b)was inspected for HcRed1-derived fluorescence, none was obvious ((c);

an insert in (b) is magnified). When another section near the (a–c)sectionwas immunostainedwith anti-Myc and anti-Cre antibodies, thearea positive for EGFP (indicated by arrows in (d)) was positive forstaining with anti-Myc antibody (indicated by arrows in (e)). However,the area negative for EGFP was negative for staining with anti-Mycantibody (indicated by arrowheads in (e)). Immunostaining using anti-Cre antibody failed to demonstrate distinct staining for NCreMH (f).


We also constructed NCrePRMH by inserting theNCrePR (Tsujita et al., 1999; Kitayama et al., 2001)sequence between theNCre gene andMyc/His-tag. ThisNCrePR is known to exhibit recombinase activity whencells carrying the NCrePR expression vector areadministratedwith the synthetic steroidRU486 (Vegetoet al., 1992). We constructed an expression vectorpCMV-NCrePRMH in which expression of NCrePRMHis controlled by the CMV promoter. Western blotting(Fig. 3) and immunohistochemical analyses (data notshown) using anti-Myc and anti-Cre antibodies failed toreveal the presence of NCrePRMH protein. However,NCrePRMHprotein (probably at low levels) appeared tobe produced in the transfected cells, because co-transfection of pCMV-NCrePRMH and plox-puro/Lucand subsequent treatment with RU486 resulted in anincrease in luc activity (Fig. 2). The inability of theNCrePRMH protein to react with these antibodies mayalso be attributed to structural alteration of theNCrePRMH protein itself, which would have disturbedthe association between the NCrePRMH protein andantibody. Interestingly, the addition of the Myc tag atthe N-terminus of the NCrePR protein may improve itsimmunoreactivity to antibodies, although that has notyet been attempted.

The protein products of NCreMH constructed in thisstudy possessed Cre recombinase activity and wereeasily detected by anti-Myc antibody. If this NCreMHgene is used in the field of Cre-loxP-mediated genemanipulation in mammals, it will be unnecessary forresearchers to use Cre-inducing reporter TG mice formonitoring localized expression of NCre. We can nowassess the tissue localization ofNCreMH gene productsin TG mice using an immunohistochemical approachwith anti-Myc antibody. Such a procedure would bothshorten the period required for the analysis of TG miceexhibiting Cre-mediated recombination, and reduce thenumber ofmice required.OurNCreMHconstructwill beuseful for performing tissue-specific genetic modifica-tion in livestock, such as pigs or cattle, becauseintroduction of a Cre-loxP system into livestock animalshas not yet been attempted. In the future, tissue-specificgene manipulation via the Cre-loxP system in theseanimals will be increasingly used for production ofdisease models, or animals secreting pharmaceuticalproteins. In this case, production of tester animalscarrying loxP-flanked marker genes for monitoring Creexpression is not necessary if NCreMH construct-incorporating transgenic animals are produced andused. Such attempt will reduce the number of animalsused and the period of animal breeding, since thelifespans of livestock animals are much longer thanmice, and the efficiency of the production of transgeniccloned animal using somatic cloning is very low(Watanabe et al., 2005).

ACKNOWLEDGMENTS

The authors thank Mrs. Sachiko Watanabe fortechnical assistance in molecular biology.

REFERENCES

Bernstein A, Breitman M. 1989. Genetic ablation in transgenic mice.Mol Biol Med 6:523–530.

FurusawaT,HosoeM,OhkoshiK, Takahashi S,KiyokawaN,FujimotoJ,AmemiyaH,SuzukiS,TokunagaT. 2003.CatalyticRAG1mutantsobstruct V(D)J recombination in vitro and in vivo. Mol Immunol39:871–878.

Hoess R, Abremski K, Sternberg N. 1984. The nature of the interactionof the P1 recombinase Cre with the recombining site loxP. ColdSpring Harb Symp Quant Biol 49:761–768.

Kitayama K, Abe M, Kakizaki T, Honma D, Natsume R, Fukaya M,Watanabe M, Miyazaki J, Mishina M, Sakimura K. 2001. Purkinjecell-specific and inducible gene recombination system generatedfrom C57BL/6 mouse ES cells. Biochem Biophys Res Commun281:1134–1140.

Kolodziej PA, Young RA. 1991. Epitope tagging and protein surveil-lance. Methods Enzymol 194:508–519.

Niwa H, Yamamura K, Miyazaki J. 1991. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene108:193–199.

Rajewsky K, Gu H, Kuhn R, Betz UA, Muller W, Roes J, SchwenkF. 1996. Conditional gene targeting. J Clin Invest 98:600–603.

Sato M. 2005. Intraoviductal introduction of plasmid DNAand subsequent electroporation for efficient in vivo genetransfer to murine oviductal epithelium. Mol Reprod Dev 71:321–330.

Sato M, Yasuoka Y, Kodama H, Watanabe T, Miyazaki JI, Kimura M.2000. New approach to cell lineage analysis in mammals using theCre-loxP system. Mol Reprod Dev 56:34–44.

Sato M, Ishikawa A, Kimura M. 2002. Direct injection of foreign DNAinto mouse testis as a possible in vivo gene transfer system viaepididymal spermatozoa. Mol Reprod Dev 61:49–56.

Sauer B. 1998. Inducible gene targeting in mice using the Cre/loxsystem. Methods 14:381–392.

Schmitt J, Hess H, Stunnenberg HG. 1993. Affinity purification ofhistidine-tagged proteins. Mol Biol Rep 18:223–230.

Schwenk F, Sauer B, Kukoc N, Hoess R, Muller W, Kocks C, Kuhn R,Rajewsky K. 1997. Generation of Cre recombinase-specific mono-clonal antibodies, able to characterize the pattern of Cre expressionin Cre-transgenic mouse strains. J Immunol Methods 207:203–212.

Sutherland JD, Witke W. 1999. Molecular genetic approaches tounderstanding the actin cytoskeleton. Curr Opin Cell Biol 11:142–151.

Taniguchi M, Yuasa S, Fujisawa H, Naruse I, Saga S, MishinaM, Yagi T. 1997. Disruption of semaphorin III/D gene causessevere abnormality in peripheral nerve projection. Neuron 19:519–530.

Taniguchi M, Sanbo M, Watanabe S, Naruse I, Mishina M, Yagi T.1998. Efficient production of Cre-mediated site-directed recombi-nants through the utilization of the puromycin resistance gene, pac:A transient gene-integration marker for ES cells. Nucleic Acids Res26:679–680.

Tsujita M, Mori H, Watanabe M, Suzuki M, Miyazaki J, Mishina M.1999. Cerebellar granule cell-specific and inducible expression of Crerecombinase in the mouse. J Neurosci 19:10318–10323.

Van Duyne GD. 2001. A structural view of cre-loxp site-specificrecombination. Annu Rev Biophys Biomol Struct 30:87–104.

Vegeto E, Allan GF, Schrader WT, Tsai MJ, McDonnell DP, O’MalleyBW. 1992. Themechanism ofRU486 antagonism is dependent on theconformation of the carboxy-terminal tail of the human progesteronereceptor. Cell 69:703–713.

Watanabe S, Kai N, YasudaM,KohmuraN, SanboM,MishinaM, YagiT. 1995. Stable production of mutant mice from double geneconverted ES cells with puromycin and neomycin. Biochem BiophysRes Commun 213:130–137.

Watanabe S, Iwamoto M, Suzuki S, Fuchimoto D, Honma D, Nagai T,Hashimoto M, Yazaki S, Sato M, Onishi A. 2005. A novel method forthe production of transgenic cloned pigs: Electroporation-mediatedgene transfer to non-cultured cells and subsequent selection withpuromycin. Biol Reprod 72:309–315.



Preparation of enzymatically active human Myc-tagged-NCre recombinase exhibiting immunoreactivity...

Documents

Transcript of Preparation of enzymatically active human Myc-tagged-NCre recombinase exhibiting immunoreactivity...