LETTER

Efficient Temporally Controlled Targeted SomaticMutagenesis in Hepatocytes of the MouseMichael Schuler,1 Andree Dierich,1,2 Pierre Chambon,1,2 and Daniel Metzger1,2*1Institut de Genetique et de Biologie Moleculaire et Cellulaire (IGBMC), Centre National de la Recherche Scientifique,Institut National de la Sante et de la Recherche Medicale, Universite Louis Pasteur, Illkirch-Cedex, France2Institut Clinique de la Souris (ICS), Illkirch-Cedex, France

Received 9 February 2004; Accepted 12 March 2004

Summary: To generate temporally controlled targetedsomatic mutations selectively and efficiently in hepato-cytes, we established SA�/CreERT2 mice in which thetamoxifen-dependent Cre-ERT2 recombinase coding se-quence preceded by an internal ribosomal entry site wasinserted in the 3� untranslated region of the serum albu-min (SA) gene. Whereas the wildtype SA allele wasstrongly expressed in the liver and at lower levels insome extrahepatic tissues, SA-Cre-ERT2 fusion tran-scripts were only detected in the liver. The Cre-ERT2

protein was expressed in most if not all hepatocytes, andtamoxifen (Tam) treatments of adult mice at variousages efficiently induced Cre-mediated recombination ofLoxP flanked (floxed) alleles in these cells, but none inother cell types or tissues. Thus, SA�/CreERT2 miceshould be of great value to analyze gene function in theliver and to establish animal models of human diseases.genesis 39:167–172, 2004. © 2004 Wiley-Liss, Inc.

Key words: serum albumin, Cre/LoxP technology, Cre-ERT2 recominase, tamoxifen, liver

The liver has crucial functions in the regulation of en-ergy homeostasis, detoxification, and the production ofserum proteins. The in vivo roles played by some of thegenes involved in these functions were partly deter-mined in germline null-mutant mice (De Feo and Lucidi,2002; Foufelle and Ferre, 2002; Akiyama and Gonzalez,2003; Arrese and Trauner, 2003). However, germlinenull mutations often induce embryonic lethality or func-tional compensation during development, thus preclud-ing the analysis of gene functions at later stages. More-over, they do not allow identification of cell autonomousand noncell autonomous actions (Metzger and Cham-bon, 2001). To circumvent these problems, spatiallycontrolled targeted somatic mutagenesis systems havebeen developed in mice by cell-specific expression ofthe bacteriophage P1 Cre recombinase, which excisesLoxP-flanked (floxed) DNA segments. Additionally, tem-poral control of Cre activity was obtained with condi-tional Cre recombinases resulting from fusions with mu-tated ligand binding domains of steroid receptors (Feil et

al., 1996; Kellendonk et al., 1999; Schwenk et al., 1998;Tannour-Louet et al., 2002). Thus, cell-specific expres-sion of such fusion proteins between Cre and mutatedligand binding domains of the human estrogen receptor� (ER�) (e.g. Cre-ERTs), whose activity is induced by theanti-estrogen tamoxifen (Tam), has allowed efficient spa-tiotemporally controlled somatic mutagenesis of floxedtarget genes in various cell types and tissues (Metzger etal., 2003).

Hepatocyte-specific recombination of floxed DNA seg-ments was obtained in transgenic mice expressing Creunder the control of the rat or mouse serum albumin(SA) promoter/enhancer (Postic and Magnuson, 2000;Yakar et al., 1999) or the mouse SA regulatory elementsand �-fetoprotein enhancers (Kellendonk et al., 2000).To perform temporally controlled hepatocyte-selectiverecombination of floxed genes, we previously estab-lished transgenic mice expressing the ligand-inducibleCre-ERT recombinase (Feil et al., 1996) under the controlof the human �1-antitrypsin promoter (Imai et al., 2000).However, mosaic transgene expression was observed inparenchymal cells, resulting in floxed DNA recombina-tion in only 40–50% of hepatocytes after Tam-treatment.Moreover, transgenic mice expressing under the controlof the transthyretin promoter another Tam-dependentCre recombinase (MerCreMer), which contains mutatedmouse ER� ligand binding domains at both Cre termini,

* Correspondence to: Daniel Metzger, Institut de Genetique et de Biolo-gie Moleculaire et Cellulaire (IGBMC), Centre National de la RechercheScientifique, Institut National de la Sante et de la Recherche Medicale,Universite Louis Pasteur, BP10142, 1 rue Laurent Fries, 67404 Illkirch-Cedex, France.

E-mail: metzger@igbmc.u-strasbg.frGrant sponsors: Marie Curie Individual Fellowship (to M.S.), Centre

National de la Recherche Scientifique, Institut National de la Sante et de laRecherche Medicale, College de France, Hopital Universitaire de Stras-bourg, Association pour la Recherche sur le Cancer, Fondation pour laRecherche Medicale, Human Frontier Science Program, Ministere del’Education Nationale de la Recherche et de la Technologie.

Published online inWiley InterScience (www.interscience.wiley.com)DOI: 10.1002/gene.20039

© 2004 Wiley-Liss, Inc. genesis 39:167–172 (2004)

induced selective floxed DNA recombination in hepato-cytes, but repetitive series of 4-hydroxytamoxifen injec-tions were required to achieve efficient Cre-mediatedrecombination (Tannour-Louet et al., 2002).

To improve temporally controlled targeted mutagene-sis in hepatocytes, the coding sequence of Cre-ERT2,which is about 10-fold more efficiently activated by Tamthan Cre-ERT (Feil et al., 1997; Indra et al., 1999), wasinserted into the mouse SA locus through homologousrecombination in ES cells. To prevent functional inacti-vation of the targeted allele, the Cre-ERT2 cDNA pre-ceded by an encephalomyocarditis virus internal ribo-somal entry site (EMCV IRES) sequence, which encodes

an RNA motif for cap-independent ribosome binding andtranslation of downstream sequences (Pestova et al.,2001), was inserted 3� to the SA stop codon. ES cellswere electroporated with the targeting construct iso-lated from pSACre-ERT2(N) (Fig. 1a), which contains a 7kb genomic segment encompassing exons 13–15 of theSA gene, in which the IRES-Cre-ERT2 expression cassetteand an FRT-flanked neo resistance gene were inserteddownstream of the SA translational termination codon.Southern blot analysis revealed that 6 out of 226 G418resistant ES cell clones (including XF136) had one tar-geted SA allele (Cre-ERT2(N), Fig. 1b, and data notshown). Transfection of clone XF136 with an Flp expres-

FIG. 1. Targeted insertion of Cre-ERT2 coding sequence into the 3� nontranslated region the SA gene. a: Diagram showing the serumalbumin (SA) locus, the targeting vector pSACre-ERT2(N), the targeted allele before [Cre-ERT2(N)] and after [Cre-ERT2] Flp-mediatedexcision of the selection cassette. Exons are represented as numbered boxes. The FRT sites (‹), the IRES-Cre-ERT2pA and theneomycin-resistance cassette, the 5�-, 3�-, and Cre probe, as well as the primers for genotyping are indicated. The size of the expected DNAsegments upon digestion of genomic DNA with BglII and PvuI and hybridization with the 5�- and 3�-probes, and upon digestion with EcoRIand hybridization with the Cre probe, are shown. Relevant restriction sites: B, BamHI; Bg, BglII; E, EcoRI; P, PvuI. b: Southern blot analysisof genomic DNA from wildtype [�/�] and targeted [�/Cre-ERT2(N) and �/Cre-ERT2] ES cells. Genomic DNA was digested with theindicated restriction enzymes and analyzed by Southern hybridization with the 5�-, 3�- and Cre probes (left, middle, and right panel). c:Representative Southern blot of genomic DNA isolated from wildtype and SA�/Cre-ERT2(N) and SA�/Cre-ERT2 animals after digestion, transfer,and hybridization with the 5�-probe (left panel). PCR analysis of tail DNA isolated from SA�/Cre-ERT2(N) and SA�/Cre-ERT2 animals (right panel).d: Detection of the SA mRNA, the SA-Cre-ERT2 fusion transcript, and a reference mRNA (HPRT) by RT-PCR analysis of RNA from variousorgans of an 8-week-old SACre-ERT2/� animal. Primers used to amplify partial cDNAs are shown. li, liver; skm, skeletal muscle; cm, cardiacmuscle; WAT, white adipose tissue; st, stomach; in, intestine; sp, spleen; ki, kidney; li –RT, li without reverse transcription.

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sion vector resulted in excision of the FRT-flanked neogene in 8 out of 85 ES cell clones (Cre-ERT2 allele, Fig.1b, and data not shown), including clone XF136.64. EScells XF136 and XF136.64 were injected into blastocyststo produce chimeric mice that transmitted the tar-geted allele through the germline, thus producingSA�/Cre-ERT2(N) and SA�/Cre-ERT2 mouse lines (Fig. 1c).Intercrosses of heterozygous animals resulted in viableand fertile offspring with the expected Mendelian distri-bution.

The wildtype (WT) SA and bicistronic SA-Cre-ERT2

transcript levels were analyzed in 8-week-oldSA�/Cre-ERT2 mice by semiquantitative RT-PCR. As previ-ously described (Nahon et al., 1988), high levels of WTSA mRNA were present in the liver and lower levels innonhepatic tissues, e.g., stomach, spleen, and kidney. Incontrast, the bicistronic SA-Cre-ERT2 mRNA was onlydetected in the liver, using two different PCR primerpairs (Fig. 1d). Northern blot analysis confirmed that thelevels of the bicistronic transcript in the liver were lowerthan those of the WT SA allele (data not shown).

To analyze Cre-ERT2 protein expression in the liver ofthe SA�/Cre-ERT2 animal, we performed immunohisto-chemical analyses using an anti-Cre antibody. After Tamtreatment of SA�/Cre-ERT2 animals, a Cre-ERT2 nuclearstaining was observed in about 80% of the cells, reflect-ing the percentage of hepatocytes in the adult liver(Saxena et al., 2002), whereas no staining was observedin Tam-treated WT animals (Fig. 2). Similar results wereobtained with SA�/Cre-ERT2(N) animals (data not shown).Thus, both SA�/Cre-ERT2 and SA�/Cre-ERT2(N) lines expressCre-ERT2 in most if not all hepatocytes.

To determine the efficiency and selectivity of DNAexcision, SA�/Cre-ERT2 mice were bred with mice bearingan RXR�af2(I) allele that contains a LoxP-flanked DNAsegment in the RXR� locus [RXR��/af2(I) mice (Mascrezet al., 1998)]. Eight-week-old SA�/Cre-ERT2/RXR��/af2(I)

offspring were daily injected with oil or Tam (1 mg) for5 days and recombination was assessed in various tissues5 days after the last injection. As expected, in oil-treatedanimals no excision of the floxed RXR�af2(I) allele wasdetected by Southern blot analysis. In contrast, in Tam-treated animals about 80% of the floxed RXR�af2(I)alleles were converted into RXR�af2(II) alleles in theliver, but none in other organs (Fig. 3aI, and data notshown). A more sensitive, but nonquantitative PCR-based assay, showed that excision of floxed DNA oc-curred exclusively in the liver of Tam-treated animals(Fig. 3aII), thus confirming the selectivity of Cre-ERT2

expression and the tight regulation of Cre recombinaseactivity in SA�/Cre-ERT2 mice. Similar results were ob-tained with the SA�/Cre-ERT2(N) line (data not shown),indicating that the insertion of the resistance marker inthe SA locus did not interfere with Cre-ERT2 expression.Although it was reported that translational reinitiationfrom IRES sequences decreases with age (Kim et al.,1992), similar recombination efficiencies were observedin the liver of SA�/Cre-ERT2(N)/RXR�af2(I)/� animals thatwere Tam-treated at 4, 12, and 36 weeks (Fig. 3b, anddata not shown). Moreover, similar Cre-ERT2-mediatedrecombination efficiencies were obtained by treatingadult SA�/Cre-ERT2(N)/RXR��/af2(I) animals for 5 days with0.1 mg Tam (Fig. 3b).

In summary, the SA�/Cre-ERT2 and SA�/Cre-ERT2(N) linesexpressing Cre-ERT2 under the control of the endoge-nous SA promoter allow efficient Tam-dependent recom-bination of floxed DNA selectively in hepatocytes. Theyare more potent than the previously described hepato-cyte selective lines (Imai et al., 2000; Tannour-Louet etal., 2002), and thus should be important tools to studythe function of selected genes specifically in hepato-cytes, and to create mouse models for metabolic andliver diseases.

MATERIALS AND METHODS

Knockin of Cre-ERT2 Into the SA LocusA mouse genomic PAC library (RPCI21, K. Osoegawa

and P. de Jong, Roswell Park Cancer Institute) wasscreened for the SA gene by PCR, as recommended bythe RZPD (Deutsches Ressourcenzentrum fuer Genom-forschung, Berlin, Germany). Briefly, primers ZV 293(5�-tgccttttcccagtatctcc-3�) and ZV294 (5�-ttggcggcagact-catcggc-3�) were used to isolate the PAC clone RCIP-711-235N12. A 7-kb BamHI DNA segment including the 3�end of the SA locus was subcloned into the BamHI site ofpBluescriptIISK� (Stratagene, La Jolla, CA), resulting inpSA7B. An IRES-Cre-ERT2 cassette and an FRT-flankedneomycin/kanamycin resistance gene were inserted 3�of the SA coding sequence by homologous recombina-tion in E. coli (Zhang et al., 1998). To this end, PCR-

FIG. 2. Immunohistochemical analysis of Cre-ERT2 expression inthe liver. Immunohistochemistry with a Cre antibody was performedon liver cryosections from 8-week-old Tam-treated wildtype (a anda�) and SA�/Cre-ERT2 animals (b, b�). The red color corresponds tothe staining of the Cre-ERT2 recombinase (b), the blue color to theDAPI signal of nuclear DNA (a�, b�). The magenta color in b� resultsfrom the superimposition of the red color of the Cre signal and theblue color of the DAPI staining.

169TARGETED SOMATIC MUTAGENESIS

amplified 5� and 3� homology DNA segments A1, 280 bp;primers ABC219 (5�-aaggaaaaaagcggccgctctgaatcattt-cacattcc-3�) and ABC220 (5�-agcaattccagtttaaacaggttagtg-gttgtgatgtgt-3�) and A2, 210 bp; primers ABC221(5�-aaggaaaaaagtcgactctcaggtaactatacttgg-3�) and ABC222(5�-catgactgaactggcgcgccctccttttgtgtaaaggcat-3�) werecloned in the PmeI and AscI restriction sites, locatedupstream and downstream of the IRES-Cre-ERT2-FRTneo-FRT cassette of pCre-ERT2.KI2kan, respectively, resultingin pA1-Cre-ERT2.KI2kan-A2. To construct pCre-ERT2.KI2kan, the plasmid pBV.IRES.lacZpA, which con-tains the bovine PGK polyA sequence, was digested withBamHI, blunt-ended, and digested with EcoRI to ex-change the IRES.lacZ fragment with the 2-kb Cre-ERT2

encoding Ecl136II-EcoRI restriction fragment of pCre-ERT2 (Feil et al., 1997), resulting in pBV.Cre-ERT2pA. Theplasmid pSVKeoXI, which contains a neomycin resis-tance gene, was digested with HindIII and SalI, intowhich the 3� FRT site (annealed oligonucleotides ZH30:5�-agctgaagttcctattctctagaaagtataggaacttcggccggccg-3�and ZH31: 5�-tcgacggccggccgaagttcctatactttctagagaatg-aaaacttc-3�) was cloned, followed by digestion with AgeIand BamHI to introduce the 5� FRT site (annealed oligo-nucleotide pairs ZH32: 5�-aaggaagttcctattctctagaaagtat-aggaacttc-3� and ZH33: 5�-gatcgaagttcctatactttctagaga-ataggaactt-3�), yielding pSVKeoF. The SalI/PacI Cre-ERT2pA DNA segment isolated from pBV.Cre-ERT2pAwas ligated into XhoI/PacI digested pSVKeoF, resultingin pCre-ERT2.KI1. A Cre 5� linker (annealed oligonucle-otides ZH34: 5�-atgtccaatctgaccgtacaccaaaatttgcctgcat-taccggtgg-3� and ZH35: 5�-cgcgccaccggtaatgcaggcaaattt-tggtgtacggtcagtaaattgga-3�) was cloned in the NcoI-AscIsites of the EMCV IRES containing plasmid pIZKeoXI.The PmeI/AgeI fragment containing the IRES-Cre-5�linker was ligated into the PmeI/AgeI sites of pCre-ERT2.KI1 plasmid, to give pCre-ERT2.KI2. The AccI-Tth111I restriction segment containing the SV40 early

promoter was replaced with the ClaI-Tth111I segment ofpGK-FRT to insert the eukaryotic PGK and the prokary-otic Tn5 promoter, resulting in pCre-ERT2.KI2kan.

The purified 6-kb EcoNI/AscI restriction fragmentfrom pA1-Cre-ERT2.KI2kan-A2 was coelectroporatedwith pSA7B in JC8679 as described previously (Zhang etal., 1998) and grown on ampicillin- and kanamycin-containing plates. Resistant clones were analyzed byrestriction enzyme mapping and sequencing.

The targeting DNA was released from pSACre-ERT2(N)

by NotI/SmaI digestion, purified on a sucrose gradient,and electroporated into 129Sv/PAS ES cells (Dierich andDolle, 1997). G418 resistant clones were expanded,genomic DNA prepared, and analyzed by Southern blothybridization with the 5� probe (nucleotides 1219 to1641 of the SA cDNA, AJ011413). Clones targeted byhomologous recombination were further analyzed withthe 3� probe (a genomic DNA fragment amplified withthe primers ABC223: 5�-aaggaaaaaagtcgacgaaaaaaaga-catgaagact-3� and ABC224: 5�-catgactgaactggcgcgccag-gcagggattcctctgagc-3�) and a neomycin probe (Chapel-lier et al., 2002). Targeted ES cells were electroporatedwith the Flp expression plasmid pCAGGS-Flpe (Schaft etal., 2001) to excise the neomycin resistance gene. Flp-mediated excision was analyzed on genomic DNA bySouthern blot hybridization with a Cre-specific probe(Feil et al., 1997).

Generation and Genotyping of Mutant Mice

Targeted ES cells were injected into C57BL/6 blasto-cysts. Chimeric male mice were bred with C57BL/6 miceand germline transmission of the targeted allele wasdetermined in agouti offspring by Southern blot analysisof tail DNA (Cammas et al., 2000) with the SA 5� probe.Genotyping of the mouse line SA�/Cre-ERT2(N) was per-formed by PCR using the primers ABT290 (5�-atcatttctt-tgttttcagg-3�) and ABV93 (5�-ggaacccaaactgatgacca-3�) to

FIG. 3. Cre-ERT2-mediated floxedDNA excision. a: Analysis ofgenomic DNA extracted from theindicated tissues of 8-week-oldSA�/Cre-ERT2/RXR��/af2(I) animalstreated either with oil or 1 mg Tam.(I) Southern blot analysis of thewildtype [�], floxed [af2(I)] and ex-cised [af2(II)] RXR� alleles. (II)Ethidium bromide stained agarosegel of PCR products of wildtypeand excised af2(II) RXR� alleles. b:Southern blot analysis of liver andtail DNA from SA�/Cre-ERT2(N)/RXR��/af2(I) animals isolated 5 daysafter 1 mg or 0.1 mg Tam treat-ments. The age of the mice at thebeginning of Tam injections is indi-cated.

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detect the 229 bp DNA segment of the WT allele, andABT292 (5�-ttcgcccccgttttaaccat-3�) and ABV93 to detectthe 500 bp DNA segment of the targeted allele.

PCR reactions for SA�/Cre-ERT2 mouse genotyping con-tained the primers ABT 294 (5�-ttaaacaagcaaaaccaaat-3�)and ABV 93 to detect the 444 bp DNA fragment of thetargeted allele, and primers ABT 290 and ABV 93 todetect the WT allele.

RT-PCR Analysis

Total RNA was isolated using RNA-Solv Reagent(Omega Biotek). Five �g RNA were converted to cDNAwith SuperScript II reverse transcriptase (Invitrogen LifeTechnologies, La Jolla, CA). Two �l of the reverse tran-scriptase reaction (1:20 dilution) were amplified for 25cycles with the following primers and annealing temper-atures: HPRT, QG197 (5�-gtaatgatcagtcaacgggggac-3�)and QG198 (5�-ccagcaagcttgcaaccttaacca-3�), 55°C; Cre,TK139 (5�-atttgcctgcattaccggtc-3�) and TK141 (5�-at-caacgttttgttttcgga-3�), 55°C; SA, AAC41 (5�-ggtccaaacctt-gtcactag-3�) and ZP146 (5�-cctagggtgttgattttaca-3�), 50°C;SA-Cre-ERT2, AAC41 (5�-ggtccaaaccttgtcactag-3�) andABV92 (5�-gcttatcatcgtgtttttca-3�), 55°C. PCR productswere resolved, transferred to a nylon membrane, andsubjected to Southern hybridization with internal, se-quence-specific end-labeled oligonucleotides (Sambrooket al., 1989): QG199 (5�-gctttccctggttaagcagtacagcccc-3�) for HPRT, RJ208 (5�-tcttgcgaacctcatcactc-3�) for Creand ABC220 (5�-agcaattccagtttaaacaggttggtggttgtgatgtgt-3�) for SA and SA-Cre-ERT2.

Cre Immunohistochemistry

Mice were daily injected with 1 mg Tam for 4 days(Metzger et al., 2004) and livers isolated 1 day after thelast injection were immediately frozen in OCT (Sakura,Torrance, CA). Ten �m cryosections were incubated atroom temperature (RT) for 30 s in phosphate-bufferedsaline (PBS) and fixed for 4 min in a 2% PBS-bufferedparaformaldehyde solution. After three washes in PBS,sections were incubated for 30 min in 5% normal goatserum diluted in PBS/0.01% Triton X-100 (5% NGS), andfor 1.5 h at RT in a 1:1,000 dilution of an anti Cre-antibody (PRB-106C; BAbCO, Richmond, CA) in 5% NGS(Weber et al., 2001). Following four washes in PBS/0.01% Triton X-100 (PBST) and 1 wash in PBS, slideswere incubated for 1 h in a 1:400 dilution of a donkeyantirabbit-Cy3 antibody (Jackson ImmunoResearch,West Grove, PA). After four washes in PBST and onewash in PBS, nuclei were stained with DAPI solution andsections were mounted in Vectashield (Vector, Burlin-game, CA; H1000; Brocard et al., 1997).

Analysis of Cre Recombinase Activity at the DNALevel

SA�/Cre-ERT2(N) and SA�/Cre-ERT2 mice were bred withRXR��/af2(I) mice (Mascrez et al., 1998). Double het-erozygous animals were injected for 5 days with vehicleor with 0.1 or 1 mg tamoxifen (Indra et al., 1999;Metzger et al., 2004). Five days after the last injection

organs were isolated and genomic DNA was analyzed bySouthern blotting (Mascrez et al., 1998). The autoradio-graphs were scanned and the signal intensities corre-sponding to both the RXR� wildtype and floxedRXR�af2(I) alleles (A value) and to the recombinedRXR�af2(II) alleles (B value) were determined with theMolecular Analyst software (Bio-Rad, Hercules, CA). Thepercentage of Cre-ERT2 mediated recombination was cal-culated according to the formula: [2B/(A�B)] � 100.PCR detection of Cre-mediated recombination was per-formed as described previously (Feil et al., 1996).

ACKNOWLEDGMENTS

We thank P.O. Angrand and F. Stewart for the pBV.I-RES.lacZpA, pSVKeoXI, pIZKeoXI, pGK-frt, andpCAGGS-Flpe plasmids, B. Chofflet and the animal facil-ity staff for excellent technical assistance, and the secre-tarial staff for typing the manuscript.

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