Generation of Smad4/Dpc4 Conditional Knockout MiceXiao Yang,1 Cuiling Li,1 Pedro-Luis Herrera,2 and Chu-Xia Deng1*1Genetics of Development and Disease Branch, NIDDK, National Institutes of Health, Bethesda, Maryland2Department of Morphology, University of Geneva Medical School, Geneva, Switzerland

Received 16 November 2001; Accepted 9 December 2001Published online 00 Month 2001

Smad4/Dpc4 is a central mediator of TGF-� signals (Mas-sague, 1998). It was previously shown that loss ofSmad4 results in lethality at embryonic (E) days 6–7 due

to impaired extraembroynic membrane formation anddecreased epiblast proliferation (Sirard et al., 1998; Yanget al., 1998). To overcome the early lethality and to studyfunctions of Smad4 at later stages, we generated aSmad4 conditional knockout allele using the Cre-loxPapproach (Nagy, 2000).

Smad4 was targeted by ploxPneoSmad4, a cotransfer-type targeting vector (Deng et al., 1993), which intro-duced a ploxPneo cassette (Yang et al., 1998) and a thirdloxP into intron 8 and 7 of the Smad4 locus, respectively(Fig. 1A). The targeted ES cell clones were identified bySouthern blot analysis using both flanking and internalprobes (Fig. 1B). Chimeras were generated by injectionsof targeted ES cells into C57BL/6 blastocysts. After germ-line transmission by crosses with Black Swiss mice, wefound that the presence of the ploxPneo in intron 8completely blocked normal splicing of Smad4 andcaused recessive lethality at around E7 (not shown), aphenotype resembling that of Smad4-null embryos(Yang et al., 1998). Therefore, we removed the plox-Pneo cassette from the germ line by crossing the het-erozygous mutant (Smad4neo-Co/�) mice with EIIa-Cretransgenic mice (Lakso et al., 1996) ( Fig. 2A). This crossgenerated three possible alleles due to Cre-mediatedrecombination among three loxP sites. The recombina-tion between loxP2 and loxP3 deleted the neo cassetteand generated the Smad4 conditional allele (Smad4Co,which contains two loxP sites flanking exon 8 of Smad4(Fig. 2B). Recombination between loxP1 and loxP3 de-leted exon 8 (Smad4�8, Fig. 2C). Since the Cre-mediatedrecombination only removed exon 8, we assessed thehomozygous (Smad4 �8/�8) phenotype to determinewhether the deletion could generate a null allele. Wefound the Smad4 �8/�8embryos died at E7–8, suggestingthat the conditional allele should be null after recombi-nation (Fig. 2E).

To determine whether the Smad4 conditional knock-out allele is active, the Smad4Co/� mice were further

Xiao Yang’s current address is: Institute of Biotechnology, 20 DongdajieStreet, Beijing, Peoples Republic of China 100071.

* Correspondence to: Chu-Xia Deng, Chief, Mammalian Genetics Section,GDDB, NIDDK, National Institutes of Health, 10/9N105, 10 Center Drive,Bethesda, MD 20892.

E-mail: chuxiad@bdg10.niddk.nih.gov†This article is a US government work and, as such, is in the public

domain in the United States of America.

FIG. 1 Introduction of loxP sites into the Smad4 locus. (A) Targetingvector was constructed by inserting a 4.8-kb HpaI-NotI fragment(the NotI site is from the polylinker of pBluescript SK, Stratagene)that is 3� to exon 8 of the Smad4 gene into the ploxPneo vector(Yang et al., 1998). The resulting construct was digested with ClaIand Asp718, followed by insertion of a 4.8-kb HpaI-Asp718 frag-ment (the HpaI site is from the polylinker of the vector). A 60bp-linker containing loxP sequence was inserted into the HpaI sitein intron 7. This vector gives about a 10% targeting frequency inTC-1 ES cells (Deng et al., 1996). (B) Targeted events were identifiedby Southern blot analysis of Ev (EcoRV)-digested genomic DNAswith a 5� flanking probe (probe a). The wild-type clones only show afragment of 9.5 kb and the targeted clones showed an additionalfragment of 5.5 kb due to the introduction of an EcoRV site. TheEcoRV-digested genomic DNAs were also blotted using an internalprobe (probe b) to verify the presence of the ploxPneo gene. In thiscase, the targeted ES clones showed a fragment of 3 kb in addi-tional to the wild-type fragment of 9.5 kb.

© 2002 Wiley-Liss, Inc. genesis 32:80–81 (2002)DOI 10.1002/gene.10029

crossed with mice that carry a Pdx-Cre transgene (Her-rera, 2000). DNA isolated from multiple tissues/organs ofSmad4Co/�;Pdx-Cre mice was subject to PCR analysis todetermine Cre-mediated recombination (Fig. 2D). Ourdata revealed Cre-mediated recombination in the pan-creas but not in a number of other tissues/organs tested,indicating that our Smad4Co allele can be recombined todelete exon 8 in a tissue-specific fashion. Therefore, webelieve that these Smad4 conditional knockout miceshould serve as a useful tool to study functions of TGF�/Smad4 signals at all stages of mouse development.

ACKNOWLEDGMENTS

We thank H. Westphal for providing EIIa-Cre transgenicmice and S. Mackem for critically readying the manu-script.

LITERATURE CITED

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FIG. 2 Generation and validation of Smad4 conditional knock-out allele. (A) Because the Smad4neo-Co/neo-Co mice were lethal,we deleted the ploxPneo cassette from the germ line by cross-ing the Smad4neo-Co/� mice with EIIa-Cre transgenic mice onan FVB genetic background using an approach describedrecently (Xu et al., 2001). (B, C) Cre-mediated recombinationbetween loxP2 and loxP3 (B) and loxP1 and loxP3 (C) areshown. (D) Tissue-specific inactivation of the Smad4 condi-tional allele by a Pdx-Cre transgene was detected by PCRanalysis using primer pair a/b, which amplifies about 450 bpfrom the conditional allele and 390 bp from the wild-type allele,and primer pair a/c, which amplifies about 500 bp from therecombined allele. Sequences for primers a, b, and c are:5�-AAGAGCCACAGGTCAAGCAG-3�, 5�-GGGCAGCGTAGC-ATATAAGA-3�, and 5�-GACCCAAACGTCACCTTCAG-3�, re-spectively. DNA was isolated from liver (1), spleen (2) andpancreas (3) of Smad4Co/�;Pdx-Cre mice. (E) Smad4�8/�8 micedied at E7, indicating that the Smad4Co is null afterrecombination.

81SMAD4/DPC4 CONDITIONAL KNOCKOUT MICE