TNF-R2 induced NF-KB-dependent reporter gene ac-tivation in 293 cells that were not transfected with TNF-R2 (H. Hsu and D. V. Goeddel, unpublished results).This observation is consistent with results obtained fromelectrophoretic mobility-shift assays (Fig. 1).

21. M. Rothe and D. V. Goeddel, unpublished results.22. M. Lewis et al., Proc. Natl. Acad. Sci. U.S.A. 88,

2830 (1991).23. Transient transfection of CT6 cells was done accord-

ing to the DEAE-dextran method [F. M. Ausubel et

al., Current Protocols in Molecular Biology (Green/Wiley, New York, 1994)]. CT6 cells (107) were trans-fected with a total of 10 [ig of plasmid DNA in DEAE-dextran (250 Vug/mI) for 90 min. Reporter gene activ-ity was assayed 40 hours after transfection (18).

24. Similarly, TNF-Rl overexpression in the absence ofexogenous TNF induces apoptosis and NF-KB acti-vation in both HeLa cells and 293 cells [M. P. Boldinet al., J. Biol. Chem. 270, 387 (1995); H. Hsu and D.V. Goeddel, unpublished results].

25. C. Laherty, H. Hu, A. Opipari, F. Wang, V. M. Dixit, J.Biol. Chem. 267, 24157 (1992); M. Rowe et al., J.Virol. 68, 5602 (1994).

26. S. Schutze et al., Cell 71, 765 (1992).27. We thank Z. Cao, V. R. Baichwal, and S. L. McKnight

for helpful comments on the manuscript. Supportedin part by National Institutes of Health grantCA64803 to V.M.D.

1 May 1995; accepted 1 1 July 1995

Inducible Gene Targeting in MiceRalf KOhn,* Frieder Schwenk, Michel Aguet,t Klaus Rajewsky

A method of gene targeting that allows the inducible inactivation of a target gene in miceis presented. The method uses an interferon-responsive promoter to control the expres-sion of Cre recombinase. Here, Cre was used to delete a segment of the DNA polymerase, gene flanked by loxP recombinase recognition sites. Deletion was complete in liver andnearly complete in lymphocytes within a few days, whereas partial deletion was obtainedin other tissues. This method can be used for the inducible inactivation of any other genein vivo.

Studies of gene function in mice ofteninvolve the analysis of embryonic stem (ES)cell-derived gene-targeted mice. Such micecarry a predesigned mutation in their germline and are devoid of a particular geneproduct throughout ontogeny (1). A strat-egy for conditional, cell type-specific genetargeting was recently developed (2) thatuses the Cre/loxP recombination system ofbacteriophage P1 (3). In this procedure, atarget gene is flanked ("floxed") with re-combinase recognition (loxP) sites intro-duced by homologous recombination in EScells; the amount of expression of the floxedallele should be the same as that in the wildtype unless it is inactivated by Cre-mediat-ed deletion of the loxP-flanked gene seg-ment. Gene inactivation can be restrictedin vivo to a particular cell type by crossinga mouse strain harboring the floxed allele toa transgenic strain expressing Cre recombi-nase constitutively under the control of acell type-specific promoter (2).

These approaches have a major draw-back. The function of the gene productmust be deduced from the phenotype ofanimals that are, either in all cells or incertain cell types from an early time pointon, constantly deficient throughout ontog-eny for the product of the disrupted gene. Amutant organism may compensate for theloss of a gene product so that no obviousdeviation from the wild type is seen, or the

R. Kuhn, F. Schwenk, K. Rajewsky, Institute for Genetics,University of Cologne, Weyertal 121, 50931 Cologne,Germany.M. Aguet, Institute of Molecular Biology I, University ofZurich, Honggerberg, 8093 Zurich, Switzerland.

*To whom correspondence should be addressed.tPresent address: Genentech Inc., 460 Point San BrunoBoulevard, South San Francisco, CA 94080, USA.

organism may react to the mutation to givea complex, secondary phenotype. More-over, if the complete loss of a gene productresults in embryonic lethality (4), genefunction at later stages of development can-not be analyzed. To overcome these limita-tions, we have developed a method to in-duce gene inactivation in mice at a giventime during ontogeny. This method uses theinducible promoter of the mouse Mxl gene(5) to control the expression of a Cre re-combinase transgene. Mxl, part of the de-fense to viral infections, is silent in healthymice. The Mxl promoter can be transientlyactivated to high amounts of transcriptionin many tissues upon application of inter-

feron x (IFN-ox) or IFN-P or of syntheticdouble-stranded RNA [polyinosinic-poly-cytidylic acid (p1-pC), an IFN inducer (6,7)1. Hence, mice harboring a floxed targetgene and a Mx-cre transgene acquire aninactivating mutation of the target geneupon treatment of the mice with IFN orpI-pC.

To assess the efficiency of induced Cre-mediated deletion of a target gene, we gen-erated mice transgenic for a Mx-cre expres-sion vector (8) and crossed them to a strainharboring an allele of the DNA polymerase1 gene that contains two directly repeatedloxP sites. This allele, pol-fl-x (2), is inacti-vated upon excision of a loxP-flanked 1.5-kb segment containing the promoter regionand first exon of the pol13 gene. Cre-medi-ated deletion of the floxed gene segment ina given tissue can be quantified by Southernblot analysis of genomic DNA, which en-ables comparison of the intensities of theband from the pol13O'x allele and the frag-ment derived from the deleted (polp3A) al-lele (Fig. 1).

Two of 42 Cre transgenic strains gener-ated (Mx-cre31a and 31b) (8) exhibitedlow background recombination, but exten-sive deletion could be induced by treatmentof adult mice with IFN or pI-pC. Groups offive mice (strain Mx-cre31a) received ei-

Fig. 1. Detection of Cre- A Nontreated pl-pC treatedmediated deletion. (A) B B , .Southern blot strategy to , Bdistinguish pol(3 wild- ok10 - * 4bdI* Floxtype (+) (upper line), B4.5kbpo/e110x (middle line), and B 1k BC a . ;WT

polo,' (lower line) alleles. ,Bam HI fragments (B) B B' ' B Floxcontaining the promoter3kAregion (ellipses), the first D 4WA1644 |;; ^ Floxtwo exons of the polo > Agene (numbered rectan-gles), and an adjacent genomic probe used for hybridization (bar) are shown. In the 1 2 3pol4fiOX allele a 1.5-kb gene segment has been flanked by two loxP sites (triangles) E[f Flox(2). Upon Bam HI digestion the wild-type, polp3fl0x, and pol/A alleles yield fragmentsof 10, 4.5, and 3 kb, respectively (2). (B through D) Southern blot analysis of DNALfAfrom liver (B) and spleen (D) of groups of pol1Oflox/+ Mx-cre transgenic mice and fromliver (C) of polflox/pol/flox Mx-cre transgenic mice (strain Mx-cre31 a) that received three intraperitoneal(IP) injections of pl-pC (250 fig) at 2-day intervals or were left untreated. Genomic DNA was prepared 2days after the last injection. The positions of the fragments derived from the polo wild-type (WT), pol3liox(Flox), and poloA (A) alleles are indicated. Each lane represents one mouse. In (C) the wild-type bands areabsent but a weak background band appears at the same position. (E) Southern blot analysis of DNAfrom liver of mice (strain Mx-cre31 a) that received three IP injections of human IFN- 2/ax1 (1 06 units; lane3) (12) or pl-pC (250 ,g; lane 2) in comparison to a nontreated mouse (lane 1). Female littermatesheterozygous for the Mx-cre transgene were used at an age of 8 weeks.

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ther three injections of pl-pC at 2-day in-tervals or were left untreated. GenomicDNA was prepared from various organs ofthese mice 2 days after the last injection.Deletion of the floxed polo gene segmentwas complete (100% deletion) in the liverof treated mice heterozygous or homozygousfor the polI300x allele, whereas the back-ground recombination was in the range of1% in nontreated mice (1 + 2%, mean ±SD; Figs. 1, B and C, and 2). In other organsthe extent of induced deletion was variable,ranging from 94% in spleen (9) to 8% inbrain (Figs. iD and 2) (10), whereas nodeletion was found in testis (1 1). This het-erogeneity may reflect differing proportionsof IFN-responsive cells or IFN availabilityin various tissues.

Similar amounts of deletion were ob-tained when recombinant human IFN-t2/jaI(12) was used for induction instead of pl-pC(Fig. lE). The efficiency of Cre-mediateddeletion was similar in organs from heterozy-gous and homozygous polflx mice (Fig. 2) aswell as hemizygous polfl'ox/pol13 mice (1 1).Background recombination in nontreatedmice was undetectable in some tissues butwas as much as 10% in spleen (Fig. 2),possibly because of the endogenous produc-tion of IFN in these mice, which were main-tained in a conventional facility. In Mx-cremice kept under specific pathogen-free(SPF) conditions, endogenous IFN produc-tion will be lower and hence this backgroundrecombination may be reduced.

In addition to their antiviral activity,IFN-oa/1 and pI-pC exert transient antipro-liferative and immunomodulatory effects invitro and in vivo (7). When the liver andkidney of mice treated three times with pl-pC were examined histologically 2 days afterthe last injection, no gross abnormalitiescompared to nontreated controls were found(13). In spleen, the populations of B and Tlymphocytes appeared normal by flow cyto-metric analysis (14) whereas the number ofthymocytes, known to be reduced by stress(15), was diminished by 50% in pl-pC-treat-ed mice and by 25% in IFN-treated mice.Although the treatment of mice with IFN-cx/p or pl-pC has side effects, we believe thatthese effects are limited to the brief exposureof the mice to the inducing agent and do notimpair the utility of the IFN-induced genetargeting system. However, to minimize po-tential side effects, we suggest the use ofIFN-o/13 as the inducer of choice becausepl-pC can stimulate the production of cyto-kines other than IFN, such as tumor necrosisfactor ox (TNF-ox) (16).A single pl-pC injection was sufficient

to induce the deletion of 97% of the pol1''xallele within 2 days in the liver of Mx-cretransgenic mice (Fig. 3). A second injectionafter 2 days did not increase the amount ofdeletion, probably because the first stimu-

1428

Fig. 2. Deletion of the po/gflOx allele in variousorgans of pol/3Ox/+ Mx-cre transgenic mice (flox+) and popfl'ox/po/flOx Mx-cre transgenic mice(flox/flox). Groups of 5 mice (8-week-old females,strain Mx-cre31 a) were treated with pi-pC as de-scribed in Fig. 1 (solid bars) or were left untreated(striped bars) and genomic DNA was preparedfrom the indicated organs 2 days after the lastinjection. Cre-mediated deletion of the polQfIOx al-lele was detected by Southern blot analysis andquantified with a Bio-lmaging analyzer (Fuji Bas1000). Percentages of deletion (mean ± SD) werecalculated by subtraction of the background ra-dioactivity measured between the bands from thepol/fpox and pol3a alleles from the activity mea-sured in these bands; areas of the same size were

100

cc 80-

X 60-

0-

.2 20-

oC)

HE

flox!+ floxiftlox

used for all measurements. The remaining activity was added and set as 100%, and the proportion of thepolao allele was calculated. This method gave values of 46 to 54% for the ratio of po/,BflOx to pol/f" allelesin the genome of polpflOx/po/,13 mice in independent experiments (11).

lation elicits a state of IFN hyporesponsive-ness that lasts longer than 48 hours (7).After a third dose of pl-pC, deletion wascomplete in liver and was increased inspleen and heart (Fig. 3). Thus, Cre-medi-ated deletion can occur rapidly and veryefficiently in differentiated cells in vivo andis almost complete in liver after a singleinjection of pl-pC. These results demon-strate that gene inactivation can be inducedwith high efficiency in mice within a fewdays and that Cre-mediated deletion isachievable in organs composed mainly ofresting cells. We have also shown that bothalleles of a loxP-modified gene can be de-leted in vivo with the same high efficiencyas a single loxP-flanked allele in heterozy-gous mice; this result establishes the feasi-bility of the conditional gene targeting

Days0 2 4 6

100.

80/

M 60-40

0

s400= 20-

pi-pC injections

Fig. 3. Cre-mediated deletion of the pol/tflox allelein relation to the number of pi-pC injections.Groups of two polBfiox/+ Mx-cre transgenic mice(8 weeks old, strain Mx-cre31 b) received one,two, or three IP injections of p1-pC (250 [ig) at2-day intervals and genomic DNA was preparedfrom liver (*), spleen (R), and heart (0) 2 days afterthe last injection (at day 2, 4, or 6). Deletion of thepolpf'Ox allele (mean + SD) was measured as de-scribed in Fig. 2; for most data points the deviationwas too small to be visualized. Values obtainedfrom nontreated mice (day 0) are shown for com-parison. Deletion in spleen of Mx-cre 31 b mice isnot as efficient as in Mx-cre31 a mice (Fig. 2).

strategy because both alleles of a target genemust be inactivated to investigate genefunction in a mutant.

The ubiquitously expressed DNA poly-merase P fills small single-stranded DNAgaps and is considered to be part of themammalian DNA repair machinery (17).Polymerase P-deficient mice derived fromgene-targeted ES cells are not viable (2).Thus, the lEN-inducible gene targeting sys-tem will be a valuable tool to investigatethe possible roles of polymerase P in vivo,such as DNA repair in liver and, potential-ly, somatic hypermutation in B lympho-cytes. The system should prove especiallyuseful in studies of gene function in liverand the immune system because extensivedeletion can be obtained in these tissues.The partial deletion obtained in other tis-sues of Mx-cre mice may be sufficient forgene inactivation experiments that result inthe gain of a new, easily detectable pheno-type and do not depend on the inactivationof a gene in all cells of a tissue. At present,Cre-mediated gene deletion in Mx-cre micecannot be restricted to certain tissues. How-ever, gene inactivation could be confinedto a certain cell type if Mx-cre mice werecrossed to IFN-o4/P receptor-deficient mice(18) reconstituted with a murine or humanIFN-a/o3 receptor gene controlled by a celltype-specific promoter.

REFERENCES AND NOTES

1. B. H. Koller and 0. Smithies, Annu. Rev. Immuno!.10, 705 (1992); D. W. Melton, Bioessays 16, 633(1994).

2. H. Gu, J. D. Marth, P. C. Orban, H. Mossmann, K.Rajewsky, Science 265,103 (1994).

3. N. Steinberg and D. Hamilton, J. Mol. Biol. 150, 467(1981); B. Sauer and N. Henderson, Proc. Natl.Acad. Sci. U.S.A. 85, 5166 (1988).

4. A. J. Copp, Trends Genet. 11, 87 (1995).5. H. Hug, M. Costas, P. Staeheli, M. Aebi, C. Weiss-

mann, Mol. Cell. Biol. 8, 3065 (1988); H. Amheiter, S.Skuntz, M. Noteborn, S. Chang, E. Meier, Cell 62, 51(1 990).

6. P. Staeheli, P. Danielson, 0. Hailer, G. Sutcliffe, Mol.Cell. Biol. 6, 4770 (1986); G. C. Sen and R. M. Ran-sohoff, Adv. Virus Res. 42, 57 (1993); B. R. G. Wil-

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liams, Eur. J. Biochem. 200,1 (1991); K.-C. Chang,G. Goldspink, J. Lida, Arch. Virol. 110, 151 (1990).

7. E. de Clerq, Antibiot. Chemother. (Basel) 27, 251(1980); I. Gresser, Cell. Immunol. 34, 406 (1977); F.D. Finkelman et al., J. Exp. Med. 174,1179 (1991).

8. To construct the Mx-cre expression vector, we ligateda 2.3-kb Eco RI-Bam HI fragment containing the Mx1gene promoter (5) to the modified coding region of Cre[H. Gu, Y. R. Zou, K. Rajewsky, Cell 73, 1155 (1993)1,which includes a nuclear localization signal, and to a2.1 -kb fragment of the human growth hormone gene,which provides splicing and polyadenylation signals[K. E. Chaffin et al., EMBO J. 9, 3821 (1990)1. Trans-genic mice were produced by pronucleus injection offertilized (C57BU6x CBA)F2 eggs. Founder mice werefirst screened for IFN-inducible expression of Cre withthe use of peripheral blood lymphocytes. Of 42 strainsgenerated, 16 showed inducible Cre expression and

were crossed to mice harboring the pol,3f'Ox allele totest for Cre-mediated deletion in vivo. Strains Mx-cre31 a and 31 b, which contain 10 transgene copiesand orginate from the same founder mouse but aredistinguished by their transgene integration pattern,showed the most extensive deletion.

9. The extent of deletion was comparable in purified Bcells, T cells, and non-B-non-T cells (11).

10. These results were reproducible in several indepen-dent experiments (R. Kuhn, unpublished data).

11. R. Kuhn, unpublished data.12. H. Weber, D. Valenzuela, G. Luijber, M. Gubler, C.

Weissmann, EMBO J. 6, 591 (1987).13. J. Lohler, unpublished data.14. Stainings were performed with antibodies to B220,

immunoglobulin M, CD3, CD4, and CD8 (R. Kuhn,unpublished data).

15. T. F. Dougherty, Physiol. Rev. 32, 379 (1952).

16. M. G. Whatelet, P. M. Berr, G. A. Huez, Eur. J.Biochem. 206, 901 (1992).

17. R. K. Singhal and S. H. Wilson, J. Biol. Chem. 268,15906 (1993); T. S.-F. Wang, Annu. Rev. Biochem.60, 513 (1991); K. Wiebauer and J. Jiricny, Proc.Natl. Acad. Sci. U.S.A. 87, 5842 (1990).

18. U. Muller et al., Science 264, 1918 (1994).19. We thank J. Lohler for the histological analysis, H. Gu

for polf gene-targeted mice, C. Weissmann for IFN,and U. Ringeisen for graphical work. Supportedby Deutsche Forschungsgemeinschaft grant SFB243, the Human Frontier Science Program, theBundesministerium fur Forschung und Technologie,and the Land Nordrhein-Westfalen. F.S. is support-ed by a stipend from the Boehringer IngelheimFonds.

17 May 1995; accepted 18 July 1995

The Chromodomain Protein Swi6: A KeyComponent at Fission Yeast Centromeres

Karl Ekwall,* Jean-Paul Javerzat,*t Axel Lorentz,Henning Schmidt, Gwen Cranston, Robin Allshiret

Centromeres attach chromosomes to the spindle during mitosis, thereby ensuring theequal distribution of chromosomes into daughter cells. Transcriptionally silent hetero-chromatin of unknown function is associated with centromeres in many organisms. In thefission yeast Schizosaccharomyces pombe, the silent mating-type loci, centromeres, andtelomeres are assembled into silent heterochromatin-like domains. The Swi6 chromo-domain protein affects this silencing, and now it is shown that Swi6p localizes with thesethree chromosomal regions. In cells lacking Swi6p, centromeres lag on the spindle duringanaphase and chromosomes are lost at high rates. Thus, Swi6p is located at fission yeastcentromeres and is required for their proper function.

Cytologically observable heterochromatinin Drosophila and mammals is, in general,incompatible with gene expression (1, 2).The function of these transcriptionally silentregions remains enigmatic (1, 2). Highereukaryotic centromeres, with their associat-ed kinetochore complex, are formed in thevicinity of centromeric heterochromatin, butwhether heterochromatin itself contributesto centromere function is unknown. Onepossibility is that transcriptional repressionat centromeres, and therefore heterochroma-tin, is a manifestation of normal centromere-kinetochore assembly. If this is true, thencertain gene products should localize withcentromeres and play a role in both theformation of silent heterochromatin andcentromere function.

In the fruit fly Drosophila melanogaster andin mammals, proteins containing chromodo-

K. Ekwall, J.-P. Javerzat, G. Cranston, R. Allshire, Medi-cal Research Council Human Genetics Unit, WesternGeneral Hospital, Crewe Road, Edinburgh EH4 2XU,Scotland, UK.A. Lorentz and H. Schmidt, Institit fur Genetik, TU Braun-schweig, Sperlmannstrasse 7, D-38106 Braunschweig,Germany.*These authors contributed equally to this work.tPresent address: Laboratoire de Genetique, Universitede Bordeaux II, Avenue de Facultes, 33405 Talence,France.tTo whom correspondence should be addressed.

mains, a motif first described as a region ofsimilarity between HP-1 and Polycomb (3),are associated with heterochromatin or im-pose repression in a temporal manner onimportant developmental genes (4-10). Mu-tations in the Drosophila gene Su(var)205,which encodes the chromodomain proteinHP-1, were originally identified as suppres-sors of variegating position effects, whichsuggested a role for HP-1 in the formation ofheterochromatin (11). In the fission yeastSchizosaccharomyces pombe, the silent mat-ing-type loci, centromeres, and telomeresshare several properties with heterochromat-ic domains (12-14). The swi6+ gene of fis-sion yeast encodes a chromodomain proteinand is required to maintain transcriptional

repression at the silent mating-type loci andcentromeres (15, 16). Mutations in the swi6gene result in a highly increased rate ofchromosome loss (16). The chromodomainof Swi6p is 46% identical to that of Drosoph-ila HP-1 across this 50-amino acid domain(15). We now present evidence that theSwi6 protein is an important functionalcomponent at fission yeast centromeres.A purified fusion protein was used to

raise a polyclonal serum and to affinity-purify Swi6p-specific antibodies (anti-Swi6p). Protein immunoblotting with anti-Swi6p detected a single band migrating at-50 kD in extracts from wild-type strains(Fig. 1A) (17). This band was absent inextracts from strains in which the swi6+gene is deleted (swi6::his 1), and only a faintsignal was seen in extracts from strains withthe missense swi6-1 15 allele (W269R).

Immunofluorescence analysis with affin-ity-purified anti-Swi6p produced a distinctpattern of staining in fixed cells (17). Sev-eral discrete spots were seen in interphasehaploid and diploid cells (red, Fig. 1, B andC). Most haploid and diploid cells con-tained three and five spots of anti-Swi6pfluorescence, respectively (Fig. 1E). Nostaining with anti-Swi6p was detected instrains harboring a complete deletion of theswi6 gene (Fig. 1D).

Mutations at swi6 cause a high rate ofchromosome loss during mitosis (Table 1)and affect silencing within the centromere(16). It therefore seemed plausible thatsome of the anti-Swi6p spots might repre-

Table 1. Frequency of chromosome lagging and loss in wild-type and swi6 mutants at 180C.

Back-Cells in late Late anaphase Rate of minichro-Back- anaphase cells with lagging mosome loss (Ch1 6)

ground (%~)0 centromeres (%)* per divisiontWild type 3.5 <1.0 (0/98) <0.08 (0/1241)swi6::his' 3.7 48.8 (39/80) 4.6 (17/370)swi6-115 2.7 24.5 (15/61) 1.3 (11/849)*Late anaphase cells were defined as cells with a spindle length greater than 5 pim. Cells were classified as havinglagging centromeres if a single centromere FISH signal was detected at least 1.5 pKm away from an anaphase spindlepole (as in Fig. 4B). For all strains, the frequency of lagging centromeres and minichromosome loss were measured incells from the same culture. Similar results were obtained in different cultures. tThe rate of minichromosome losswas determined as described (16).

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(5229), 1427-1429. [doi: 10.1126/science.7660125]269Science R Kuhn, F Schwenk, M Aguet and K Rajewsky (September 8, 1995) Inducible gene targeting in mice

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