Proc. Natl. Acad. Sci. USAVol. 92, pp. 160-164, January 1995Genetics

Site-specific recombination of a transgene in fertilized eggs bytransient expression of Cre recombinase

(microinjection/chicken j3-actin promoter/(3-galactosidase)

KIMI ARAKI*t, MASATAKE ARAKI*t, JUN-ICHI MIYAZAKI4, AND PIERRE VASSALLI*§*Department of Pathology, Centre Medical Universitaire, University of Geneva, CH 1211, Geneva 4, Switzerland; and *Department of Disease-Related GeneRegulation Research (Sandoz), University of Tokyo, Faculty of Medicine, Hongo 7-3-1, Bunkyo-ku Tokyo 113, Japan

Communicated by K Rajewsky, Institut fiir Genetik der Universitat zu Koin, Cologne, Germany, September 26, 1994

ABSTRACT An efficient method oftransgene modulationin fertilized eggs has been developed that uses the Cre/loxPrecombination system. Twelve transgenic mouse lines carry-ing a chicken P-actin promoter-4oxP-chloramphenicol acetyl-transferase (CAT) gene-oxP-j3-galactosidase gene constructwere produced. After selection of the line showing the highestexpression of the CAT gene in a variety of tissues, eggs of thisline were injected in the male or female pronucleus with a Creexpression vector placed under the control of the chickenf8-actin promoter and kept in a circular form to avoid genomicintegration. This resulted in a transient expression of Cre inthe eggs, leading to recombination ofthe transgene as detectedby galactosidase expression and DNA analysis. Recombina-tion was completed before the morula stage with both types ofpronuclear injections and occurred with a very high fre-quency; no mosaicism, no incomplete recombination, and nointegration of the Cre sequence were observed in 18 mice bornwith this modified transgene. The 8-galactosidase gene wasexpressed in various tissues at levels comparable to thosefound for the CAT gene in the founder line. This Cre transientexpression system should be useful for breeding transgeniclines in which transgene expression leads to sterility orlethality--in particular, for selecting transgenic lines withhigh expression of a potentially lethal transgene whose fullactivity is difficult to explore in a conventional transgenicsystem because of the risk of selecting for transgenic linescarrying only poorly expressed transgenes.

Development of the technology for gene transfer in the germline (1), leading to the production of transgenic animals, hashad an enormous impact in biology. When the transgeneresults from random integration of a DNA fragment injectedinto a fertilized egg's pronucleus, its level of expression in theresulting transgenic animal varies and depends upon factorswhich cannot be controlled, such as the integration site and thetransgene copy number. Usually, several transgenic mouselines are created to ensure that the phenotype resulting fromthe presence of a transgene is reproducible and/or to selectlines with high expression of the transgene. However, specialdifficulties arise when expression of the transgene results insterility or is lethal before the age of reproduction. Thissituation requires the repeated production of new transgenicmice, each with a different transgene integration site and copynumber, resulting in unwanted variations in experimentalconditions. An additional complication may arise when highexpression of a transgene is lethal, since transgenic founderembryos with weak expression of the transgene will be se-lected. Thus, observation of a partial phenotype may result inerroneous interpretations concerning the exact functions ofthe explored gene.

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

To overcome these difficulties, expression of a potentiallytoxic transgene should be suppressed. This would allow the useof a single founder line stably transmitting the same transgene,provided reexpression of the transgene can be achieved underconditions controlled by the investigator. This could be ob-tained by a DNA recombination event mediated by the bac-teriophage P1 cre gene, which encodes a recombinase, Cre,cleaving DNA at loxP sequences. The transgene may beintroduced into a mouse in a "silenced" form by placing aloxP-flanked DNA sequence between its promoter and itscoding sequence, thus preventing transcription. The transgenemay be "reactivated" by mating mice of this line with mice ofanother transgenic line, expressing a cre transgene. Applica-tion of the Cre/loxP system to transgenic mice has alreadybeen reported, indicating efficient excision of the DNA lo-cated between the two loxP sequences in doubly transgenicmice, at least in tissues expressing the cre gene (2, 3). The aimof the present study was to develop a technique which allowsthe rapid and reproducible removal of the transgene silencingsequence in all cells from a mouse, without introducing anyother permanent genetic modification. This was achieved bymicroinjecting a cre gene into fertilized eggs, where it is onlytransiently expressed. In addition, we have used, as a reporter/silencer sequence, a loxP-flanked chloramphenicol acetyl-transferase (CAT) gene; expression of this reporter gene canthus be used for the selection of transgenic founder lines withthe highest expression level.

MATERIALS AND METHODSPlasmid Constructions and Production ofTransgenic Mice.

The Cre expression vector pCAGGS-Cre was constructed byinserting the cre gene, obtained as a 1.1-kb Xho I-Mlu Ifragment from pBS185 (ref. 4; gift from B. Sauer), into theEcoRI site of pCAGGS (5). The transgene vector pCAG-CAT-Z, containing a chicken 3-actin gene (CAG) promoter-loxP-CAT gene-4oxP-4acZ region, was constructed frompCAG-lacZ (5) by inserting a CAT gene [derived frompBLCAT3 (6)] flanked by directly repeated loxP sequences[derived from pBS64 (7), gift from B. Sauer] into the HindIIIsite located between the CAG promoter and lacZ. This 6.9-kbconstruct (Fig. 1) was purified by electrophoresis and elutionfrom NACS PREPAC (BRL) and used for microinjection.The plasmid pCAGGS-Cre was purified by NACS PREPAC,suspended in 1 mM Tris-HCl, pH 7.5/0.1 mM EDTA) at 5ng/,ul, and used for microinjection. Transgenic mice wereproduced as described (8).

Abbreviations: CAG, chicken ,-actin promoter; CAT, chloramphen-icol acetyltransferase; X-Gal, 5-bromo-4-chloro-3-indolyl P-D-galactopyranoside.TPresent address: Institute of Molecular Embryology and Genetics,Kumamoto University School of Medicine, Kuhonji 4-24-1, Kum-amoto 862, Japan.§To whom reprint requests should be addressed.

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Southern Blot Analysis and PCR. Tails and tissues werelysed with NaDodSO4/proteinase K (Boehringer Mannheim)and then treated with phenol/chloroform, 1:1 (vol/vol), pre-cipitated with ethanol, and dissolved in 10 mM Tris HCl, pH7.5/1 mM EDTA). DNAs (5 ,ug) were subjected to electro-phoresis in a 1% agarose gel. After transfer to nylon mem-branes (Boehringer Mannheim), Southern hybridizations wereperformed with probes 32p labeled by random priming: (9).For PCR analysis, DNAs (0.5-1 jig) were submitted to 27 or32 cycles of amplification [with each cycle consisting of 1 minat 94°C, 2 min at 55°C (or 65°C for cre primers), and 2 min at72°C] on a thermal cycler. The 5' primers and 3' primers wereCAT2 (5'-CAGTCAGTTGCTCAATGTACC-3') and CAT3(5'-ACTGGTGAAACTCACCCA-3'), respectively, for theCAT gene; Zl (5'-GCGTTACCCAACTTAATCG-3') andZ2 (5'-TGTGAGCGAGTAACAACC-3'), respectively, forthe lacZ gene (2); and Crel (5'-GGACATGTTCAGG-GATCGCCAGGCG-3') and Cre2 (5'-GCATAACCAGT-GAAACAGCATTGCTG-3'), respectively, for the cre gene.The 5'primer and 3'primer for exploring the junction betweenCAG promoter and lacZ were AG-2 (5'-CTGCTAACCAT-GTTCATGCC-3') and Z3 (5'-GGCCTCTTCGCTATTACG-3'), respectively. Half of the reaction volume was analyzed byelectrophoresis in a 1.4% agarose gel.

Detection of CAT and 18-Galactosidase Activities. Organswere homogenized in extraction buffer (15 mM Tris HCl, pH8.0/60 mM KCl/15 mM NaCl/2 mM EDTA/1 mM dithioth-reitol/0.4 mM phenylmethylsulfonyl fluoride) (10) with a glasshomogenizer. After centrifugation at 13,000 x g for 15 min at4°C, the protein concentration of the supernatant was deter-mined with a kit from Bio-Rad (based on the Bradfordmethod), and the amount of CAT in the samples was deter-mined with a CAT-ELISA kit from Boehringer Mannheim.,B-Galactosidase activity in the sample was measured by incu-bation with o-nitrophenyl 3-D-galactopyranoside (11). Fordetection of 3-galactosidase activity in early-stage (two- orfour-cell) embryos, embryos were fixed in 2% glutaraldehydesolution for 5 min, treated with 0.1% Triton X-100 for 5 min,washed in phosphate-buffered saline, and incubated in stainsolution [8.4mM KCl/84 mM phosphate buffer, pH 7.5/1 mMMgCl2/3 mM K4Fe(CN)6/3 mM K3Fe(CN)6/3% 5-bromo-4-chloro-3-indolyl 13-D-galactopyranoside (X-Gal)] overnight atroom temperature. Whole embryos (13th day of gestation)were stained for f3-galactosidase activity with X-Gal accordingto the method of Allen et al. (12).

RESULTSExperimental Design. The experimental design is outlined

in Fig. 1. The first step is the production of transgenic lines inwhich the coding region of the transgene of interest is pre-ceded by a reporter/silencer transgene surrounded by loxsequences. The line(s) are then screened to determine whichone(s) shows the strongest expression of the reporter/silencer.In this study, in order to easily monitor the expression of thetwo transgenes, we used the CAT and lacZ genes, and theinjected construct was thus CAG promoter-1oxP-CAT-4oxP-lacZ (CAG-CAT-Z). In the next step, eggs are collected frommice of the transgenic line(s) and submitted to pronuclearinjection of the plasmid pCAGGS-Cre in a circular form; thisresults in transient Cre expression and recombination at theloxP sequences. In embryos carrying the "recombined" trans-gene, 13-galactosidase activity is now detectable; the pCAGGS-Cre plasmids disappear rapidly by dilution during embryonicdevelopment.The Cre Transient Expression System. To confirm that the

CAG promoter works efficiently in fertilized eggs, we firstinjected circular pCAG-lacZ into fertilized eggs and stainedthem by X-Gal at the two-cell stage. All cells were stained verystrongly (data not shown), showing that the CAG promoter

CAG promoter CAT pA lacZ pA

Transgene CAG-CAT-Z

Production of CAG-CATZ transgenic lines

4Selection of high expression lines using CAT assay

Egfotngimice Injection of pCAGGS-CREEggs from transgenic mice (circular form)

Transient expression of CRE

Deletion of the CAT geneI

Expression of f-gal

FIG. 1. Description of the Cre transient expression system. Thestructure of the CAG-CAT-Z transgene construct is shown at the top;large arrows represent loxP sequences; pA, polyadenylylation signals.After the transgenic (Tg) mouse lines are produced and those withhigh CAT expression are selected, the CAT gene between the two loxPsites is removed by injection of oocytes with the Cre expression plasmidin a circular form. Recombination results in ,B-galactosidase (,3-gal)expression, and ,8-gal activity is detected by staining early-stageembryos.

has strong activity in eggs. We compared injection into thefemale or male pronucleus and found that at the two-cell stagethe ,B-galactosidase staining was stronger with those injected inthe female pronucleus, which may reflect the difference in thestarting time of expression. The important point was that bothpronuclear injections led to constant expression of the injectedplasmid, which probably indicates that the CAG promoter isespecially strong; indeed, other investigators, who injectedlacZ plasmids placed under the control of different promotersinto mouse eggs, have reported f3-galactosidase expression inonly 50% (12) or less (13) of the injected eggs.

Production and Selection of Transgenic Lines. The CAG-CAT-Z construct was injected into fertilized eggs fromB6D2F1 x B6D2F1 mice, and 12 transgenic founders wereobtained. F1 mice were then obtained from each founder, andCAT activity present in the hearts of these F1 mice wasdetermined. Three lines were found to display a high, com-parable levels of CAT activity (data not shown). For furtherexperiments, we chose one of these lines, CAG-CAT-Z 34,which carries about four copies of the transgene in head-to-tailarrangement (see Fig. 3c), since it gave offspring more rapidly

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FIG. 2. X-Gal staining of CAG-CAT-Z 34 embryos, recombined ornot recombined. Fertilized eggs were collected from superovulatedfemales mated with CAG-CAT-Z 34 males. After pronuclear injectionof pCAGG-Cre (5 ng/,ul), eggs were cultured in a CO2 incubator (5%CO2, 37°C), and stained at the two- or four-cell stage (24 or 48 hr ofincubation). The injected eggs were also transferred into pseudopreg-nant foster mothers, which were killed at day 13 of gestation; theembryos were stained for 13-galactosidase expression. (a and b)Tlwo-cell stage, uninjected (a) or injected (b) eggs. (c) Four-cell stage,injected eggs. (d) Embryos derived from injected eggs, at day 13 ofgestation. Uninjected eggs (a) never showed any staining. In b and c,stained eggs were selected to show differences in the staining pattern,which are probably related to variations in time and/or cell stage ofrecombination. In d, the placental DNAs from the correspondingembryos were analyzed by PCR (Fig. 3); the stained embryo on the lefthad a recombined transgene and the unstained embryo on the righthad no transgene (embryos with unrecombined transgene had anappearance similar to that of the embryo on the right).

than the others. Transmission of this transgene followedmendelian rules and never showed rearrangement or segre-gation.Cre Expressed in Fertilized Eggs Recombines the lox-

Containing Transgene. Eggs from B6D2F1 females mated withCAG-CAT-Z 34 males were collected, and a solution (5 ng/,ul)of pCAGGS-Cre was injected into the male or female pronu-cleus. Injected eggs were then incubated and stained withX-Gal at the two-cell or four-cell stage. As shown in Fig. 2 andTable 1, about half of the four-cell-stage embryos obtainedafter either male or female pronuclear injections were stained,whereas noninjected eggs were never stained. This resultshows that the Cre transient expression system removes veryefficiently the lox-surrounded CAT reporter gene in early-stage embryos, since about half of the eggs are expected tocontain the transgene. After male pronuclear injection, thepercentage of stained two-cell embryos was less than thatobserved after female pronuclear injection, but since no

difference was observed at the four-cell stage after either typeof injection, this probably indicates that injection into the malepronucleus results in a later start of Cre expression. In oneexperiment, the pCAGGS-Cre plasmid was injected at 1 ng/l.dinstead of 5 ng/jl; about half of the eggs were stained,indicating that the plasmid concentration used in all otherexperiments was in excess. With both types of injection,however, in some embryos only half of the cells were stained.This might reflect a difference in the level of expression of13-galactosidase or may suggest that recombination is a lateevent occurring in only one cell of the two-cell-stage embryo.To explore the second possibility, injected eggs were. trans-ferred into foster mothers, and the resulting embryos werestained at 13 days of gestation; three embryos out of five werestained (Fig. 2), with a ubiquitous and strong pattern ofstaining, suggesting that the recombination event had in factoccurred at the single-cell stage or eventually did occur in allcells; exploration of the placenta of these embryos by PCRshowed that only the placentas of the stained embryo con-tained a lacZ gene, in the absence of the CAT gene (see belowand Fig. 3). These experiments indicated that Cre worksefficiently in eggs and probably leads to transgene recombi-nation in all eggs injected with the Cre plasmid, since thefrequency of eggs expressing B3-galactosidase activity corre-sponds to the expected frequency of eggs carrying the trans-gene.Mice Born with a Transgene Recombination Show Absence

of Mosaicism, Complete Recombination, and Lack of CreIntegration. We transferred into pseudopregnant mice 60 and146 eggs injected with the Cre plasmid in the female or malepronucleus and obtained 16 and 31 mice, respectively, whichwere studied at 4-6 weeks of age. First, tail DNAs wereexamined by PCR using four kinds of primer sets. As shown inFig. 3a, the Zl and Z2 primers were designed to explorewhether the lacZ gene had been transmitted or not, the Z3 andAG2 primers to detect the recombination event, and the CAT2and CAT3 primers to explore whether the CAT gene had beendeleted completely. In the case of incomplete recombinationand/or of mosaicism, the DNA samples should show PCR-amplified DNA sequences with the use of CAT2/CAT3primers and Z3/AG2 primers. Primers Crel and Cre2 wereused to detect cre gene integration.As shown in Table 2, 18 of these 47 mice carried the

transgene. Irrespective of the site of pronucleus injection, in allthese 18 transgenic mice (which expressed lacZ; see below),DNA amplification using Z3 and AG2 primers gave a DNAsegment of the expected size, whereas no amplified DNAsegment was seen when the CAT2/CAT3 or Crel/Cre2primer pairs were used (Fig. 3b). Liver and kidney DNAs fromsome of these recombined-transgene-carrying mice gave thesame pattern as tail DNAs (data not shown). This indicatesthat the expression of Cre in the injected transgenic eggs ledto the excision of all CAT genes present in head-to-tailarrangement, that the cre gene was not integrated in thegenome, and that the recombination event probably occurredvery early in the embryos, since no mosaicism was detected.

Southern blot analysis of tail DNAs confirmed the existenceof the expected recombination. As shown in Fig. 3a, recom-bination should reduce the four copies of the CAG-CAT-Ztransgene array of transgenic line 34 to a recombined allele

Table 1. Number of stained eggs after pCAGGS-Cre injectionParental origin Two-cell stage Four-cell stage

of injectedpronucleus Injected eggs Stained Unstained Injected eggs Stained UnstainedFemale 15 7 (47%) 8 16 8 (50%) 8Male 17 4 (24%) 13 16 7 (44%) 9

The eggs were obtained as described in Fig. 2, and pCAGG-Cre was injected into the male or femalepronucleus. After staining with X-Gal, stained eggs were counted.

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a Transgene CAG-CATZ

CAG promoter CAT pA lacZ A=tw

~~~4-AG2 CAT2 CAT3 Z3 z2 22 PROBE

360 bp 320 bp

I

690 bp 320 bp

E 3.1 kb E

bCAG-CAT-Z 34 Fl INJECTEDIZ1 CAT2 Z3 IIZ1 CAT2 z3

Z2 CAT3 AG3 Z2 CAT3 AG3ICAG-CAT-Z

34 Fl INJECTED

4.. b^

4.4 kb_:... .......

3.1 kb

FIG. 3. PCR and Southern analyses of tail DNAs from CAG-CAT-Z 34 Fl mice or cre-injected transgenic mice. (a) PCR primersand expected results of Southern blot analysis. The upper map showsthe transgene before recombination, and the lower shows it after. Thesmall arrows indicate the positions and directions of the primers. Theexpected sizes of PCR products are indicated. EcoRI (E) sites in thetransgene are indicated. pA, polyadenylylation signal. (b) PCR analysisof tail DNAs from two transgenic mice before recombination (CAG-CAT-Z 34 F1) and after recombination (injected). The primers usedare indicated above each lane. (c) Southern blot analysis of EcoRI-digested tail DNAs from the same animals as in b. The 3.1-kb band

corresponds to a single-copy EcoRI fragment of the recombinedtransgene, indicating that CAT-pA sequences were excised; the 4.4-kbband corresponds to EcoRI fragments of the unrecombined trans-genes (about four copies). A thick bar below the maps in a correspondsto the probe used for this Southern analysis.

consisting of a single copy of the CAG promoter, the residualloxP site, and the lacZ gene. In agreement with this expecta-tion, the recombined transgenic DNAs did not show the 4.4-kbband observed with the intact transgene (Fig. 3c Left) butinstead showed the 3.1-kb band expected from the recombinedDNA, with an intensity compatible with a single copy (Fig. 3cRight).The lacZ Transgene in the Recombined Mice Shows a Tissue

Distribution and Expression Level Comparable to Those ofthe Reporter/Silencer CAT Gene in the Parent Mice. Expres-sion of the intact or recombined transgene was determined inthe liver, kidney, spleen, heart, lung, and muscle (Table 3). Inmice bearing the nonrecombined transgene, the CAT gene wasexpressed at very high levels in heart and muscle and only at

C

low levels in other tissues. In these mice, the transgene had no

detectable f-galactosidase activity. In mice bearing a recom-

bined transgene, 3-galactosidase activity was very high inmuscle and heart; in the other tissues, it was not above thebackground level. This probably reflects the difference insensitivity of the assays used, the CAT assay being moresensitive because of the lack of endogenous activity.

Transmission of the recombined transgene to progeny

showed no evidence of instability.

DISCUSSIONThese results establish a highly efficient method of genomicrecombination whose product is subsequently expressed in alltissues and cells of the resulting animals. This method is basedon the injection of a circular pCAGGS-Cre plasmid, whichallows transient expression of Cre, into the pronucleus of eggsobtained from transgenic mice bearing a transgene containingtwo loxP sequences. The method presents the following fea-tures. (i) It is rapid: many eggs can be obtained from a singletransgenic mouse, avoiding the requirement of prolongedbreeding and mating of two transgenic lines otherwise neces-sary for the expression of the selected transgene and of the cre

gene within the same mouse. (ii) It is very efficient, since itappears that, provided the injection of pCAGGS-Cre into a

pronucleus is successful, recombination takes place in all cases.(iii) The recombination takes place shortly after injection:since the recombination event occurs at a very early stage ofthe embryo, the risk of mosaicism appears to be very low. (iv)The method does not further modify the genome, since thecircular plasmid is not integrated.

This system has a number of useful applications. (a) Breed-ing transgenic lines in which transgene expression leads tolethality, sterility, or any source of inability to have progeny. Inutero lethality should, however, occur later than the 10th day,since earlier lethality might not be easily recognized from eggdamage resulting from microinjection. (b) Selecting transgeniclines with high expression (as done in the present work), an

especially useful procedure when there is a chance that hightransgene expression might induce in utero lethality. However,several transgenic lines should be selected, because transgeneexpression level may be related, in some integration sites, tocopy number of the transgene; since the recombination eventreduces the copy number to 1, the expression level may be, insuch cases, reduced after recombination. (c) Gene targetingintroducing loxP sequences. A variety of strategies can beconsidered for the selective removal of fragment of genes(instead of complete "knockouts"), using loxP sequencessurrounding the fragment to be removed. In an experimentaldesign based on this principle, a targeted neomycin-resistancegene was deleted by Cre transient expression in embryonicstem cells (14, 15). Instead of screening embryonic stem cellsfor successful recombination after transient expression of cre

introduced by electroporation, nonrecombined cells can beused to prepare mice (provided the nondeleted gene alterationis not toxic) in whose eggs recombination could be achieved.

Table 2. PCR analysis of transgene status

No. of mice positive by PCR

Parental origin No. of lacZ+ Recombined CAT Creof injected injected No. of (Z1/Z2 (AG2/Z3 (CAT2/CAT3 (Crel/Cre2pronucleus eggs newborns PCR) PCR) PCR) PCR)Female 60 16 7 7 0 0Male 146 31 11 11 0 0

The injected eggs were transferred into foster mothers, and mice born as the result of this procedurewere used when 3-4 weeks old. Tail DNAs were examined by PCR and the results with various primers(see Fig. 3 and Materials and Methods) are tabulated. Positive control for PCR detection of the cresequence consisted of 1 pg of the cre plasmid and 1 ,ug of DNA from the tail of a nontransgenic mouse.

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Table 3. Detection of CAT protein and ,B-galactosidase activity from various tissues of transgenic(Tg) mice with and without transgene recombination

Assay Mice Liver Kidney Spleen Heart Lung Muscle

CAT, pg/50 ,ug of protein

CAT CAG-CAT-Z Tg 1.3 1.4 1.4 200 8.0 960Non-Tg littermates 0.2 0.2 0.3 0.2 0.2 0.2

,B-Galactosidase, units/200 jig of protein

,3-Galactosidase CAG-CAT-Z Tg 0 0.01 0 0.11 0 0.04Recombined Tg 0 0.29 0 25.4 0.13 14.9Non-Tg littermates 0 0.07 0 0.04 0 0.04

Homogenates were prepared and CAT activity was measured by a CAT-ELISA kit (BoehringerMannheim), and ,B-galactosidase activity by the o-nitrophenyl ,B-D-galactopyranoside method (1 unit of13-galactosidase is defined as the amount hydrolyzing 1 nmol of substrate per minute at 28°C). Values areaverages for three mice.

We thank Dr. Brian Sauer for the gift of the cre-containing plasmid,Jean-Dominique Vassalli and Dominique Belin for comments on themanuscript, Mr. Jean-Claude Rumbeli and Mr. Etienne Denkinger forphotographic work, and Ms. Jacqueline Ntah for typing the manu-script. This research was supported by the Swiss National Foundation(31-37516.93). KA. was supported by Glaxo Institute of MolecularBiology (Geneva).

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