969Research Article

IntroductionProgeroid disorders can provide valuable insights into the geneticmechanisms underlying aging (Bohr, 2002; Martin and Oshima,2000). Hutchinson-Gilford progeria syndrome (HGPS, progeria) isa rare genetic disorder affecting children with features suggestiveof premature or accelerated aging. HGPS children appear normalat birth, but begin to display features of the disease within theirfirst year(s) of life. Affected children experience delayed growth,are short in stature, have universal alopecia and suffer fromrestricted joint mobility and osteoporosis. Other key abnormalitiesinclude prominent scalp veins, delayed eruption of teeth, impairedsexual maturation, and a thin and high-pitched voice (DeBusk,1972). Most children die in their early teens from heart attack orstroke, attributable to rapidly progressive atherosclerosis (see theProgeria Research Foundation’s medical and research database atwww.progeriaresearch.org).

The skin of children with progeria is described as having an agedlook. Consistent progressive clinical findings include atrophicepidermis, dermal fibrosis (scleroderma-like with thickening,hyalinization and disorganization of collagen bundles), thin or absenthypodermis and a complete loss of skin appendages or decrease innumber of hair follicles and sebaceous glands (Ackerman andGilbert-Barness, 2002; DeBusk, 1972; Erdem et al., 1994; Gillaret al., 1991; Hutchison et al., 2001; Jansen and Romiti, 2000;Rodriguez et al., 1999; Sevenants et al., 2005; Stables and Morley,

1994; Plasilova et al., 2004) (the GENEReviews’ database atwww.geneclinics.org). Hypoplastic eccrine glands and spotty skinpigmentation have also been reported (DeBusk, 1972; Jansen andRomiti, 2000).

The inheritance pattern of HGPS is autosomal dominant. It isreproductive lethal. At least 90% of the cases are due to a de novomutation in a single nucleotide in exon 11 of the LMNA gene,1824C>T (G608G). The mutation partially activates a cryptic splicesite and produces a mRNA that codes for lamin A protein with aninternal deletion of 50 amino acids (De Sandre-Giovannoli et al.,2003; Eriksson et al., 2003). The LMNA gene encodes lamin A,lamin C, lamin AΔ10 and lamin C2 (Burke and Stewart, 2002; Fisheret al., 1986). Lamin A and lamin C are major proteins of the innernuclear lamina, located beneath the nuclear envelope. The laminais believed to give the nucleus its shape and strength and to playsignificant roles in DNA replication (Burke and Stewart, 2002; Moirand Spann, 2001). The inner nuclear lamina is also essential indefining higher order structure by providing anchoring sites forchromatin domains and various proteins at the nuclear periphery(Burke and Stewart, 2002). Lamin A is synthesized as a precursorprotein, prelamin A, which is rapidly subjected to posttranslationalprocessing to produce mature lamin A. The processing is initiatedby farnesylation of the C-terminal CAAX motif (CSIM), removalof the last three amino acids (–SIM) and methyl esterification ofthe cysteine. The final step to yield mature lamin A, cleavage by

Hutchinson-Gilford progeria syndrome (HGPS) is a rare humangenetic disorder characterized by striking progeroid features.Clinical findings in the skin include scleroderma, alopecia andloss of subcutaneous fat. HGPS is usually caused by a dominant-negative mutation in LMNA, a gene that encodes two majorproteins of the inner nuclear lamina: lamin A and lamin C. Wehave generated tetracycline-inducible transgenic lines thatcarry a minigene of human LMNA under the control of a tet-operon. Two mouse lines were created: one carrying the wild-type sequence of LMNA and the other carrying the mostcommon HGPS mutation. Targeted expression of the HGPSmutation in keratin-5-expressing tissues led to abnormalities inthe skin and teeth, including fibrosis, loss of hypodermal

adipocytes, structural defects in the hair follicles and sebaceousglands, and abnormal incisors. The severity of the defects wasrelated to the level of expression of the transgene in differentmouse lines. These transgenic mice appear to be good modelsfor studies of the molecular mechanisms of skin abnormalitiesin HGPS and other related disorders.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/121/7/969/DC1

Key words: Hutchinson-Gilford Progeria Syndrome, Progerin, LMNAgene, Lamin A/C, Prelamin A, Tet-off system, K5tTA, Epidermalhyperplasia

Summary

Targeted transgenic expression of the mutationcausing Hutchinson-Gilford progeria syndrome leadsto proliferative and degenerative epidermal diseaseHanna Sagelius1, Ylva Rosengardten1, Mubashir Hanif1, Michael R. Erdos2, Björn Rozell3, Francis S. Collins2

and Maria Eriksson1,*1Department of Biosciences and Nutrition, Karolinska Institutet, Karolinska University Hospital, Huddinge, Novum, SE-14186 Stockholm, Sweden2Genome Technology Branch, National Human Genome Research Institute, National Institutes of Health, 50 South Drive, Bethesda, MD 20892,USA3Clinical Research Center, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, Huddinge, SE-14186Stockholm, Sweden*Author for correspondence (e-mail: maria.eriksson@mednut.ki.se)

Accepted 19 January 2008Journal of Cell Science 121, 969-978 Published by The Company of Biologists 2008doi:10.1242/jcs.022913

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the metalloproteinase ZMPSTE24, discards the 15 C-terminal residues of the prelamin A molecule previouslyinserted in the inner nuclear lamina (Bergo et al., 2002;Hutchison et al., 2001; Sasseville and Raymond, 1995;Stuurman et al., 1998). In progeria, the exon 11 deletionremoves 50 amino acids, including the recognition site forZMPSTE24, and therefore progeria children accumulatea indigestible farnesylated prelamin A molecule (referredto as progerin or LAdel50). Without the ability to bereleased from its lipid tether, progerin apparently interactsaberrantly within the nuclear lamina, interfering with itsstructure, intranuclear architecture and macro-molecularinteractions, and collectively producing major impact onnuclear functions. The presence of progerin has beenshown to lead to lobulated nuclei, thickening of thelamina, loss of peripheral heterochromatin, clustering ofnuclear pores and derangement of normal mitosis (Cao etal., 2007; Dechat et al., 2007; Eriksson et al., 2003;Goldman et al., 2004).

There are at least nine different autosomal recessive anddominant genetic diseases linked to mutations in the LMNAgene, collectively called laminopathies (Capell and Collins,2006; Worman and Courvalin, 2004). Several mousemodels, particular useful for studies on laminopathies, havebeen published (Bergo et al., 2002; Mounkes et al., 2003;Pendas et al., 2002; Sullivan et al., 1999; Varga et al., 2006;Yang et al., 2006). Mice homozygous for the Emery-Dreifuss muscular dystrophy point mutation, L530P, anddefective splicing of lamin A and lamin C transcripts,develop severe growth retardation and die within 4 to 5weeks. These mice have a slight waddling gait, small jaws,abnormal dentition, thickened epidermal layer with regionsof hyperkeratosis, thinning of dermis, absence ofsubcutaneous fat and osteoporosis (Mounkes et al., 2003).A BAC transgenic mouse that carries the G608G mutatedhuman LMNA shows no external phenotype, butdemonstrates progressive abnormalities of large arteries thatclosely resemble the most lethal aspect of the humanphenotype (Varga et al., 2006). A recent report from Yang and co-workers presents evidence of severe growth retardation, bone diseaseand a reduction of subcutaneous fat in a knock-in mouse model ofprogeria (Yang et al., 2005; Yang et al., 2006).

In the present study we describe the development of inducibleLMNA transgenic mouse lines based on the tet-on/off system(Gossen and Bujard, 1992; Zhu et al., 2002). This tissue-specificexpression system is particularly useful for the study of geneproducts that might be toxic when expressed early duringdevelopment, or products that could have a negative effect onreproduction. We constructed two tet-operon driven transgenic lines,one containing a minigene of wild-type human LMNA, and the othercarrying the most common HGPS mutation (1824C>T). By breedingwith K5tTA mice (Diamond et al., 2000), we obtain an induciblesystem that expresses human lamin A (LA) and progerin (LAdel50)in epidermal keratinocytes. In this study, we show that expressionof the mutant LMNA allele in postnatal epidermis replicates severalfeatures of the HGPS skin phenotype.

ResultsGeneration of tetop-LAwt and tetop-LAG608G transgenic miceMinigenes of human Lamin A (LAwt and LAG608G) were constructedby PCR amplification of human genomic DNA and cDNA,

subsequently digested with a unique restriction site in exon 11(DrdI), purified and ligated to a tetop vector (gift of P. Scacheri).The human LA minigenes contained the complete coding region oflamin A, including exon 1-11, intron 11 and exon 12 (downstreamof the stop codon) (Fig. 1). The tetop-LAwt and tetop-LAG608G

transgenic lines were created by injecting a fragment of 4165 bpcontaining the lamin A minigene, and the upstream tet-operon(tetop), downstream internal ribosomal entry site (IRES), thecoding region for eGFP and a SV40 poly A site. Twenty foundersof each minigene, as determined by PCR genotyping, were bornand bred to obtain F1 lines.

Screening for minigene expression and expression regulatedby the K5tTAF1 animals were intercrossed to K5tTA transgenic mice (Fig. 1C),and minigene expression from different F1 lines was evaluated bywestern blot and immunofluorescence (Figs 2, 3). Skin biopsiesfrom single transgenic tetop-LAwt and tetop-LAG608G littermateswere also analyzed by western blot to confirm protein expressionfrom the minigene only in the presence of the K5 transactivator.Bi-transgenic offspring were obtained from 11 F1 lines (from sevenfounders) of tetop-LAwt and eight F1 lines (from six founders) oftetop-LAG608G. Expression of GFP was detected in bi-transgenic

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Fig. 1. Minigenes of human lamin A. (A,B) Constructs used for tetop-LAwt (A) andtetop-LAG608G (B) transgenic mice. Both constructs contain a minigene, including thecoding region of lamin A (exons 1-10, intron 11 and exon 12), and differ only innucleotide 1824, where tetop-LAwt+ carries the wild-type lamin A sequence, and thetetop-LAG608G+ carries the most common HGPS mutation, 1824C>T. tetop, Tet-operon;IRES, internal ribosomal entry site; eGFP, coding region for enhanced green fluorescentprotein. (C) The tet-off system used in this study. Bi-transgenic animals, tetop-LAG608G+;K5tTA+, express human LA, LAdel50 (progerin) and green fluorescent protein (GFP).In the presence of doxycycline (DOX) the transcription is turned off.

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offspring from eight of the 11 F1 lines of tetop-LAwt, and expressionin five of the GFP positive F1 lines demonstrated tight regulationby the tet-operon. The remaining three F1 lines showed expressionin non-K5tTA containing tissues and were discarded from the study(data not shown). Expression of GFP was detected in bi-transgenicoffspring from seven out of the eight F1 lines of tetop-LAG608G.One tetop-LAG608G+; K5tTA– F1 line was excluded owing to GFPexpression in protein extracts in the absence of the transactivator(data not shown). Minigene expression [human LA and LAdel50(progerin)], were also screened by western blot. LA was present infour bi-transgenic F1 lines of tetop-LAwt (SF1-04, SF1-02, EF1-02and EF1-03) (Fig. 2B, data not shown). LA and LAdel50 werepresent in three bi-transgenic F1 lines of tetop-LAG608G (VF1-07,CF1-05 and DF1-03) (Fig. 2B, data not shown). To evaluate thetransgenic expression pattern in the skin, we performedimmunofluorescence with an antibody specific for human lamin

A/C on skin sections from all F1 lines that were positive for GFPon western blot, and that did not show expression in singletransgenic animals (Fig. 3). Minigene expression was detected inthe hair follicle and the interfollicular epidermis in one F1 line (VF1-07) of tetop-LAG608G (Fig. 3A-I). Bi-transgenic animals of the otherF1 lines show expression in only a few cells of the hair follicle,although the same F1 lines were previously positive by westernblot with human lamin A/C antibody (CF1-05 and DF1-03) (datanot shown). Lamin A minigene expression was detected in the hairfollicle and the interfollicular epidermis in two F1 lines [SF1-04and SF1-02 (from the same founder, S)] of tetop-LAwt (Fig. 3M-O). The other three F1 lines showed expression in only a few cellsmainly of the hair follicle (EF1-02 and EF1-03 from founder E) orno expression (data not shown), confirming the previous results fromthe western blots.

Other tissues known to contain keratin 5-expressing cells werealso investigated for minigene expression. Immunofluorescencewith human lamin A/C antibody was performed on sections fromesophagus, salivary glands, stomach and tongue of 9-week-old bi-transgenic animals of F1 line VF1-07, tetop-LAG608G. Expressionwas detected in myoepithelial cells of the salivary gland, basal andsuprabasal cells of esophagus, stomach and tongue, indicatingcorrect targeting of the transgene to keratin 5-expressing tissue(supplementary material Fig. S1, data not shown). No minigeneexpression was detected in tissues from tetop-LAG608G+; K5tTA–

transgenic mice, indicating tight regulation from the tet-operon (datanot shown). In mice receiving doxycycline in their drinking water,no minigene expression was seen in dorsal skin sections. Minigeneexpression was first noted in dorsal skin sections 7 days post-doxycycline removal.

Transgenic expression, levels and prelamin A accumulationPCR with primers specific for human LA and lamin Adel150(LAdel150) was performed on cDNA from skin of bi-transgenicand control mice from F1 lines (Fig. 2A, data not shown).Amplification of human LA and progerin (LAdel150) gives twofragments of 276 bp and 123 bp, respectively (Fig. 2A, lanes 1, 2).All bi-transgenic animals from the selected F1 lines have a fragmentcorresponding in size to human LA (Fig. 2A, lanes 4-7, 9-11),indicating correct splicing of the minigene. In bi-transgenic animalsfrom F1 lines of tetop-LAG608G, there is also an additional fragment

Fig. 2. Transgene expression in bi-transgenic mice. (A) RT-PCR ofdorsal/ventral skin from bi-transgenic mice and controls using a human LAand LAdel150-specific assay. Presence and absence of transgene is indicatedwith + and –, respectively. Lanes 1 and 2 are RT-PCR from cell line of HGPSparent AG03504 and HGPS AG03506, respectively. Lane 14 contains cDNAfrom ventral skin of FVB/N wild-type mice. Lanes 3, 12 and 13 are no-RTcontrol samples for AG03506, tetop-LAG608G+; K5tTA+ and tetop-LAwt+;K5tTA+, respectively. Lane 15 is a no-template control. M, 100 bp ladder(Invitrogen). RT-PCR for TATA-binding protein (TBP) served as a control forthe RT (bottom panel). (B,C) Western blot on protein extracts fromdorsal/ventral skin of bi-transgenic animals and controls. (B) Filters weresimultaneously incubated with antibodies for detection of human lamin A/C(mab3211) and β-actin. Separate western blots, including the same amount ofprotein extract, were run for detection of green fluorescent protein (GFP,ab290). (C) Enhanced protein separation western blots. An antibody to the N-terminal region of lamin A/C detects lamin A/C of both human and mouseorigin, sc-6215 (left). sc-6214 (right) detects prelamin A. (B,C) Same filterswere incubated with an antibody to β-actin. F1 line and PCR genotype areindicated above each lane. + and – indicate presence of LA minigene orK5tTA, and absence of K5tTA, respectively. Lane 1 contains protein extractsfrom HGPS cell line AG03506. Lane 8 is protein extract from a FVB/N wild-type mouse.

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corresponding in size to progerin (Fig. 2A, lanes 4-7), indicatingthat the 1824C>T mutation is recognized by the mouse splicingapparatus. Protein extracts from skin of bi-transgenic and controlanimals of different F1 lines were analyzed by western blot.Approximately equal levels of LA transgenic expression wereidentified in bi-transgenic animals of F1 lines VF1-07 and SF1-04when compared with β-actin (Fig. 2B). Lower levels of minigeneexpression were seen in bi-transgenic animals from F1 lines EF1-03, DF1-03 and CF1-05 (Fig. 2B, data not shown), which correlatedwith the expression seen on immunofluorescence.

Relative levels of human LA to mouse lamin A were quantifiedusing densitometry on western filters hybridized with an antibodythat recognize both human and mouse lamin A/C (Fig. 2C, data notshown). The average overexpression of lamin A in dorsal skinsamples from bi-transgenic animals of VF1-07 was 0.88 and forSF1-04 it was 0.73.

A protein corresponding in size to prelamin A was identified inlanes with protein extracts from bi-transgenic F1 lines, VF1-07 andSF1-04 using the human lamin A/C and the prelamin A antibodies

[Fig. 2C (right filter), data not shown]. Additional westernexperiments with increased separation between lamin A andprelamin A confirmed the presence of prelamin A accumulation inboth lines [Fig. 2C (left filter)].

Skin histopathology of F1 line VF1-07, tetop-LAG608G+; K5tTA+

transgenic miceTetop-LAG608G, F1 line VF1-07, transgenic mice were intercrossedto K5tTA transgenic mice. Doxycycline was removed on postnatalday 21 (dox day 21). Six weeks after doxycycline removal, skinabnormalities were apparent (Fig. 4B-G). At this stage, lesions werein patches of varying severity (Fig. 4B). The less affected regionswere characterized by a slight to moderate hyperplasia of theinterfollicular epidermis associated with hypergranulosis andhyperkeratosis. There were dystrophic changes in hair follicles whilethe associated sebaceous glands were beginning to show hyperplasiaand irregular maturation of the sebocytes [Fig. 4B (right), D,G]. Atthis stage, there were increased numbers of inflammatory cells,including polymorphonuclear granulocytes in the dermis. In the

more severely affected regions, there were papillarychanges with severe epidermal hyperplasia,hyperparakeratosis and enlargement and displacement ofsebaceous glands [Fig. 4B (left part), C-F]. The structureof the sebaceous glands was immature and did not appearto be fully differentiated (Fig. 4F,I). Dermal changesincluded fibrosis and moderate to severe inflammatorycell invasion. The end-stage, within 17 weeks of turn-onof expression of the transgene, was characterized by lossof hypodermis, fibrosis of the dermis, hypoplasticsebaceous glands and small hyperchromatic nucleioriented in parallel with the basement membrane; theepithelium still shows stratification with maturation of thecornified layer (Fig. 4J). Dorsal skin of F1 line of VF1-07, tetop-LAG608G+; K5tTA–, showed a normal structureof the skin up to 1 year and 9 months (Fig. 4A,K, datanot shown).

Skin phenotype in the tetop-LAG608G mouse linesexpressing low levels of the transgenes.Dorsal skin of bi-transgenic animals for F1 line CF1-05of tetop-LAG608G is normal and, even at 1 year and 9months of age, is indistinguishable from wild-typecontrols (data not shown). The dorsal skin of bi-transgenicanimals for tetop-LAG608G F1 line DF1-03, show apossible slight hypoplasia of the epidermis, but otherwisenormal structure of the other layers of the skin at 2 yearsof age (data not shown). The phenotypes of these mouselines suggest that sustained lower expression levels oflamin A and progerin (within only a few cells of the hairfollicle), does not have any significant effect on thestructure of the epidermis.

Skin histopathology of tetop-LAwt+; K5tTA+ transgenicmiceThe mouse lines overexpressing wild-type human LAwere created as a control for our experiments.Histopathology of the skin from bi-transgenic tetop-LAwt

F1 lines SF1-04, SF1-02, EF1-03 and EF1-02 wereanalyzed for histopathology. No significant skin lesionswere noted in bi-transgenic animals of F1 lines SF1-04,EF1-02 or EF1-03 older than 1 year of age (Fig. 4L, data

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Fig. 3. Transgenic minigene expression is present in keratinocytes of the interfollicularepidermis and the hair follicle. (A-I) F1 line VF1-07 tetop-LAG608G+; K5tTA+.(J-L) FvB/N wild-type mice. (M-O) F1 line SF1-04 tetop-LAwt+; K5tTA+.Immunofluorescence with the cytokeratin 5 (CK5) antibody in green (A,G,J), humanlamin A/C antibody in red (B,E,H,K,N) and DAPI (D,M). (C,F,I,L,O) Merged panels,including DAPI. Dorsal skin tissue sections from different ages of mice: 18 weeks(A-C), 9 weeks (D-F), 9 weeks end-stage (G-I), 4 weeks (J-L) and 22 weeks (M-O).(K) No staining with the human lamin A/C antibody to FVB/N wild-type dorsal skinshows that the antibody is specific to human lamin A/C. Transgenic mice were suppliedwith doxycycline until time of weaning – day 21. Scale bars: 50 μm.

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not shown). Bi-transgenic animals of F1 line SF1-02 (dox day 21)show a slight to moderate epidermal hyperplasia and

hyperparakeratosis, with hyperplasia of the sebaceous glands, andfibrotic dermis, already present at 6 weeks (data not shown).However, higher levels of LA (average human LA relative to mouselamin A were 1.5) and significant amounts of progerin weredetected in this line of mice (data not shown), and makes it hardto interpret. In the single transgenic animals for tetop-LAwt, of thesame F1 lines skin structure is normal. Taken together, these datasuggests that overexpression of tetop-LAwt in the skin is not resultingin any pathological changes.

Hyperproliferation in dorsal skin of tetop-LAG608G+; K5tTA+

transgenic miceImmunohistochemistry with cytokeratin 5 and cytokeratin 6antibodies on sections from dorsal skin tetop-LAG608G+; K5tTA+

F1 line VF1-07 with severe epidermal hyperplasia showmislocalized keratin 5 and keratin 6 expression (Fig. 5C-E). Anti-phospho-histone H3 (a marker for hyperproliferation) showedincreased proliferation especially in regions of severe epidermalhyperplasia (Fig. 5G,H). Increased apoptosis, measured with cleavedcaspase 3 antibodies, was not seen in these areas or in sections fromend-stage disease of the same transgenic line (data not shown).

Epidermal differentiation in dorsal skin of tetop-LAG608G+;K5tTA+ transgenic miceWe used immunofluorescence with antibodies against keratin 1,keratin 10, filaggrin and loricrin to examine whether epidermal

Fig. 4. Progressive pathological changes from epidermal overexpression oflamin A and lamin Adel50 in sections from dorsal skin. (A-G,J-L)Haematoxylin and Eosin staining. (A) Normal structure of the skin in lateanagen phase is shown in a section from dorsal skin of FVB/N wild-type mice.(B-G) F1 line VF1-07, tetop-LAG608G+; K5tTA+, 9-week-old mice.Intermediate severe and mild stage evident in the same section (B; left andright, respectively). Intermediate severe stage with severe epidermalhyperplasia, extensive immature hyperparakeratosis, and enlargement anddisplacement of sebaceous glands (B, enlarged in C,E,F), and mild stage(B,D,G). (H,I) Immunohistochemistry using and antibody to adipophilin showstaining of lipid-containing cells of the sebaceous unit in FVB/N wild-type (H)and F1 line VF1-07, tetop-LAG608G+; K5tTA+ (I) 17-week-old mice. (J) End-stage disease characterized by epidermal hypoplasia, resting hair follicle,associated hypoplastic sebaceous gland, absence of hypodermis and a well-developed fibrosis of the dermis. (K) F1 line VF1-07 tetop-LAG608G+; K5tTA–

at 11 months (for comparison with J). (L) Bi-transgenic animals from the SF1-04 line show a well-defined structure of the skin, as shown in a dorsal skinsection from a 24.5-week-old mouse. Transgenic mice were supplied withdoxycycline until time of weaning – day 21. Scale bars: A,C-G,J-L, 100 μm;B, 500 μm; H,I, 50 μm.

Fig. 5. Epidermal overexpression of lamin A and lamin Adel50 leads toaberrant expression of keratin 5 and keratin 6, and increased proliferation, asshown by immunohistochemistry with antibodies to keratin 5 (CK5), keratin 6(CK6) and phospho-histone H3 (PHH3). (A,B,F) FVB/N wild type in lateanagen stage. (C-E,G,H) Bi-transgenic animals from F1 line VF1-07 of 9 and18 weeks. (A) CK5 normal expression pattern in the basal cells of theinterfollicular epidermis, the outer root sheet and the peripheral cells of thesebaceous glands. (B) CK6 normal expression pattern in the inner root sheet.(C) In progerin-expressing mice, CK5 expression is also detected in thesuprabasal population of keratinocytes and is expressed equally across thesebaceous glands. (D,E) CK6 expression is also detected in the interfollicularepidermis and sebaceous glands. (F) Normal PHH3 expression in the bulb of alate anagen hair follicle. (G,H) Increased proliferation in cells of theinterfollicular epidermis and the hair follicle. Transgenic mice were suppliedwith doxycycline until time of weaning – day 21. Scale bars: C-E, 100 μm;A,B,F-H, 50 μm.

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differentiation was altered in regions of epidermal hyperplasia inbi-transgenic animals of the F1 line VF1-07. Similar to wild-typeskin, keratin 1- and keratin 10-positive keratinocytes resided in thesuprabasal layers of bi-transgenic epidermis (Fig. 6A,B,D,E).Although the total expression of keratin 1 and keratin 10 wasincreased in regions of epidermal hyperplasia, the majority of theexpression was seen in the spinous layers and only a few cells ofthe basal layer were seen to express keratin 1 and keratin 10 (Fig.6C,F). Immunostaining using antibodies to filaggrin and loricrinshowed expression in the granular layer of wild-type and bi-transgenic epidermis (Fig. 6G-L). Similar to the spinous markers,filaggrin expression was seen in multiple layers of the thickenedepidermis (Fig. 6I). Filaggrin-expressing cells were also seen in thedermis of bi-transgenic skin (Fig. 6H,I).

Premature death and external phenotypeHair-thinning, growth retardation and premature death were notedin bi-transgenic animals from F1 line VF1-07 (tetop-LAG608G+;K5tTA+) (Fig. 7). Intercross breeding pairs were supplied withdoxycycline, which was removed at weaning, postnatal day 21 (doxday 21) or postnatal day 0 (dox day 0). Hair follicle density wasmeasured by counting hair follicles in sections from dorsal skinfrom bi-transgenic and wild-type mice. Although bi-transgenic miceexperienced hair thinning on gross examination, there was noreduction in the number of hair follicles compared with wild-typemice. Weights were recorded weekly starting at postnatal week 1.Growth retardation and early death were noted in bi-transgenicanimals with a median survival of 14 weeks for dox day 21 (Fig.7A,D) and median survival of 7 weeks for dox day 0 (Fig. 7B,E).

In an effort to identify causes of premature death in these animals,we identified dental problems (described below), which wesuspected could have significant effect on their lack of growth. Totest whether this was the case, we fed the mice a softer diet ofdissolved pellets on the cage floor. With this special feeding themedian survival increased from 7 to 29 weeks (compare Fig. 7B,Ewith 7C,F). Weights were also recorded for bi-transgenic mice ofF1 line DF1-03, of tetop-LAG608G, but were indistinguishable fromcontrol mice (data not shown). These mice did not show any externalphenotype, except for a slight hair thinning at 2 years of age (datanot shown). Weights were also monitored on bi-transgenic animalsfrom F1 line SF1-04, tetop-LAwt (dox day 21), but no prematuredeath or abnormal weights were seen for this line (+/+, n=9) (datanot shown). However, all bi-transgenic animals of the SF1-04 lineshowed partial hair loss, and regions with crusting of the skin firstnoted at 7 weeks post dox removal (+/+, n=10) (data not shown).No premature death or significant changes in weight were noted inbi-transgenic animals of F1 line EF1-02 of tetop-LAwt (data notshown). Premature death with a median survival of 8.7 weeks, andpartial hair loss with regions of skin crusting were also noted in bi-transgenic animals of F1 line SF1-02 (tetop-LAwt+; K5tTA+) (datanot shown). Higher levels of LA (average relative to mouse laminA were 1.5) and significant amounts of progerin were detected inthis line of mice, which makes it hard to interpretate the phenotype(data not shown).

Histopathology of organs other than skinWe examined the gastrointestinal system, liver, pancreas, spleen,thymus, mammary glands, salivary glands, tear glands, brown fat,kidneys, adrenal glands, reproductive organs, skull, tongue, brain,the respiratory mucosa of the nasal cavity, trachea and lungs onbi-transgenic animals of F1 line VF1-07 (tetop-LAG608G) and F1line SF1-04 (tetop-LAwt) of different ages. Bi-transgenic animals(with dox stopped at day 21) of F1 line VF1-07 were sacrificedfor histopathology at 9 weeks (n=3) and at 17-20 weeks (n=4),and bi-transgenic animals of F1 line SF1-04 were sacrificed at 22-24 weeks (n=3). In bi-transgenic mice from F1 line VF1-07, theonly significant recurrent changes included a possibly mildhyperplasia in the stomach and more extensive changes involvingboth the lower incisors and surrounding tissues. The latter changesranged from increased impaction of food or bedding material inthe gingival sulcus with associated acute inflammatory reaction toovert necrosis of the pulp. In the latter cases, foreign material withthe appearance of cellulose was found in the pulp (supplementarymaterial Fig. S2B). Inflammation spread into the periodontalligament and surrounding bony structures but no changes were seenin the upper jaws. No significant recurrent changes were observedin bi-transgenic animals of tetop-LAwt. Single transgenic littermatesfor the respective minigene of different ages, were also analyzed(n=7), and no significant abnormalities were observed in thesemice.

DiscussionFor diseases like HGPS that are both devastating and rare, animalmodels are essential to understand the molecular basis of the diseaseand investigate what kinds of interventions may benefit patients.Transgenic and knockout mice have been used to shed light on thegenetics behind laminopathies and aging in general. In the transgenicmouse model described here, we decided to leave the mouse Lmnagene intact and to generate transgenic mice that express minigenesof wild-type and HGPS human LA under the control of an inducible

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Fig. 6. Expression of epidermal differentiation markers. Dorsal skin sectionfrom FVB/N wild type in late anagen phase (A,D,G,J), and bi-transgenicanimals of the F1 line VF1-07 in intermediate stage (B,C,E,F,H,I,K,L).Immunofluorescence with the cytokeratin 1 (CK1), cytokeratin 10 (CK10),filaggrin and loricrin antibodies in green and merged with DAPI (blue). High-magnification view of interfollicular epidermis show expression of CK1 andCK10 in a few cells of the basal layer (C and F, respectively). Transgenic micewere supplied with doxycyline until time of weaning – day 21. BL, basallayer; D, dermis. Scale bars: 50 μm.

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promotor, providing the opportunity to limit expression to a specifictissue. Using the tissue specific tet-on/off system, we developedtwo lines carrying minigenes of human LA, differing only innucleotide 1824, where one transgene carries the wild-type sequenceof LMNA, and the other carries the most common HGPS mutation(G608G, 1824C>T). The clinical picture in HGPS involves multipleorgans and tissues, and skin is one of the first organs to show typicalsigns of disease, including scleroderma, loss of subcutaneous fatand alopecia. Based on this, we decided to use the K5tTA (Diamondet al., 2000) as the transactivator, which is predicted to direct theexpression of the minigenes to basal cells of the interfollicularepidermis and hair follicle. Screening multiple bi-transgenicoffspring from multiple F1 lines from different founder animalsidentified lines that showed different levels and pattern ofexpression, but only bi-transgenic animals from the F1 line VF1-07 of the tetop-LAG608G, and SF1-04 of tetop-LAwt, showedexpression in cells of the interfollicular epidermis and hair follicle.Accurate intron splicing and partial splice site activation of the1824C>T mutation in the minigenes was demonstrated by RT-PCR,and confirmed by direct sequencing (data not shown). Proteinexpression of human LA and LAdel50 was detected with westernblot and immunofluorescence in keratin 5-expressing tissues. TheVF1-07 and SF1-04 lines show similarly high levels of human LAprotein, whereas bi-transgenic animals of other F1 lines have lowerexpression levels. In bi-transgenic animals expressing progerin,there was a progressive phenotype beginning with regions of mildto severe epidermal hyperplasia, hyperparakeratosis and hyperplasiaof the sebaceous glands, and progressing to an end stagecharacterized by loss of subcutaneous fat, fibrosis of dermis andhypoplastic sebaceous glands.

We are unsure what causes the observed variation in phenotypewithin the intermediate stage (with severe and mild regions even

within the same section, Fig. 4B). However, immunofluorescenceusing the anti-human lamin A/C antibody on sections with regionsof phenotypic variability showed staining of cells of theinterfollicular epidermis and the hair follicle of both mildly andmore severely affected regions, which excludes the possibility ofmosaic expression of the transgene (data not shown). Our progerin-expressing mice also had an external phenotype with hair-thinning,low weight and premature death, with a median survival of 7 or 14weeks, depending on when the transgene was turned on. Severeabnormalities include a well-developed dermal fibrosis, absenceof detectable hypodermal adipocytes and premature death, allclinical features that are found in children with HGPS.Immunohistochemistry with antibodies to keratin 5, keratin 6 andphospho-histone H3 show mislocalized expression of keratin 5 and6, with increased proliferation in regions of severe epidermalhyperplasia. This is in agreement with prior studies on mouse skinwere suprabasal expression of keratin 5 and expression of keratin6 in the interfollicular epidermis associates with hyperproliferativedisease (Fuchs, 1995; HogenEsch et al., 1999). Analysis of theexpression pattern of additional markers for epidermaldifferentiation (keratin 1, keratin 10, loricrin and filaggrin) indicatedthat regions with epidermal hyperplasia and increased proliferationcontained layers of differentiated keratinocytes. Although there wasan increase in the thickness of the spinous and granular layerscompared with wild-type skin, the terminal differentiation ofkeratinocytes was essentially similar to what was seen in normalskin. Hyperproliferation and apoptosis has been reported in HGPSfibroblasts previously (Bridger and Kill, 2004). However, we didnot detect any increased apoptosis in skin from these animals (datanot shown).

Considering that nutritional problems might contribute to growthfailures and early death, we looked into the possibility that these

Fig. 7. Reduced growth rates andpremature death of tetop-LAG608G bi-transgenic animals. Body weights (grams,A-C) or percent survival to age (weeks,D-F) were plotted for F1 line VF1-07.Each panel includes data from threedifferent genotypes: (A) tetop-LAG608G+;K5tTA+ (+/+); (B) tetop-LAG608G+;K5tTA– (+/–); and (C) tetop-LAG608G–;K5tTA– (–/–). The difference in survivalfor bi-transgenic animals is noted: (D) +/+(n=12, median survival=14 weeks) and+/– (n=10), χ2=12.79, P=0.0003. (E) +/+(n=5, median survival=7 weeks) and +/–(n=9), χ2=13.4, P=0.0003. (F) +/+ (n=6,median survival=29 weeks) and +/–(n=9), χ2=7.943, P=0.0048. Nostatistically significant difference insurvival was noted between +/– and –/–animals (D-F). Intercross breeding pairswere supplied with doxycycline that wasremoved at day of weaning – day 21(A,D) or day of birth – day 0 (B,C,E,F).(C,F) Results when animals were fed onthe cage floor with water-dissolved pelletsfrom postnatal day 21 and onwards.(G) Photograph of 7-week-old bi-transgenic and single transgenic animals.

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mice had problems with eating. The stomach of a few animalsshowed presence of mild hyperkeratosis (data not shown) butmalnutrition was most probably due to dental abnormalities(inflammation and foreign material present within the pulp of theteeth) in the lower jaws of these mice. Feeding the animals a softerdiet increased their median survival (from 7 to 29 weeks). Eventhough complete necropsies were performed and correspondingorgans examined histopathologically, other potential contributingchanges to the early mortality were not found, even in internal cellsand organs known to express keratin 5.

Bi-transgenic mice of the SF1-04 line, with an averageoverexpression of human LA of 0.73, were used as a controls.Animals in the SF1-04 line did not show any weight loss, prematuredeath or any histopathological abnormalities of the skin or otherorgans that were investigated, though all animals had an externalphenotype of partial hair loss, with regions of skin crusting (notapparent on histopathology). It is interesting to note that a modestamount of prelamin A accumulates in this line, presumably owingto high levels of expression. But the mildness of the phenotypeargues against major toxicity of this amount of prelamin Aaccumulation in the skin. This contrasts with the results reportedin the Zmpste24-deficient mice (Bergo et al., 2002; Fong et al.,2004; Pendas et al., 2002), where virtually all of the mouse laminA remains as prelamin A.

Surprisingly, bi-transgenic mice of an additional F1 line, SF1-02, of tetop-LAwt, with an higher overexpression of normal humanLA (average relative to mouse lamin A of 1.5 compared with 0.73for SF1-04), were also found to express a significant amount ofprogerin (data not shown). Prior studies have shown that progerinsplicing occurs at a low level in human wild-type lamin A, withthe G608G mutation greatly increasing the usage of this crypticsplice donor (Cao et al., 2007; Scaffidi and Misteli, 2006). Forreasons that are not clear, this particular line used that splice donorquite efficiently, despite the wild-type sequence. These miceshowed premature death, partial hair loss, skin crusting and somehistopathological abnormalities of the dorsal skin similar to thosepresent in bi-transgenic mice from the VF1-07 line (data notshown).

In summary, this study shows that expression of progerin in theskin of transgenic mice induces a progressive disease that beginswith hyperproliferation and ends in a phenotype that resembles manyof the clinical features of the skin reported in children with HGPS.This transgenic mouse model should therefore be useful in studyingthe pathogenesis and potential treatment of HGPS.

Materials and MethodsGeneration of tetop-LAwt and tetop-LAG608G transgenic miceThe lamin A (LAwt and LAG608G) minigenes were constructed by PCR amplificationof human genomic DNA (AG11498) (Eriksson et al., 2003) with primers 5�-GCTCTTCTGCCTCCAGTGTC-3� and BamHI-GTCCCAGATTACATGATGCTG,and PCR amplification of human cDNA from an individual with HGPS (AG01972)(Eriksson et al., 2003) with primers EcoR1-ACTCCGAGCAGTCTCTGTCC and5�-GGTCCCAGATTACATGATGCT-3�. Both PCR products were purified(QIAquick, Qiagen) and the genomic DNA PCR product were digested with DrdIand BamHI, and the cDNA PCR product were digested with DrdI and EcoR1.Following gel purification (Wizard SV Gel and PCR Clean-Up system, Promega),the LA minigene fragments were ligated to a vector that had been generatedpreviously for another transgenic construct (tetop-Men1, a gift from P. Scacheri).The Men1 transgenic vector was constructed as follows: the complete codingsequence of the mouse Men1 gene was inserted into a tetracycline responsive cloningvector containing the mp-1 intron poly(A) sequence (gift of G. Fisher and H. Varmus,Sloan-Kettering). A 2.6 kb fragment containing the tet-operon and Men1 gene wasPCR amplified from this construct and cloned into pIRES2-EGFP (Clontech). Priorto insertion of the LA minigene fragments, the Men1 gene was removed by an initialgel purification of a digest with BamHI, followed by a second gel purification of a

digest with EcoR1. PCR screening of clones, with EcoR1-ACTCCGAGC -AGTCTCTGTCC and BamHI-GTCCCAGATTACATGATGCTG, identified cloneswhere the LA minigene had replaced the Men1 gene downstream of the tet-operon(tetop) and upstream of the eGFP. Sequencing of the clones with LMNA-specificprimers identified minigenes of tetop-LAwt (wild-type sequence in codon 608) andtetop-LAG608G [G608G (1824C>T)]. Double CsCl banding (Lofstrand Laboratories,Gaithersburg, MD) was used to purify the vectors. The transgenic constructs (4165bp) were released from its carrier by digests with AseI and NotI, gel purified andinjected into the male pronucleus of recently fertilized Fvb/N embryos. Thegeneration of tetop-LAG608G and tetop-LAwt transgenic mice was approved by theAnimal care and Use Committee of the National Human Genome Research Instituteand the National Institutes of Health, G-03-5. The animal studies were approved bythe Stockholm South Ethical review board, Dnr. S148-03, S139-05, S111-05 andS141-06.

Mouse genotypingDNA was extracted from tail biopsies using standard phenol-chloroform protocol.Genotyping was performed with PCR for the LA minigenes (tetop-LAG608G and tetop-LAwt) (Eriksson et al., 2003) and K5tTA (Diamond et al., 2000). PCR with Mycprimers was used as positive control for presence of DNA. Southern blotting wasperformed according to standard protocol using SacI. The probe was created by PCRamplification of human LMNA (exon 11, intron 11 and exon 12 to the stop codon)with primers 5�-ACCCCGCTGAGTACAACCT-3� and ACATGATGCTGCAGTTC -TGG-3�. The PCR product were TA cloned (TOPO TA-cloning kit, Invitrogen) anddigested with EcoRI, to release a fragment of 609 bp that was gel purified and usedas probe for the LA minigenes. For single transgenic integration, the probe hybridizesto a fragment larger than 3387 bp, depending on the next SacI site at the transgenicintegration site. For multiple tandem integrations, the probe hybridizes to anadditional fragment of 3690 bp. All filters contained a SacI digest of human genomicDNA and the probe hybridized to a fragment of 4449 bp. Estimation of transgeniccopy number was made by comparison with SacI digests of non-transgenic genomicDNA spiked with different amounts of plasmid (1�, 5�, 10� and 20�) that containedthe tetop-LAG608G transgenic construct, described above. The probe hybridizes to afragment of 7034 bp in the spiked DNA. Calculations for estimation of transgeniccopy number were in accordance with standard method (Gene Targeting andTransgenic Facility, University of Virginia Health System). F1 lines of tetop-LAwt:SF1-04, SF1-02 and EF1-03 each have an estimated copy number of 4. F1 lines oftetop-LAG608G: VF1-07, CF1-05 and DF1-03 have an estimated copy number of 1,4 and 2, respectively (data not shown).

Animal housingAnimals were housed at the experimental unit animal housing facility at theKarolinska Hospital, Huddinge, Sweden. Animals were kept in open Makrolon 2 or3 community cages, with a maximum number of five and 10 mice per cage,respectively. The housing conditions included a 12 hour light/dark cycle, a temperatureof 20-22°C and 50-75% air humidity. Mice were supplied with R36 pellets (Lactamin,Sweden) and drinking water ad libitum.

Animal breeding and screening of transgenic linesColonies were maintained with mating to FVB/NCrl. During intercross breeding andup to postnatal day 0 or 21, the animals’ drinking water was supplemented with 100μg/ml doxycycline (Sigma) and 2.5% sucrose. The drinking bottles were covered infoil, and the water was changed every third day. Small skin biopsies were obtainedfrom dorsal skin and/or tail skin of F1 and F2 that were positive for both the laminA minigene and K5tTA (Diamond et al., 2000) (tetop-LAwt+; K5tTA+ and tetop-LAG608G+; K5tTA+). Skin biopsies were also obtained from tetop-LAwt+; K5tTA– andtetop-LAG608G+; K5tTA– transgenic mice, and served as negative controls forexpression analysis. Weights were recorded weekly. In accordance with the KarolinskaInstitute guidelines for animal care, animals were sacrificed when a sudden weightdrop was recorded (sudden weight loss of 25%) and/or if their general health statuswere compromised. Weight and survival curves, and statistical analysis wereperformed using the GraphPad Prism, version 4 software. Immunofluorescence withthe human lamin A/C antibody showed that withdrawal of doxycycline from thedrinking water resulted in transgenic expression first noted at day 7 post-doxycyclineremoval (n=7 skin samples).

Analysis of transgene expression (RNA and protein)RNA was extracted from mouse tissues with Trizol or Micro-FastTrack 2.0 mRNAisolation kit (Invitrogen), using Lysing Matrix D and Fastprep 220A (Qbiogene).DNase treatment and cDNA synthesis were performed in accordance with previouslypublished procedure (Eriksson et al., 2003). Expression of the LA minigenes wereanalyzed by PCR with 5�-AGTTCTGGGGGCTCTGGGT-3�, 5�-ACTGCAGCA -GCTCGGGG-3� and 5�-TCTGGGGGCTCTGGGC-3�. Expression of human LAgives a fragment of 276 bp, and expression of human LAdel150 gives a fragment of123 bp, respectively. PCR with TBP-specific primers was performed on all samplesas control (Eriksson et al., 2000).

Protein was extracted from mouse tissues in RIPA buffer or 8 M UREA, 5%RIPA (including a cocktail of proteinase inhibitors, Roche) and homogenized with

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Lysing Matrix D and Fastprep 220A (Qbiogene). Mini-gel western blots wereperformed in accordance with previously published procedures (Eriksson et al.,2003). A larger western blot system was used to enhance the separation of proteins(PROTEAN II xi Cell, BIORAD). Enhanced protein separation were accomplishedusing 1 mm thick 20 cm long 4%/7.5% discontinuous Laemmli slab gel. The gelswere run for 12 hours at 25 mA using a cooling system at 2°C, according to themanufacturer’s recommendation. Protein transfer was performed according tostandard procedure for the Semidry Transfer Cell (BIORAD). Protein markers wereBenchMark Pre-stained protein ladder (10748-101, Invitrogen) and PageRulerPrestained protein ladder (#SM0671, Fermentas). Primary antibodies used forwestern blot were: mouse monoclonal anti-human lamin A+C (mab3211,Chemicon), goat polyclonal anti-human prelamin A (sc-6214, Santa CruzBiotechnology), goat polyclonal anti-lamin A/C (sc-6215, Santa CruzBiotechnology), rabbit polyclonal anti-gfp (ab290, Abcam Ltd), mouse monoclonalanti-gfp (Sc-9996, Santa Cruz Biotechnology) and mouse monoclonal anti-β-actin(A5441, Sigma).

Protein quantification was performed on western filters, with enhanced proteinseparation, hybridized with an antibody to the N-terminal region of lamin A/C (sc-6215). The antibody recognized lamin A and C of both human and mouse origin.Relative band intensities of human LA to mouse lamin A in the same lane werequantified in protein extracts from dorsal skin samples from three bi-transgenicanimals of F1 lines VF1-07, SF1-04 and SF1-02. The average relative levels wereused to assess the degree of lamin A overexpression. Densitometry was performedusing Versa Doc Imaging System (BIORAD) and analyzed using the Quantity Onesoftware.

ImmunohistochemistryAnimals were sacrificed using an overdose of isoflurane and tissues were collectedand fixed at +4°C overnight in 4% paraformaldehyde (pH 7.4). Following fixation,the tissues were dehydrated in ethanol and embedded in paraffin. All assays wereperformed on 4 μm sections. Sections were stained with Haematoxylin and Eosinaccording to standard procedures. Immunohistochemistry for cytokeratin 5,cytokeratin 6, phospho-histone H3 and cleaved caspase 3 were performed inaccordance with previously published procedure (Svard et al., 2006).Immunohistochemistry for adipophilin (1:300; Progen), using a pressure cookerfor antigen retrieval. Immunofluorescence experiments for human lamin A/C (1:30;mab3211, Chemicon), loricrin (1:500; PRB-145P, Covance), filaggrin (1:1000;PRB-417P, Covance), cytokeratin 1 (1:500; PRB-165P, Covance), cytokeratin 10(1:500; PRB-159P, Covance) and cytokeratin 5 (1:1000; PRB-160P, Covance)were performed in accordance with previously described procedures (Varga et al.,2006).

Hair follicle densityMice were intercrossed on doxycycline, which was removed at day of birth. Frompostnatal day 21 and onwards, the animals were supplied with a soft diet of dissolvedpellets ad libitum. Animals were sacrificed at 7 weeks of age, and dorsal skin sampleswere collected and processed for Haematoxylin and Eosin staining. Number of folliclesper mm section were counted in sections from two different anatomical dorsal skinregions (forehead and mid-dorsal region) in wild-type (n=3) and bi-transgenic animalsof the VF1-07 line (n=3). Statistical analysis was performed using unpaired two-tailed Student’s t-test (Prism, GraphPad).

We acknowledge the technical assistance of Jun Cheng, CarinLundmark, Eva Schmidt, Anton Paier and Sofia Rodriguez. We alsothank Åsa Bergström for technical consultation, Margaret Warner forconstructive editing of the manuscript, and Adam Glick for kindlysharing his K5tTA mice. This work was supported by grants from theTore Nilsson Foundation, the Åke Wiberg Foundation, the HagelenFoundation, the Loo and Hans Osterman Foundation, the Torsten andRagnar Söderberg Foundations, the Jeansson Foundations, the SwedishResearch Council, and the Swedish Foundation for Strategic Research.M.H. is supported by a postdoctoral fellowship from the Wennergrenskasamfundet.

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