Proc. Nati. Acad. Sci. USAVol. 86, pp. 1563-1567, March 1989Cell Biology

Tissue-specific and differentiation-specific expression of a humanK14 keratin gene in transgenic mice

(stratified squamous epithelia)

ROBERT VASSAR*, MARJORIE ROSENBERG*, SUSAN ROSSt, ANGELA TYNER**, AND ELAINE FUCHS*Departments of *Molecular Genetics and Celi Biology and of Biochemistry and Molecular Biology, and the Howard Hughes Medical Institute, The Universityof Chicago, Chicago, IL 60637; and tDepartment of Biochemistry, The University of Illinois Medical School, Chicago, IL 60612

Communicated by A. A. Moscona, November 28, 1988

ABSTRACT A construct containing -2500 base pairs (bp)of 5' upstream and -700 bp of 3' downstream sequence wasused to drive the expression of an intronless human K14 genein vitro and in vivo. To track the expression of the gene, a smallsequence encoding the antigenic portion of neuropeptide sub-stance P was inserted in frame 5' to the TGA translation stopcodon of the gene. Surprisingly, this gene was expressedpromiscuously in a wide variety of cultured cells transientlytransfected with the construct. In contrast, when introducedinto the germ line of transgenic mice, the construct wasexpressed in a fashion analogous to the endogenous K14gene-namely, in the basal layer of stratified squamousepithelia. Our results suggest that some regulatory mechanismis overridden as a consequence oftransient transfection but thatsequences that can control proper K14 expression are presentin the construct. The appropriate tissue-specific and differen-tiation-specific expression of K14 P in transgenic mice is animportant first step in characterizing a promoter that could beemployed to drive the foreign expression of drug-related genesin the epidermis of skin grafts.

Among the major proteins exclusive to epithelial tissues arekeratins, a family of >20 proteins whose members are differ-entially expressed in a tissue-, differentiation-, and develop-mental-specific fashion (for review, see ref. 1). Based onsequence homologies, these proteins can be subdivided intotwo distinct groups: type I keratins are smaller (40-56.5 kDa)and acidic (pKi = 4.5-5.5), and type II keratins are larger (53-67 kDa) and more basic (pKi = 5.5-7.5) (2). Both types areessential for the formation of 8-nm keratin filaments (3, 4).Type I and type II keratins are expressed as specific pairs

(5), and filaments composed of different pairs can havewidely different properties (3, 4, 6). K5 (type II; 58 kDa) andK14 (type I; 50 kDa) are the pair expressed in the basal layerof all stratified squamous epithelia (1, 5, 7). In epidermis,these proteins can account for up to 30o of the total protein(1, 2, 5). As a basal cell undergoes a commitment todifferentiate, it down-regulates the expression of basal-specific keratins (7) and induces the expression of a new setof keratins whose identity is dependent upon the particularstratified squamous epithelium: terminally differentiatingepidermal cells express K1, K2, K10, and K11 (1, 2, 5);differentiating corneal cells express K3 and K12 (5); esoph-ageal cells express K4 and K13 (8). Simple epithelial cellsexpress an entirely different set of keratins (1, 9).

Elucidating the molecular mechanisms underlying tissue-specific and differentiation-specific expression of keratingenes is an important step in understanding the regulatorsthat control epithelial differentiation. The promoters ofkeratin genes may also provide useful tools for drivingexpression of foreign genes in an epithelial cell-specific

manner. This might be particularly valuable for epidermis,where grafting after propagation by culture in vitro hasalready been shown to be a viable and useful medicaltechnique (10). To this end, we have focused on the regula-tion of the human gene encoding K14, since this keratin andits partner K5 are faithful markers of the mitotically activecells in epidermis. We have used transient transfection invitro and transgenic mice in vivo to investigate K14 geneexpression. Our findings have important implications for themechanisms involved in the differential expression of K14,and they provide a valuable first step in defining a stratifiedsquamous epithelial cell-specific promoter.

MATERIALS AND METHODSPreparation of pH3cK14 P. pJK14'P contains the sequence

corresponding to human K14 mRNA, extending from thetranscription initiation site of the K14 gene to an Ava II site6 amino acid codons 5' from the TGA stop codon (11).Inserted in frame at the Ava II site of this plasmid is asequence coding for an antigenic portion of neuropeptidesubstance P (11). Five base pairs downstream from the TGAstop codon is a Sma I site. Two EcoRI sites, one immediately3' of the Sma I site and one 1.5 kilobases (kb) 3' downstreamfrom this site, are present in the plasmid.pJK14-P was digested with Sma I and EcoRI to excise

unwanted sequences, and a Stu I-EcoRI fragment extendingfrom 21 base pairs (bp) 3' of the TGA stop codon to -700 bp3' of the poly(A) signal of the human K14 gene (12) wasinserted. A fragment extending from a unique Kpn I sitewithin exon I of the K14 sequence to the EcoRI site =700 bp3' from the K14 poly(A) signal was then excised and ligatedto a HindIII/Kpn I fragment of the K14 gene (12). TheHindIII site was located -2500 bp 5' from the transcriptioninitiation site of the K14 gene, and the Kpn I site wasrecreated upon ligation. The fragment was then subclonedinto the HindIII/EcoRI site of plasmid pGEM2 (PromegaBiotec). The completed construct is shown in Fig. 1. For somegene transfection studies, supercoiled plasmid pH3cK14-Pwas used. For other gene transfection studies and for alltransgenic mouse studies, the H3cK14 P insert was isolatedand purified from vector sequences prior to use.

Preparation of Plasmids Used in Southern and NorthernHybridizations. 3SP. 3SP contains a 1090-bp insert encodinga major portion of the coding region ofhuman K14 subclonedinto pSP65 (7). This sequence shares 91% identity with thecorresponding portion of the mouse K14 cDNA (13), with 63bp being the longest stretch of identity. Radiolabeled com-plementary RNA (cRNA) probe was prepared by linearizingthe plasmid with HindIII and using SP6 RNA polymerase inthe presence of [32P]UTP and unlabeled ribonucleotides.

Abbreviation: cRNA, complementary RNA.*Present address: Department of Molecular Biology, PrincetonUniversity, Princeton, NJ 08540.

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pH3cK14 P

Hind III ATG TGA pA Eco RI

300 bp

FIG. 1. Genetic map of plasmid pH3cK14 P. The components ofplasmid pH3cK14 P are as follows: thin lines, pGEM2 vectorsequences; thick black line, 5' upstream sequence of the human K14gene (12) extending from the ATG translation initiation codon to theHindIII site located -2.5 kb 5' to the TATA box; open box, thecomplete coding sequence of the human K14 gene (12); stippled box,the sequence coding for the substance P tag (11); vertical striped box,K14 3' untranslated sequence extending from 21 bp 3' from the TGAstop codon to the poly(A) signal (pA); diagonal striped box, K14 genesequence extending -700 bp 3' from pA.

NSP. A 326-bp BamHI/EcoRV fragment of pH3cK14 P,encompassing the sequence from 4 bp 3' into the substanceP sequence to 170 bp 3' from the K14 poly(A) signal, wassubcloned into the HincII/BamHI sites of pGEM3 blue(Promega Biotec). A 135-nucleotide stretch is perfectlycomplementary to the human K14 mRNA; a 184-nucleotidestretch is perfectly complementary to the K14 P mRNA; thelongest stretch of sequence identity with the correspondingregion of the mouse K14 mRNA is 9 nucleotides. Radiola-beled probe was prepared by linearizing the plasmid withBamHI and using SP6 RNA polymerase in the presence of[32P]UTP and unlabeled ribonucleotides.

Cell Lines and Transient Transfections. SCC-13 was de-rived from a human squamous cell carcinoma of the skin (14)and was a gift from James Rheinwald (Dana-Farber CancerInstitute). Primary mouse epidermal keratinocytes werecultured from shaved mouse back skin as described byHennings et al. (15). Other cell lines were obtained from theAmerican Tissue Culture Collection. All cells were tran-siently transfected with DNA using the calcium phosphateprecipitation method, followed by a 15% glycerol shock (16).Cells were fixed in methanol at 65 hr posttransfection andsubjected to indirect immunofluorescence using the antibodyNCI/34 against substance P (17), followed by a fluorescein-conjugated secondary antibody.

Transgenic Mice. An outbred strain (CD-1) of mice (CharlesRiver Breeding Laboratories) was used for generation oftransgenic mice. Embryos were isolated and microinjected atthe single cell stage. Procedures used were essentially ac-cording to Choi et al. (18).

RESULTSThe Modified Human K14 Gene Construct and Its Expres-

sion in Tissue Culture Cells. In the human genome, there arethree K14 genes, but only one appears to be functional (16).To follow the expression of this gene immunologically, weinserted a small tag, encoding an antigenic portion of neuro-peptide substance P, just 5' to the TGA stop codon of thehuman K14 cDNA. Approximately 2500 bp of 5' K14 genesequence was used to drive the expression of this intronless,P-tagged gene. The construct, referred to as pH3cK14P, isillustrated in Fig. 1.To examine the expression of pH3cK14-P in vitro, we

transfected the construct into a variety of cultured cells,including cells of stratified squamous epithelial origin, wherethe endogenous K14 gene is expressed, and simple epithelialand fibroblast cells, which are negative for K14. Surprisingly,transfected cells from all tissue culture lines showed stainingwith the anti-P antibody (Fig. 2 A-F). Though the levels ofstaining varied somewhat for individual transfected cells, nomajor cell type or species-related differences in staining wereobserved. Hence, even though endogenous K14 expressionwas limited to squamous cell carcinoma cells and primaryepidermal keratinocytes, K14-P expression was detected inall transiently transfected cell types examined.A priori, it was possible that promiscuous expression of

K14*P in tissue culture cells was due to sequences locatedwithin the vector DNA rather than the K14 gene itself. Toexclude this possibility, we repeated the expression studies,this time using purified H3cK14-P DNA insert instead ofsupercoiled plasmid DNA. Although the efficiency of trans-fection was somewhat lower, transfected cells expressingK14*P were still readily detected by anti-P staining in allpopulations of cultured cells tested (Fig. 2 G and H).Moreover, widespread K14*P expression could not be ex-plained by the presence of a cryptic promoter within the 5'flanking region of the gene, since (i) a DNA with only 94 bp5' upstream from the transcription start site of the K14 genewas still expressed in cells of non-epithelial origin (notshown) and (ii) Northern blot analyses have shown that K14mRNAs of the appropriate size are produced from constructscontaining -400 bp of 5' K14 gene sequence (20).

Introduction of the K14-P Gene Construct into the GermLine of Mice. A number of possible explanations couldaccount for the failure of the K14 P gene to exhibit tissue-specific expression in transiently transfected cell lines. Itcould be that suppression of K14 gene expression in cells ofnonstratified squamous epithelial origin is maintained bynegative regulatory sequences that were missing in the K14-P

FIG. 2. Promiscuous expression of pH3cK14 P intransiently transfected cultured cells. Cells were trans-fected with pH3cK14 P (A-F) or purified H3cK14 Pinsert (G and H). Transfected cells were fixed andstained with an antibody specific for the substance Ptag (17): (A) SCC-13 cells (K5+, K14+). (B) Primarymouse keratinocytes (ref. 6 and 15; K5+, K14+). (Cand G) 3T3, a mouse fibroblast line (ref. 1; expressesvimentin). (D) HeLa, a human cervical carcinoma line(ref. 19; expresses simple epithelial keratins and vi-mentin). (E and H) PtK2, a potoroo kidney (simpleepithelial) cell line (ref. 19; expresses simple epithelialkeratins and vimentin). (F) MCF-7, a human breastadenocarcinoma line (ref. 19; expresses simple epithe-lial keratins). For cells of epidermal and simple epi-thelial origin, K14*P expression resulted in its integra-tion into the endogenous keratin filament network.With no endogenous keratin filament network, trans-fected fibroblasts showed an accumulation of aggre-gates of K14'P protein. (Bar = 20 am.)

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construct. Alternatively, regulation may be conferred bytissue-specific differences in the chromatin structure of theK14 gene, which were not manifested in the transientlytransfected DNA. Though other explanations are also pos-sible, these two general hypotheses could be readily distin-guished by introducing the K14 P gene into the germ line ofmouse embryos and examining foreign expression in thetissues of the transgenic offspring.Copies ofpurified H3cK14P insert were microinjected into

male pronuclei of single cell mouse embryos. At 3 weeks afterbirth, mouse tail DNAs were isolated and assayed for thepresence ofhuman K14-P sequences (Fig. 3A). Digestion withBgi II generated a 965-bp diagnostic fragment in DNAs frommice harboring the K14-P gene (see restriction map sitesmarked in Fig. 3B). In a Southern blot analysis (21), this bandwas detected with a 32P-labeled cRNA probe (3SP) tosequences partially contained within this fragment (Fig. 3A,lanes 1-6). Asjudged by linearized control DNAs (H3cK14 Pinserts) diluted as ifpresent 0.5x, 1 x, 2x, 5x, lOx, and 50xin the mouse genome, the number of integrated human K14-Pgenes ranged from =0.5 (lane 2) to >20 copies (lane 3).

In addition to the 965-bp diagnostic band, a hybridizingband of 3.6 kb was seen in many of the transgenic DNAsamples. The variability in intensity of this band correlatedwell with that seen for the diagnostic band. The simplestinterpretation of this band is that it is due to the tandem,head-to-tail integration of multiple K14'P DNAs into a singlesite within the mouse genome (Fig. 3B). This form oftransgenic integration has been noted before (22). The ap-pearance of additional, less intense radiolabeled bands (see,for example, lane 5) probably reflects hybrid DNA bands

A 1 2 3 4 5 6 c

10,000-

5,000 -4,000 -

3,000 -

2,000-

1,000- _

B

Bgl 11 Bgl 11 Bgl 11. Bgl 11 Bgl 11 Bgl 11

3SP

1 kb

FIG. 3. Southern analysis of DNAs from transgenic mice har-boring H3cK14'P sequences. DNAs from potential transgenic micewere digested with Bgl II, resolved by agarose gel electrophoresis,denatured, and transferred to nitrocellulose by blotting (21). Blotswere hybridized with radiolabeled cRNA from 3SP, containing theK14 coding sequence. (A) Lanes 1-6, DNAs from transgenic mice;lane C, DNA from control mouse. Molecular markers are indicatedin bp. (Re-runs of samples in lanes 1-4 revealed that the 3.3-kb bandseen here is actually 3.6 kb, as it is in lanes 5 and 6.) (B) Hypotheticaldiagram oftandem head-to-tail integration ofthe H3cK14 P sequenceinto a single site of the mouse genome. Three copies of H3cK14 Psequence are indicated by the thick arrows. Boxes with diagonal barsindicate mouse DNA. The two Bgl II sites within H3cK14-P areindicated as is the sequence encompassed by the 3SP probe.

containing the junction of the- 5' end of K14-P DNA and theintegration site(s) within the mouse genome. Since the sizesof these hybrid DNA bands varied among trangenic mice, itis likely that the integration sites of the K14P gene weredifferent and sometimes multiple.

Expression of K14-P Protein in Tail Skin of Transgenic Mice.To rapidly screen for K14 P expression, we stained frozensections of tail skins with anti-P antibody. Each transgenicmouse tail showed proper expression ofthe human K14-P genein the basal layer of the epidermis and in the outer root sheathcells of the hair follicles (Fig. 4). For the mouse whose DNAwas analyzed in lane 1 of Fig. 3A, K14 P expression was seenin every basal epidermal cell, suggesting that the transgene hadbeen stably integrated prior to the first embryonic cell division(Fig. 4A). In contrast, mice whose DNAs were analyzed inlanes 2-4 and 6 of Fig. 3A showed K14 P expression only inclusters of basal cells (Fig. 4C). In these cases, the transgenewas most likely integrated after the first embryonic celldivision. Although mosaic mice were not studied further, theyprovided a valuable insight into the movement of terminallydifferentiating epidermal cells: the persistence ofa low level ofK14*P in the suprabasal cells demonstrated a columnar migra-tion perpendicular to the skin surface.The persistence of K14*P expression in the suprabasal

layers seemed to be more pronounced for some transgenicmice than others. An anti-P-stained section of tail skin fromthe mouse that showed the least down-regulation is shown inFig. 4D. The variability in degree of down-regulation was

AU

FIG. 4. Rapid analysis oftransgenic mice to check for integrationof the K14P gene at the single cell stage and correct expression inskin. Expression of K14'P in frozen sections of transgenic mice tailskins was detected by indirect immunofluorescence using anti-P anda fluorescein isothiocyanate-conjugated secondary antibody.Sources of tails were as follows: (A) The lower expressing transgenicmouse whose DNA is shown in Fig. 3A, lane 1. (B) A control(nontransgenic) mouse. (C) A mosaic transgenic mouse whose DNAis shown in Fig. 3A, lane 3. (D) The higher expressing transgenicmouse whose DNA is shown in Fig. 3A, lane 5. The dashed linedepicts the position of the basal lamina; the solid line delineates theexternal boundary of the stratum corneum. bl, Basal layer; ors, outerroot sheath of hair follicle. (Bar = 30 gm.)

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most likely due to variation in the site(s) of integration of theK14 P gene and/or transgene copy number.

Expression of K14-P mRNA: Proper Tissue-Specific Expres-sion. To examine K14-P expression in other tissues, wesacrificed transgenic offspring of the mice whose DNAs areshown in lanes 1 and 5 of Fig. 3A. We chose six tissues forour studies: liver, kidney, brain, tongue, esophagus, andskin. For each tissue, some samples were used directly forRNA extraction; others were either fixed in 4% paraformal-dehyde or frozen and prepared for immunohistochemistry.Equal amounts of control and transgenic RNAs from each

tissue were subjected to Northern blot analyses using radio-labeled cRNA probes to either (i) 3SP, complementary to theK14 coding sequence (Fig. 5A, left) or (ii) NSP, complemen-tary to the substance P sequence and 3' noncoding portion ofhuman K14 mRNA (Fig. SA, right, and B). Under theseconditions, a strongly hybridizing band of 1.6 kb was de-tected with the 3SP probe for control tissues from tongue(lane T), esophagus (lane E), and skin (lane S) but not in tissuefrom liver (lane L), kidney (lane K), or brain (lane B). Thisband comigrated with K14 mRNA in human skin (lane H) andcorresponded in size and tissue specificity to mouse K14mRNA (13). With the exception of a slight nonspecificcross-hybridization with 18S rRNA (-41.65 kb; Fig. SA, right;see also Fig. SB, left, lanes L, K, and B), the NSP probeshowed no hybridization with any mouse RNAs.

H LK B T E S

B T E S

*. is

1.6-3

B H L K.-...

..

1.6-

L' K' B' T' E'....... : C

In contrast to RNAs from control mice tissues, RNAs fromstratified squamous epithelial tissues of either the lowerexpressing transgenic mouse or the higher expressing trans-genic mouse showed marked hybridization with the NSPprobe (Fig. SB, left and right blots, respectively). A 1.6-kbband was readily detected in tongue (lane T) and skin (lane S)samples of the lower expressing transgenic mouse and in thetongue (lane T'), esophagus (lane E'), and skin (lane S')samples of the higher expressing transgenic mouse. Althoughthe mRNA from the esophageal tissue ofthe lower expressingtransgenic mouse did not seem to transfer well in the regionof the K14 P mRNA, the existence ofK14 P in this tissue waslater confirmed by immunohistochemistry. No specific hy-bridization was detected in RNAs from liver, kidney, andbrain, suggesting that K14 P mRNA was expressed properlyin the transgenic animals.Taking into account the difference in length of hybridizing

sequence for K14 and K14-P mRNAs (135 and 184 nucleo-tides, respectively), it was estimated that the amount of K14mRNA present in the human foreskin sample was l'1Oxhigher than the amount ofK14 P mRNA present in the higherexpressing transgenic mouse skin sample.

Expression of K14-P Protein: Proper Down-Regulation inSuprabasal Cells. Since skin, tongue, and esophagus containa heterogeneous mixture of cell types, verification that K14-Pis expressed only in stratified squamous epithelial cellsrequired investigation of individual cells within each tissue.Appropriate tissue sections (5 ,um) were thus examined byimmunohistochemistry, using anti-P to localize K14-P intransgenic tissues and anti-K14 to localize endogenous K14in control tissues (Fig. 6). Similar to that seen previously forthe parental mouse, prominent anti-P staining was observed

A B

I' -

L' K' B' T' E'..- ....{E C...

O'fi /D

FIG. 5. Northern analysis of K14-P mRNA expression in trans-genic mice tissues. Tissues from control mice (A) or transgenicoffspring (B) were frozen in liquid nitrogen and homogenized in asolution of 4 M guanidinium thiocyanate for RNA extraction (23).RNAs were resolved by formaldehyde gel electrophoresis, trans-ferred to nitrocellulose by blotting (24), and hybridized with 32p_labeled cRNA complementary to either 3SP (which recognizesmouse and human K14 mRNA) or NSP (which recognizes onlyhuman K14 and K14-P mRNA). For the 3SP probe, wash conditionswere chosen at 2-5°C below the melting temperature of a hybridbetween the probe and the highly homologous mouse K14 mRNA.For the NSP probe, wash conditions were chosen such that onlyhybrids between the probe and either K14-P mRNA (forming a184-bp hybrid) or human K14 mRNA (forming a 135-bp hybrid) weredetected. (A) Northern analysis of control mouse RNAs hybridizedwith radiolabeled 3SP (left) or NSP (right). RNA sources were asfollows: lane H, human foreskin; lane L, mouse liver; lane K, mousekidney; lane B, mouse brain; lane T, mouse tongue; lane E, mouseesophagus; lane S, mouse skin. The size of the hybridizing band isindicated in kb. (B) Northern analysis of transgenic mouse RNAshybridized with radiolabeled NSP. RNAs were from the tissuesindicated in A. The left blot shows RNAs from the lower expressingtransgenic mouse; the right blot shows RNAs from the higherexpressing transgenic mouse.

E F

FIG. 6. Comparison of immunolocalization of K14-P and endoge-nous K14 in transgenic mice harboring a hybrid human K14-P gene.Tissues shown are from offspring ofthe transgenic mouse whoseDNAis shown in lane 1 of Fig. 3 (A, C, and E) and from a control mouse(B, D, and F). Tissues from nonstratified squamous epithelial tissueswere negative for K14 P and for endogenous K14 (not shown). Tissuesfrom tail skin (A and B), tongue (C and D), and esophagus (E and F)were fixed in 4% paraformaldehyde and sectioned (5 ,um). Sectionswere incubated with either anti-P (17) (A, C, and E) or the rabbitpolyclonal antiserum (no. 199) specific for the carboxyl-terminalsequence of K14 (25) (B, D, and F). Antibody binding was visualizedby staining with 5-nm gold particle-conjugated goat anti-rat IgG andgoat anti-rabbit IgG, respectively, followed by the silver enhancementtechnique of Janssen Pharmaceutical. (Bar = 20 a.tm.)

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in the basal epidermal layer from skin of a transgenicoffspring (Fig. 6A). The pattern of basal cell-specific stainingwas indistiguishable from that seen in control mouse skinstained with anti-K14 antiserum (Fig. 6B). For both trans-genic and control mice, suprabasal cells were distinguishedby a marked decline in the levels of K14P and K14, respec-tively. Only occasional regions of skin sections showedprominent suprabasal staining with anti-P or anti-K14, and thiswas most likely due to the angle of the section, rather thancontinued elevated expression in suprabasal cells. Stainingwas not seen in dermal fibroblasts or in any other nonepithelialcells of the skin. Collectively, these data agree with previousknowledge of K14 expression in human and in rodent skin (1).The dorsal side of tongue also showed indistinguishable

staining patterns with anti-P and anti-K14 (Fig. 6 C and D,respectively). Though expression was predominantly basal,some elevated expression was seen in the ridges betweenfiliform papillae. The esophagus also showed more prominentstaining of the basal layer, although in this case, the dimi-nution in anti-P and anti-K14 staining in suprabasal cells wasnot as pronounced as it was in skin or tongue (Fig. 6 E andF). Hence in all cases, K14-P expression paralleled that ofendogenous K14 protein and was down-regulated to varyingdegrees as cells moved to the suprabasal layers.

DISCUSSIONLittle is known about the molecular mechanisms underlyingexpression of genes in stratified squamous epithelial tissues.Such knowledge is of major importance, not only for enhanc-ing our understanding of epithelial differentiation but also forexploring the possibility of directing the foreign expression ofa variety of medically useful agents in the epidermis of skingrafts. Recent studies showed that retrovirus-mediated genetransfer could be used to introduce a recombinant humangrowth hormone gene into cultured human keratinocytes,which were subsequently grafted as a sheet onto athymicmice (10). Although these investigations have not yet dem-onstrated the ability of keratinocyte-produced hormone toenter the bloodstream of grafted mice, they have providedencouraging preliminary evidence that transfected epidermalcells might be a valuable tool in drug delivery. Such findingsclearly merit further investigation, and the use of a naturalepidermal promoter for graft-mediated administration ofgeneproducts would obviously be desirable. Since previous stud-ies showed that K14 expression is a faithful and abundantbiochemical marker of stratified squamous epithelia, the K14gene was a good candidate for a strong epidermal promoter.Our present studies were aimed at elucidating the biochem-

ical features of K14 gene expression. Thus far, our resultssuggest that non-intron sequences extending from 2500 bp 5'upstream from the transcription initiation site to -700 bp 3'downstream from the poly(A) signal of the human K14 geneare sufficient to regulate K14 expression. At least for mouseand human, these sequences appear to transcend speciesdifferences, since proper expression of the human K14 genewas observed in mice. Moreover, the regulatory mechanismsthat operate on these sequences do not seem to be affectedgreatly by chromosomal position.The promiscuous expression of K14 in transiently trans-

fected cells of nonstratified squamous epithelial origin wassomewhat surprising, since most genes are properly ex-pressed in transient and in permanent cell lines. Since theH3cK14 P construct was expressed properly in transgenicmice, it seems that at least part of the differential control ofK14 must reside at the transcriptional level and that sometype of negative regulation must normally take place in cellsof nonstratified squamous epithelial origin. We have not yetruled out the possibility that a negative regulatory factorplays a role in this process and that promiscuous expression

arises when the K14 gene is present in high copy number,thereby titrating this factor. However, if they exist, thesequences mediating the effects of such a factor must bepresent in our H3cK14 P construct, and the factor cannot belimiting in transgenic mice harboring >20 copies of the K14 Pgene. Another possibility is that a negative control involvingchromatin structure or methylation pattern normally sup-presses K14 in nonstratified squamous epithelia, but thismechanism is overridden in transiently transfected cells.Whatever the molecular details of K14 gene regulation, it

must be consistent with our findings that (i) all of thetranscriptional factors necessary to drive the expression ofthe naked K14 gene are present in a wide variety ofcell types,including fibroblasts and simple epithelial cells, and (ii) atleast some of the sequences necessary for tissue-specific anddifferentiation-specific expression of the K14 gene residewithin our K14-P construct. As we begin to define thesequences involved in the regulation of the K14 gene, theprecise molecular pathways leading to tissue-specific anddifferentiation-specific expression should become clearer.For the moment, the proper expression exhibited by theK14*P construct in transgenic mice is an exciting first step indemonstrating that these sequences can be used to expressforeign genes in stratified squamous epithelia and, perhaps inthe future, to correct genetic disorders of these tissues.

We extend our thanks to Ms. Grazina Traska for her excellenttechnical assistance in tissue culture and to Mr. Philip Galiga for hisartful presentation of our data. This work was funded by grants toE.F. from the National Institutes of Health and from the CancerResearch Foundation. E.F. is the recipient of a Presidential YoungInvestigator Award from the National Science Foundation. R.V. isa National Institutes of Health Predoctoral Trainee.

1. Moll, R., Franke, W., Schiller, D., Geiger, B. & Krepler, R. (1982)Cell 31, 11-24.

2. Fuchs, E., Tyner, A. L., Giudice, G. J., Marchuk, D., RayChaud-hury, A. & Rosenberg, M. (1987) Curr. Top. Dev. Biol. 22, 5-34.

3. Hatzfeld, M. & Franke, W. W. (1985) J. Cell Biol. 101, 1826-1841.4. Eichner, R., Sun, T.-T. & Aebi, U. (1986) J. Cell Biol. 102, 1767-

1777.5. Sun, T.-T., Eichner, R., Schermer, A., Cooper, D., Nelson, W. G.

& Weiss, R. A. (1984) in The Cancer Cell, eds. Levine, A., Topp,W., van de Woude, G. & Watson, J. D. (Cold Spring Harbor Lab.,Cold Spring Harbor, NY), Vol. 1, pp. 169-176.

6. Steinert, P. M., Steven, A. C. & Roop, D. R. (1985) Cell 42, 411-419.7. Tyner, A. L. & Fuchs, E. (1986) J. Cell Biol. 103, 1945-1955.8. van Muijen, G. N. P., Ruiter, D. J., Franke, W. W., Achtstatter,

T., Haasnoot, W. H. B., Ponec, M. & Warnaar, S. 0. (1986) Exp.Cell Res. 162, 97-113.

9. Wu, Y.-J., Parker, L. M., Binder, N. E., Beckett, M. A., Sinard,J. H., Griffiths, C. T. & Rheinwald, J. G. (1982) Cell 31, 693-703.

10. Morgan, J. R., Barrandon, Y., Green, H. & Mulligan, R. C. (1987)Science 237, 1476-1479.

11. Albers, K. & Fuchs, E. (1987) J. Cell Biol. 105, 791-806.12. Marchuk, D., McCrohon, S. & Fuchs, E. (1985) Proc. Natl. Acad.

Sci. USA 82, 1609-1613.13. Knapp, B., Rentrop, M., Schweizer, J. & Winter, H. (1987) J. Biol.

Chem. 262, 938-945.14. Wu, Y.-J. & Rheinwald, J. G. (1981) Cell 25, 627-635.15. Hennings, H. D., Cheng, M. C., Steinert, P., Holbrook, K. &

Yuspa, S. H. (1980) Cell 19, 245-254.16. Graham, F. L. & van der Eb, A. J. (1973) Virology 52, 456-467.17. Cuello, A. C., Galfre, G. & Milstein, C. (1979) Proc. Natl. Acad.

Sci. USA 76, 3532-3536.18. Choi, Y., Henrard, D., Lee, I. & Ross, S. R. (1987) J. Virol. 61,

3013-3019.19. Moll, R., Krepler, R. & Franke, W. W. (1983) Differentiation 23,

256-269.20. Giudice, G. J. & Fuchs, E. (1987) Cell 48, 453-463.21. Southern, E. M. (1975) J. Mol. Biol. 98, 503-517.22. Palmiter, R. D., Chen, H. Y. & I3rinster, R. L. (1982) Cell 29, 701-

710.23. Chomczynski, P. & Sacchi, N. (1987) Anal. Biochem. 162, 156-159.24. Thomas, P. (1980) Proc. Natl. Acad. Sci. USA 77, 5201-5205.25. Stoler, A., Kopan, R., Duvic, M. & Fuchs, E. (1988) J. Cell Biol.

107, 427-446.

Cell Biology: Vassar et al.

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