Sequence and Chromosomal Context Effects on Variegated Expression of Keratin 5/lacZ Constructs in...

Copyright 2001 by the Genetics Society of America

Sequence and Chromosomal Context Effects on Variegated Expression ofKeratin 5/lacZ Constructs in Stratified Epithelia of Transgenic Mice

Angel Ramırez,* Eric Milot,† Immaculada Ponsa,‡ Camelia Marcos-Gutierrez,§ Angustias Page,*Mirentxu Santos,* Jose Jorcano* and Miguel Vidal§

*Cell and Molecular Biology, Centro Investigaciones Medio Ambientales y Energeticas (CIEMAT), 28040 Madrid, Spain, †Department of CellBiology, Medical Genetics Center, Erasmus University, 3000 DR Rotterdam, The Netherlands, ‡Departament de Biologia Cellular,

Fisiologia i Immunologia, Facultat de Medicina, Universitat Autonoma de Barcelona, E-08193 Bellaterra, Spain and§Department of Developmental and Cell Biology, Centro Investigaciones Biologicas, 28006 Madrid, Spain

Manuscript received June 1, 2000Accepted for publication January 30, 2001

ABSTRACTThe expression of transgene loci in mammals often occurs in a heterocellular fashion resulting in

variegated patterns of expression. We have examined the effect of chromosomal integration site, copynumber, and transcriptionally activating sequences on the variegation of a keratin 5-lacZ (K5Z) constructin the stratified epithelia of transgenic mice. lacZ expression in these mice is always mosaic, and the b-galactivity per cell is usually higher in the lines with a higher proportion of expressing cells. Similar constructs,in which cDNAs were exchanged by lacZ sequences, showed no variegation. Also, when a strongly active,nonvariegating construct was coinjected with K5Z, most transgenic lines showed an almost homogeneouslacZ expression. The comparison of transgene arrays of different copies inserted at the same locus (obtainedby using a lox/Cre system) showed that the reduction of copy number does not lead to an increase inthe proportion of cells that express the transgene. Finally, in most of the variegating or nonexpressinglines the transgenes were located both at intermediate positions and at peritelomeric regions in the longchromosome arms. These findings suggest that the probability and efficiency of expression of K5Z genesdepend on both long range chromosomal influences and on sequences in the transgene array.

GERMLINE transformation of mice has been invalu- et al. 1993), although the mechanisms responsible forsuch silencing events are still poorly understood.able in understanding processes that control gene

expression and has led to the identification of tissue- Chromosomal context is important for variegation,shown by the mosaic expression patterns of genes lo-specific control elements (Grosveld and Kollias 1992).cated near or at pericentromeric heterochromatin inHowever, a common feature of transgenic mice is theDrosophila (Henikoff 1990). No systematic analysis ofwide variation in expression levels among individualsthe integration site of DNA constructs has been carriedcarrying the same transgene (Grosveld and Kolliasout in transgenic mice, although integration at pericen-1992). In addition, quite often DNA constructs expresstromeric regions has been reported with unexpectedin a mosaic or variegated fashion, by which transgenefrequency (Dobie et al. 1996; Festenstein et al. 1996;expression occurs in some, but not all, the cells withinMilot et al. 1996). As observed before in Drosophilaa population of equivalent transgene-bearing cells (Tho-(Dorer and Henikoff 1994) and in plants (Assaad etrey et al. 1993; Porter and Meyer 1994; Ramırez et al.al. 1993), variegation of DNA constructs in mice is re-1994; Elliott et al. 1995; Dobie et al. 1996; Festensteinlated to the repeated nature of multicopy transgeneet al. 1996; Milot et al. 1996; Morley et al. 1996; Grau-arrays (Garrick et al. 1998), although a direct relationbert et al. 1998). Thus, the transcriptional potential ofbetween copy number and proportion of expressinga given construct is controlled by more than just its cis-cells is not always observed (Festenstein et al. 1996;control elements. Recent work by Whitelaw, Martin, andZhuma et al. 1999). Also, variegation correlates withcolleagues suggest that the variability of expression lev-chromatin accessibility but not with DNA methylationels among transgenic lines results from the differences(Garrick et al. 1996). However, the presence in trans-in the proportion of cells that express the transgenegenic constructs of intact locus control regions (LCRs),(Robertson et al. 1995). The variegated expression alsowhich have a dominant activity, usually results in homo-occurs in Drosophila (Henikoff 1990), yeasts (Gottsch-geneous, nonmosaic expression patterns irrespective ofling et al. 1990; Allshire et al. 1994), and plants (Meyerthe integration site (Milot et al. 1996; Kioussis andFestenstein 1997).

The above features of variegating transgenes in miceCorresponding author: Miguel Vidal, Department of Developmental

derive mostly from constructs expressed in hematopoi-and Cell Biology, Centro de Investigaciones Biologicas, Velazquez144, 28006 Madrid, Spain. E-mail: [email protected] etic tissues (Elliott et al. 1995; Robertson et al. 1995;

Genetics 158: 341–350 (May 2001)

342 A. Ramırez et al.

DNA fluorescence in situ hybridization (FISH) analysis:Festenstein et al. 1996; Garrick et al. 1998; GraubertDNA FISH was performed as described (Milot et al. 1996).et al. 1998). In this report we have studied transgene

variegation in stratified epithelia, such as those that linethe surface of the skin and parts of the digestive tract

RESULTS(Fuchs and Weber 1994). We have used the lacZ gene

Variegated expression of K5Z constructs in transgenicof Escherichia coli fused to sequences from the keratin K5mice: The basic K5Z construct used consists of 5.3 kbgene, which encodes one of the intermediate filamentof 59 flanking and promoter sequences of the bovinepolypeptides specific to the basal layer of stratified epi-K5 gene fused to a lacZ gene of E. coli (Figure 1). Onlythelia. The remarkable tendency of these K5-lacZ (K5Z)three lines (B4, B7, and B38) showed a detectable ex-constructs to express in a mosaic fashion allowed us topression and were used to derive stable lines. Analysisask whether copy number and chromosomal integrationof sections of adult tissues (4- to 8-week-old animals)site are major determinants of variegation and how suchshowed restricted expression of the transgene to strati-variegation can be affected by the presence of transcrip-fied epithelia and their appendages, like that of thetionally active sequences. The results show that copy-endogenous K5 gene. A discrepancy, however, was thenumber reductions do not affect variegation of K5Zmosaic expression of the transgene in all epithelia ofconstructs and that position effects are imposed by telo-every animal of lines B4, B38 (Figure 2A), and B7 (notmeric regions. We also find a variability in b-galactosi-shown). Detection of b-gal activity was done using vari-dase (b-gal) activity levels per cell between differentous conditions that allowed us to rule out that mosaicvariegating lines and that the presence of strong activat-expression was due to trivial reasons, such as differentialing sequences turns variegation into a homogeneousaccessibility of substrate or others. In addition, the pro-and efficient expression of lacZ in almost all cells.portion of expressing cells varied among the three lines.Thus, line B38 showed b-gal activity in a larger numberof cells in each epithelium than lines B4 or B7 did.MATERIALS AND METHODSThere were exceptions, such as the esophagus, where

Transgenic mice: The K5Z construct was obtained by liga- the proportion of expressing cells was consistentlytion of sequences from the bovine K5 locus (25300 bp to higher in line B4 than in B38. Moreover, b-gal activity1140 bp) to a lacZ gene, as previously described (Ramırez et al.

per cell was also higher in line B38 than in the other1994). To construct K5ZMAR the 59A element of the chickentwo lines, as indicated by the intensity of X-gal staining oflysozyme gene in plasmid pB-1-X1 (Stief et al. 1989) was ligated

to the 59 end of K5Z. The K5Zlox construct was obtained by b-gal-positive cells after simultaneous, timed incubationinsertion of an oligonucleotide containing a single copy of with the substrate. Figure 2B shows the results for palatethe 34-bp lox site at the 39 end of K5Z. The K5ZHK10 construct epithelium, but similar results were observed in thecontains sequences from the bovine K5 locus (25300 bp to

tongue and tailskin of these lines and in other lines150 bp) fused to a genomic fragment spanning the entiredescribed below (not shown).coding region of the human K10 (HK10) locus (Rieger and

Such a variegated expression of K5Z transgenes con-Franke 1988). Transgenic mice were generated and geno-typed as described (Ramırez et al. 1994). For Cre recombina- trasts with the usually nonmosaic expression from simi-tion, circular Cre-expression vector pCAGGS-Cre (Araki et al. lar constructs in which a number of cDNAs are driven1995) was injected into mouse eggs at 5 ng/ml. by the same K5 regulatory sequences used in the K5Z

b-Galactosidase activity and immunofluorescence: Tissuesconstruct. Figure 3 shows examples of the expressionwere dissected and frozen in OCT medium. Histochemistryof both wild-type and truncated cDNAs encodingand immunodetection were done on 7-mm cryosections loaded

on poly-l-lysine-coated slides as described (Ramırez et al. HEGFR. In the three lines analyzed, the protein product1994). The mouse K5 was detected using a rabbit antipeptide of the transgenes is detected in an even, nonmosaicantiserum (BAbCo), HK10 with monoclonal antibody AE2 fashion in the basal layer of stratified epithelia. Similar(ICN Biochemicals), and b-gal with a rabbit antiserum

results have been obtained by other researchers who(5Prime-3Prime, Boulder, CO). Human epidermal growth fac-have used the K5 regulatory sequences present in thetor receptor (HEGFR) was detected with monoclonal antibody

EGFR528 (Santa Cruz Biotechnology). Secondary antibodies K5Z construct to drive expression of a variety of cDNAs.were Texas-red-conjugated donkey IgG anti-rabbit IgG and We conclude that the variegated expression of K5Z micefluorescein-conjugated goat IgG anti-mouse IgG from Jackson is neither due to lack of cis-acting sequences nor to aImmunoResearch Labs (West Grove, PA).

consequence of a heterogeneity among epithelial cells.Southern and Northern blot analyses: These were per-Instead, it appears most likely that it is the consequenceformed as described (Casanova et al. 1995). To normalizeof a process in which transgene activity occurs only infor loading differences in Southern blots, we used a Thy1

probe. Known amounts of plasmids were used to help deter- a subset of cells and in their descendants.mine copy numbers of the various transgenes. HK10 and The reduction of transgene copies has no effect onmouse K5 (mK5) mRNAs were detected in Northern blot using variegation of K5/lacZ constructs: To investigate thegenomic fragments spanning the last exon of the HK10 and

effect of copy number on K5Z variegation we used theof the mK5 genes, respectively. Hybridization signals werelox/Cre system of specific recombination that allowsquantitated using a Phosphorimager and Imagequant software

(Molecular Dynamics, Sunnyvale, CA). the generation of transgenic mice with different copies

343Variegation of K5 Transgenes in Stratified Epithelia

Figure 1.—Structure of K5 constructs used to generatetransgenic mice. Keratin K5 denotes the 59 flanking and pro-moter sequences of the bovine K5 locus. The boxes labeledlacZ represent the protein coding region of the trpS-lacZ geneand pA, a SV40 polyadenylation signal. White boxes indicateexon 1 sequences of the K5 gene, whereas black boxes denotethe exons of the HK10 gene. The box labeled MAR corre-sponds to the 59A element from the chicken lysozyme locus,and the box labeled loxP represents the site recognized bythe Cre recombinase of bacteriophage P1. Arrows denote thetranscription initiation site and the direction of transcription.

of the construct at the same chromosomal location(Araki et al. 1995). We used K5Zlox, a construct identicalto K5Z except for the addition of a single copy of the34-bp recognition site (loxP) of the Cre recombinaseof bacteriophage P1 (Figure 1). Since transgenes usuallyintegrate in multimeric arrays, whenever a head-to-tail Figure 2.—Expression properties of K5Z mice. (A) Com-arrangement occurs Cre-mediated recombination be- parison of lacZ expression and endogenous K5 localization

in adult tissues of K5Z mice. Tissue sections of transgenic linestween loxP sites in individual K5Zlox transgenes willB4 and B38 were incubated overnight at 378 with X-gal toreduce copies in the array. We generated six stabledetect the b-gal activity and counterstained briefly with eosin.K5Zlox transgenic lines. Two of them showed no b-gal The mouse K5 was detected by indirect immunofluorescence

activity (or only in a few cells) whereas the other four with an anti-K5 serum. (B) Comparison of b-gal activity pershowed variegated expression similar to that of K5Z cell. Palate tissue sections of lines B4 and B38 stained at 378

with X-gal for the indicated periods of time.constructs (Table 1). Cre-mediated recombination wascarried out by microinjection of a Cre expression vectorin zygotes obtained from mating wild-type female mice cells in double K5Cre/K5Zlox transgenic mice derivedwith hemizygous K5Zlox males. We used line 4734 (7 from lines 4715 (mouse 6603) and 4736 (mice 6483 andcopies) with a reduced proportion of nonexpressing 6484), compared with the absence of recombination incells and line 4712 (14 copies) with many nonexpressing single K5Zlox transgenic mice (mice 6607 and 5419),cells. The offspring from microinjected oocytes that are shown in Figure 4B.showed a reduced transgene copy number were mated Tissue sections of the parental line 4712 and of itswith wild-type animals to establish lines hemizygous for reduced-copy progeny line 4712rec.c, which retainedthe recombined transgenic loci. Cre recombination one to two copies, showed no significant alteration ofwithin the 4734 parent line produced two indepen- the b-gal activity after copy-number reduction (see Fig-dent reduced-copy progeny founders, 4734rec.a and ure 4C, I and II). A similar result was obtained for mice4734rec.b (Figure 4A, lanes 1 and 2). Similarly, from line 4712rec.a and 4712rec.b (not shown), which retained4712 we obtained three lines with a reduced number of two copies and one copy, respectively. Figure 4C, III–copies, 4712rec.a, 4712rec.b, and 4712rec.c (Figure 4A, VIII, shows tissue sections from the 4734 parent linelanes 4–6). Cre-mediated recombination was carried out and from line 4734rec.a, in which the only perceivedalso by crossing hemizygous K5Zlox mice (lines 4712, alteration was the reduced intensity in b-gal staining, but4715, 4734, and 4736) with a mouse that expressed the not in the proportion of expressing cells. Also, doubleCre recombinase in all stratified epithelia. Examples of K5Cre-K5lox mice showed no alterations in their expres-

sion patterns upon copy-number reduction (not shown).copy-number reduction specific for stratified epithelial


terns contrasted with that of mice bearing the K5Z con-struct alone. Thus, the b-gal expression pattern in sixof the lines (3578a, 3578b, 3668, 4460, 4472, and 4483)was almost indistinguishable from that of the endoge-nous K5 gene (Figure 6A), except for very small clustersof cells lacking b-gal activity (see below). Of the othertwo lines, one (4290) expressed b-gal in most epithelialcells, although not as extensively as in the precedentlines, whereas the other (4464) showed few b-gal-express-ing cells. The expression of HK10 was almost identicalto that of lacZ (not shown). Table 1 summarizes thesedata.We conclude that the presence of sequences withLCR-type properties (K5HK10) enhances significantlythe expression of variegating constructs (K5Z) in thesame transgene array.

Heterogeneity of expression of individual copieswithin a transgene array: The detailed study of the fewcases of mosaic expression in K5Z 1 K5ZHK10 lines,

Figure 3.—Expression of K5 HEGFR transgenes. Immuno- which was done by double immunofluorescence withfluorescence analysis of sagital sections of tails from mouse

rabbit anti-b-gal antibodies and with a mouse anti-HK10lines bearing wild-type (A) and truncated (B) cDNAs for themonoclonal antibody, led to some unexpected observa-HEGFR. The protein is detected continuously throughout the

basal layer of the epidermis and in the hair follicles that appear tions. Thus, although line 3668 expressed in a nonvari-as circular structures due to the section angle. gated fashion in most epithelia, patches of cells in the

epithelium of the ventral tongue did not express lacZbut expressed HK10 (Figure 6B, III and IV). Conversely,

Taken together, these results indicate that copy-number in areas of the tongue and tailskin epithelia of linereduction has little effect on the variegation of K5Z 3578b some cells expressed lacZ but not HK10 (nottransgenes. shown). The most perplexing pattern was seen in small

The presence of an LCR-type sequence in K5Z trans- regions of various epithelia of line 4290. Here, somegenic arrays overcomes variegation: The tendency of cells expressed b-gal only, while other cells expressedK5Z transgenes to express in a variegated fashion did HK10 only and yet other cells expressed both or neithernot change when the 59A element of the chicken lysozyme of the two genes (Figure 6B, V and VI). In contrast, agene, a piece of DNA that reportedly increases efficiency more conventional scenario was observed in the lineof expression of heterologous genes in transgenic mice with more extensive variegation (4464), in which cells(McKnight et al. 1992), was included in the K5Z con- expressed either both or neither of the genes (Figure 6B,struct (Table 1). In contrast, a construct consisting of VII and VIII). These data indicate a differential expres-a genomic fragment spanning all exons of the human sion of some of the copies within the transgene array.keratin K10 gene fused to the same K5 sequences that Chromosomal position of transgenes: To determinedrive lacZ expression in K5Z mice (K5HK10 ; see Figure whether the variegated expression of K5Z constructs1) showed homogeneous expression of HK10 in four correlates with chromosomal position, we performedstable lines (665, 821, 1213, and 1227). Figure 5A shows FISH on metaphase chromosomes. Only a subset oftissue sections from line 821 analyzed with an antibody transgenic lines was available when we did this analysis.that recognizes human K10. Note that although K10 is The insertion site of the transgene was localized byspecific to suprabasal cells, it is expressed in the basal hybridization to the K5Z construct. Nine lines had thecells of K5HK10 mice, suggesting that the HK10 moiety transgenes inserted at several positions throughout theof the construct does not alter the tissue-specific expres- long chromosomal arm, of which five showed peritelom-sion properties of the K5 moiety. A fifth line (825) eric insertions. Another two lines had pericentromericshowed limited but significant variegation (not shown). transgenes. Additional FISH analyses were done forQuantitative expression analysis of these lines showed seven of the lines using the K5Z construct together witha direct correlation between HK10 mRNA levels and either mouse g-satellite sequences, which recognize cen-copy number, a feature commonly associated with LCRs tromeric regions, or with a probe for telomeric repeats.(see Figure 5B). A representative transgene-bearing chromosome from

To investigate the interaction between a variegating each transgenic line is shown in Figure 7 and the local-and a nonvariegating transgene at the same integration ization data are summarized in Table 1. In two variegat-site we coinjected K5Z and K5HK10 constructs in mouse ing K5Z lines, B4 and B38, the transgene was integratedembryos. Eight stable transgenic lines were obtained into middle positions in the long chromosomal arm.

Similar integration sites were found for the nonexpress-bearing both constructs, and their lacZ expression pat-


TABLE 1

Characteristics of K5 transgenic lines

Constructs Lines Copies Expressiona Integration siteb

K5Z B4 21 Variegated (111) Middle armB7 15 Variegated (111) NDB15 36 2ve NDB22 18 2ve NDB38 4 Variegated (1) Middle armB41 18 2ve ND

K5ZMAR 1722 30 2ve ND1727 5 Variegated (1) ND1736 9 Variegated (11) ND1739 35 2ve Middle arm1740 30 2ve ND

K5HK10 665 4 Nonmosaic ND821 10 Nonmosaic ND825 6 Variegated (1) Pericentromeric

1213 20 Nonmosaic ND1227 26 Nonmosaic ND

K5Zlox 4712 14 Variegated (111) Telomeric4715 75 2ve Pericentromeric4716 40 Variegated (11) Telomeric4734 7 Hardly variegated Telomeric4736 45 2ve ND4751 3 Variegated (11) ND

K5Z 1 K5HK10 3578a 1, 2c Nonmosaic ND3578b 5, 5 Nonmosaic ND3668 13, 13 Nonmosaic Pericentromeric4290 1, 1 Variegated (1) Telomeric4460 3, 1-2 Nonmosaic ND4464 52, 28 Variegated (111) Telomeric4472 10, 4 Nonmosaic ND4483 1, 1 Nonmosaic ND

a Expression in adult tissues as detected by X-gal staining. Lack of expression, or expression in very few cells,is indicated as 2ve. The degree of variegation is indicated from few nonexpressing cells (1) to a high proportionof nonexpressing cells (111).

b Middle arm indicates lack of co-localization of transgene and centromeric or telomeric signals; telomericindicates co-localization of transgene and telomeric signals on the long chromosomal arm; pericentromericindicates that the signal of the transgene is on the centromere or very close to the telomere of the short arm.ND, not done.

c Copies of K5Z and K5HK10, respectively.

ing K5ZMAR line 1739 and for the nonmosaic K5Z 1 are the two variegating K5Z 1 K5ZHK10 lines, suggestingthat lack of expression in them could be related, atK5HK10 line 3668. The only variegating K5HK10 line

(825) and the nonexpressing K5Zlox line 4715 have least in part, to position effects imposed by telomereproximity.their transgenes inserted at pericentromeric positions.

Because of the acrocentric nature of mouse chromo-somes, we did not attempt to distinguish whether the

DISCUSSIONintegration site is centromeric or telomeric on the shortarm in these two lines, because in our hands both signals The wide occurrence of variegated expression among

transgenic lines has been noted recently. The mecha-do overlap (not shown). Among the lines with peritel-omeric insertions were K5Z 1 K5HK10 with a high nisms of appearance of variegation are likely multiple.

Some insight into these mechanisms has been obtained(4290) and low (4464) proportion of expressing cells.Similarly, K5Zlox lines with a reduced (4734) or exten- from analysis of transgene expression in erythroid and

lymphoid tissues. In this report we study transgene varie-sive (4712 and 4716) variegation showed insertions atperitelomeric regions. Most interestingly, 4290 and 4464 gation in stratified epithelia.


Figure 4.—Effect of copynumber on K5Z expression.(A) Southern blot analysisof K5Zlox mice with reducedcopy number obtained byinjecting a Cre-expressingplasmid in K5Zlox zygotes.Tail DNA was hybridized toa lacZ probe and to a Thy-1probe to normalize for load-ing differences. ParentalK5Zlox lines are shown abovethe lanes. A copy-numberreduction, or the lack of it,is indicated by 1 and 2,respectively. The asterisksshow truncated lacZ genes.(B) Southern blot analysisof genomic DNA isolatedfrom liver (L), epidermis(E), and dermis (D) fromoffspring of crosses betweenK5Zlox mice and a mouseexpressing Cre in the basallayer of stratified epithelia(K5Cre). Among the off-spring of line 4715 that carryK5Zlox, mouse 6603, but not6607, is hemizygous forK5Cre. Similarly, among theK5Zlox offspring of line 4736,mouse 6483 and mouse 6484,but not mouse 5419, arehemizygous for K5Cre. Blotswere hybridized to a lacZ

probe and to a Thy-1 probe. Specific copy-number reduction occurred in the epidermis of double transgenic mice. (C) b-Galactivity of tissue sections from parental and reduced-copy-number mice from lines 4712 and 4712rec.c (I and II, respectively)and from lines 4734 (III, V, and VII) and 4734rec.a (IV, VI, and VIII).

DNA sequences and silencing: Some sequences can the lacZ gene, because it could be argued that it is due,instead, to the LCR-like properties of HK10 alone oract as foci for gene silencing, as has been proposed to

explain the poor expression of ovine b-lactoglobulin with K5 sequences. In any case, when the transgenearrays consisted of a mixture of K5Z and K5HK10 se-genes in transgenic mice (Clark et al. 1997). Here, we

describe 17 K5Z mouse lines that either express in a quences most of the lines showed a nonmosaic expres-sion pattern. This is reminiscent of the lack of variega-variegated fashion or do not express at all. This adds

to the cumulative evidence of unreliable expression of tion often associated with transgenes harboring LCRs(Kioussis and Festenstein 1997).many lacZ-containing constructs, particularly in adult

tissues (Paldi et al. 1993; Thorey et al. 1993; Cui et al. Using model (simplified) constructs, it has beenshown that variegation varies depending on whether an1994; Guy et al. 1996). Interestingly, we show that when

cDNA fragments are exchanged by the lacZ gene, in enhancer is present or not. Thus, it has been proposedthat enhancers increase the probability of expressionequivalent K5 constructs, they express more efficiently,

with little or no variegation. Other researchers have by preventing variegation alone, without affecting thetranscription rate of the promoter (Walters et al. 1996;used our K5 promoter sequences to drive expression of

a number of cDNAs, and in most cases they report Sutherland et al. 1997) or by affecting both (Bouhas-sira et al. 1997). Our data on K5Z, a construct with anonmosaic expression (Robles et al. 1996; Pierce et al.

1998; Wang et al. 2000). This suggests that variegation complex set of regulatory sequences, support the lattermodel because the differences in b-gal activity per cell(or even lack of expression) of K5Z constructs in mice

can be due, at least in part, to lacZ sequences, rather among our K5Z lines, which are higher in the lines witha higher proportion of expressing cells, could indicatethan to the absence of cis-acting sequences required for

appropriate expression in the basal layer of stratified a variable transcription rate. An alternative possibility,however, takes into account possible differences in ac-epithelia. Nevertheless, the efficient, nonmosaic (al-

most) expression of the K5HK10 construct cannot be tive copies in the array, as indicated by the intriguingobservation in some of the K5Z 1 K5HK10 lines thatinterpreted as an indication of the negative effects of


Figure 6.—Expression of K5Z 1 K5HK10 transgenes. (A)Nonvariegated lacZ expression in sections of tongue (I), tail-Figure 5.—Expression of K5HK10 transgenes. (A) Immu-skin (II), esophagus (III), palate (IV), and footsole (V) of anofluorescence analysis of HK10 in tissue sections of a repre-representative transgenic line. (B) Double immunofluores-sentative K5HK10 line. (B) Copy-number-dependent expres-cence of HK10 (green) and b-gal (red) in sections of dorsalsion of K5HK10 in transgenic mice. Ten micrograms of total(B, I and II and V–VIII) and ventral tongue (B, III and IV)tailskin RNA isolated from a nontransgenic mouse (lane 1)from the indicated lines.and from K5HK10 lines (lanes 2–6) was analyzed by Northern

blot using a HK10 probe. To correct for loading differences,a K5 probe was used. The normalized values of K10 mRNAare plotted against gene copy number.

gating lines are located at peritelomeric sites. Whilemosaic silencing of genes inserted at telomeric positionshas been observed for both yeast (Gottschling et al.

not all copies in the transgene array are active within 1990) and Drosophila (Cryderman et al. 1999), well-the same cell. This is consistent with previous evidence documented evidence of such silencing was lacking inabout chromatin heterogeneity within multicopy trans- mammals. Because of the potential silencing effects ofgene arrays in mouse lines that showed either varie- lacZ sequences it could be argued that variegation ofgated/position-dependent or efficient expression (Huber transgenes inserted into peritelomeric sites is not neces-et al. 1996; Whitelaw and Webster 1998). sarily only a reflection of a telomeric position effect.

Variegation and chromosomal position: The influ- However, the observation that, among the lines in whichence of the integration site for variegated expression of the effects due to lacZ sequences can be overcome (bytransgenes is well established for constitutively hetero- K5HK10 sequences), the two variegating ones, but notchromatic regions at or near centromeres (Dobie et al. the one that expresses in a nonmosaic fashion, bear1996; Festenstein et al. 1996; Milot et al. 1996). In- peritelomeric transgenes supports telomeric positiondeed, the two lines we find having pericentromeric in- effects in transgenic mice. This is consistent with recentsertions correspond to variegated (K5HK10 825 line) observations of telomeric position effects on replicationor nonexpressing (K5Zlox 4715 line) conditions. How- timing of human loci (Ofir et al. 1999) and indicatesever, in this study we provide evidence that transgene that both gene expression and DNA replication can bevariegation does not require that the transgenes are affected by telomeric sequences in mammals.located in or close to the centromere, in agreement Transgene repeat arrays and silencing: Examinationwith a previous report (Graubert et al. 1998). In partic- of Table 1 suggests that very-high-copy-number lines

tend to either express in a highly variegated manner orular, we show that the transgenes in a number of varie-


Figure 7.—FISH of transgenic lines. (A–N)Bone marrow metaphase chromosomes werehybridized to a mixture of K5Z (pink) andmouse g-satellite probes (green) or to K5ZDNA (yellow) and a probe specific for mousetelomeric repeats (pink; Hanish et al. 1994).Chromosomes were also stained with 496-dia-midino-2-phenylindole (blue). (O–R) Bonemarrow metaphase chromosomes hybridizedto the K5Z construct alone (pale green). Forthe K5ZMAR 1739 line, the transgene signalis yellow and chromosomes were stained withpropidium iodide. Transgenic lines are indi-cated.

not at all. However, our analysis of the effect of gene tween variegation and transgene localization to hetero-chromatic regions of tissue culture cells (Francastelrepetition on variegation shows that a reduction in copy

number of K5Z transgenes does not increase the propor- et al. 1999). In transgenic mice, it has been reportedthat a silent transgene integrated into a peritelomeriction of expressing cells, which suggests that repeated

induced gene silencing is unnecessary for transgene region co-localizes with centromeric satellite DNA onthe nucleolus (Manuelidis 1991). It seems, however,repression. This is in contrast to results by Garrick et

al. (1998) who studied the expression properties of an that the size of the transgenic insert is critical for sucha nuclear relocalization (Manuelidis 1991). If this werea-globin/lacZ construct. The discrepancy could be ex-

plained by several differences between the two trans- the case, the a-globin/lacZ transgene arrays, which seemlarger than the K5Zlox transgenes we have studied, maygenic systems, such as the distinct cell types involved or

the possibility that subsets of chromosomal sites are find themselves in singular heterochromatic compart-ments within the interphasic nuclei from which theymore favorable to repression than others. Thus, it is

conceivable that in three of the K5Zlox lines we used can be mobilized upon copy-number reduction. Any-how, our conclusion that K5Z variegation does not de-for copy-number reduction, the peritelomeric (two) or

pericentromeric (one) position of their transgenes may pend on the copies of the transgene is consistent withother reports of no apparent correlation between trans-impose a silencing effect regardless of copy number. An

alternative explanation lies in the compartmentalization gene copy number and degree of variegation (Zhumaet al. 1999).model of gene silencing. This model, put forward to

explain the silencing of transgenes located far away from In summary, we have shown that variegation of K5Ztransgenes in stratified epithelia of transgenic mice isconstitutive heterochromatin, proposes that the varie-

gating locus, after local heterochromatinization, is mis- not due to the lack of regulatory sequences. Instead,our data suggest that variegation can be related to pro-localized to a constitutive heterochromatic compart-

ment within the nucleus (Sabl and Henikoff 1996). karyotic lacZ sequences that may cooperate with otherrepressor influences such as chromosomal position ef-Recently, a similar correlation has been observed be-


Garrick, D., H. Sutherland, G. Robertson and E. Whitelaw,fects. This silencing effect, however, can be efficiently1996 Variegated expression of a globin transgene correlates

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