�‘ol. 4. 45l-4�9, June 1993 Cell Growth & Differentiation 451

High Frequency Expression of Integrated ProvirusesDerived from Enhancer Trap Retroviruses’

Fred Sablitzky,2 Jan-lngvar J#{246}nsson,Brenda 1. Cohen,and Robert A. Phillips3

Departments ot Molecular and Medical Geneti.s and of Immunology,

Univt’rsitv of Toronto ER. A. P.), and Division of Immunology andCani�er Research, Hospital For Sick Children [F. S., I-I. I.. B. L. C.,

R. A. P.1. Toronto, Ontario M5G 1X8, Canada

AbstractSince retroviruses integrate preferentially intotranscniptionally active loci, the provirus may comeunder the control of regulatory elements of the geneinto which it integrated and thus become a functionaltag for that gene. In order to determine the frequencyof retroviral integration near active endogenousenhancer elements, a retroviral enhancer trap vectorwas constructed. Lacking the long terminal repeatenhancer, expression of the neomycin resistance (neo)gene, used as a reporter, is dependent uponendogenous enhancer elements able to activate thelong terminal repeat promoter. Infection of murinefibroblast cells indicated that a high proportion of theproviral copies expressed the neo gene. Infection ofhematopoietic lines confirmed this high frequency ofexpression of integrated proviruses. Overall, between43 and 74% of proviruses integrated into severaldifferent cell lines expressed the neo gene. These datasuggest that retroviral integration is not only dependentupon transcriptional activity of the genomic targetsites, but, more specifically, retroviruses preferentiallyintegrate near active enhancer elements which areoften associated with developmentally regulated genes.

IntroductionA crucial step in the life cycle of retroviruses is theintegration into the host genome, which occurs by anefficient and specific recombination event. Characteriza-tion of the genetic and molecular requirements for therecombination event indicate that only viral DNA asso-ciated with viral core proteins serves as a functionalintegration precursor (1-3). Sequence analysis of DNAflanking the proviral integration sites has not revealedany sequence specificity (4), suggesting that retroviralintegration occurs randomly in sites distributed over the

Received 9/1 4/92; revised 3/1 2/93; accepted 3/1 7/93.‘ This research was supported by grants from the Medical Research

Council and the National Cancer Institute of Canada with funds from theCanadian Cancer Society. F. S. was the recipient of a fellowship from theDeutsche Forschungsgemeinschaft. Germany. i-I. I. was supported in partby fellowships from The Hospital for Sick Children Foundation and fromMedicinska Forskningr#{224}det, Stockholm, Sweden.2 Present address: Max-Delbruck-Laboratorium in der MPG, Carl-von-

Linn#{233}-Weg 10, 5000 K#{246}ln30, Germany.3 To whom requests for reprints should be addressed, at Division of

Immunology and Cancer Research, The Hospital for Sick Children, 555

University Avenue, Toronto, Ontario M5G 1 X8, Canada.

entire genome. However, as reviewed by Sandmeyer etal. (5), there is convincing evidence that retrovirus inte-gration is nonrandom. For example, chromatin structureapparently influences the sites of retroviral integration;DNase I-hypersensitive sites indicating open chromatinare found invariably close to Mo-MuLV4 integration sites(6) and, as recently demonstrated, a high proportion ofrandom, nonselected integration sites of Mo-MuLV liewithin transcriptionally active loci (7). Furthermore, theavian genome contains 500-1000 sites that are highlypreferred targets for Rous sarcoma virus integration (8).These data clearly demonstrate that there are hot spotsfor retroviral integration and that the selection of genomictarget sites is influenced by the accessibility and activityof the DNA rather than its nucleotide sequence.

In recent years, promoter and enhancer trap plasmidvectors have been used to identify and to isolate inter-esting, developmentally regulated genes (9-11). Simi-larly, retroviruses have been engineered to be promotertrap vectors, suitable for identification of specific genes(12-14). When retroviral DNA integrates preferentiallyinto transcriptionally active regions, the provirus maycome under the control of the regulatory elements of thegene into which it integrated (15). Such an event couldbe used to identify developmentally regulated genes incomplex developmental systems. To test the possibilityof using retroviruses to identify developmentally regu-lated genes, we have constructed an enhancer trap retro-virus in which an internal reporter gene for neomycinresistance (neo) is driven from an LTR promoter whichlacks the LTR enhancer. Other investigators have shownthat the LTR promoter requires an enhancer for its activity(16-20). Therefore, it is likely that such a retrovirus willexpress the reporter gene only when it integrates closeenough to an active cellular enhancer to activate the LTRpromoter.

When retroviral enhancer trap vectors were used toinfect lines of murine fibroblasts and T-cells or human T-cells and macrophages, we observed that a high propor-tion of integration sites, between 43 and 74%, led toexpression of the neo gene. This result supports thepreviously obtained data discussed above, that retroviralintegration does not occur at random. In addition, ourdata suggest that open, transcriptionally active loci arenot sufficient to explain preferential integration of retro-viral DNA. It seems that transcribed regions close toactive enhancer elements are the preferred targets forretroviral integration.

ResultsEnhancer Trap Retrovirus vM/NEO. The retroviral vector

4 The abbreviations used are: Mo-MuLV, Moloney murine leukemia virus;LTR, long terminal repeat; kb, kilobase(s); bp, base pair(s); PCR. polym.erase chain reaction; G418#{176},G418 resistant.

A) PIasmId:

pAFJNE()

B) Retrovlrus:

vAE/NEO

C) Provlrus:

bp

L,/yzz�

V/AZ//I

- 293

-267

�. -243

#{190}’;�-. �.,

;�: -174

� -142

12 3456789

Fig. 2. Characterization of the LTR in retroviral and proviral RNA. Retro-

viral RNA isolated from virus-containing supernatant (Lanes 4 and 5) andcytoplasmic RNA isolated from infected Rat-2 cells selected for 2 weeksin G418 (400 pg/mI) (Lanes 6 and 7) were analyzed by RNase protection

using an in vitro transcribed RNA probe complementary to the enhancerelement (Lane 1 ). MoTN is a retrovirus containing the LTR enhancer (Lane3); IRNA (Lane 2) and noninfected Rat-2 cells (Lane 8) served as negative

controls. Lane 9 contained molecular weight markers (M).

452 Enhancer Trap Retroviral Expression

Fig. 1. Schematic diagram of the retroviral enhancer trap vector vM/NEO. The plasmid p.�E/NEO (A) consists of a functional 5’ LTR, the neo

gene, and a 3’ LTR lacking a 186-bp fragment encompassing the viralenhancer element and the GC-rich region. The retroviral RNA vM/NEO(B) and, subsequently, the proviral DNA (C) lack the enhancer elements

in both LTRs. v.�EF/NEO (not shown) is basically identical except that it

contains the supF gene in place of the LTR enhancer.

p�EINEO, schematically shown in Fig. 1, consists of LTRelements derived from Mo-MuLV and the bacterial neo-mycin resistance (neo) gene. The 3’ LTR lacks a 186-basepair fragment containing the viral enhancer and GC-richelements (19) essential for the activity of the viral pro-moter (16-20). The viral RNA and subsequently the pro-viral DNA lack the enhancer element in both 5’ and 3’LTRs. Since the overall level of transcription from proviralDNA is dependent upon the presence or absence of theenhancer element (16-20), cells infected with the retro-virus produced by this vector are not able to express theneo gene unless the retroviral DNA integrates close to anactive enhancer element which is able to initiate tran-scription at the 5’ LTR promoter. Alternatively, transcrip-tion initiated at endogenous promoter elements couldalso lead to the expression of the rieo gene.

The packaging cell line PA317 (21) was transfectedwith p�\E/NEO, and the virus production of individualcell clones was measured. One cell clone (PA/�E/NEO)produced iO� virus particles (v�E/NEO)/ml supernatantas estimated in a quantitative slot blot assay (data notshown). Infection of Rat-2 fibroblast cells (22) with vM/NEO from this clone gave a virus titer (measured at aG418 concentration of 500 jzg active compound/mI) of 2x iO� G418� Rat-2 cells/mi culture supernatant.

Since retroviral DNA frequently recombines during thetransfection process and could result in the restorationof the wild-type LTR (23), we examined the viral RNA ofv�E/NEO for the enhancer element. Viral RNA, directlyisolated from virus-containing supernatant, was analyzedby the RNase protection assay (24) (Fig. 2). A riboprobecomplementary to the LTR including half of the enhancerelement (see “Materials and Methods”; Fig. 3) was used.The riboprobe was fully protected by RNA from retrovi-rus MoTN (22) containing the LTR enhancer (Fig. 2, Lane3). In contrast, no full length protected fragment couldbe detected in the retroviral RNA from v.�E/NEO (Fig. 2,Lane 4 and 5). Since the RNase protection assay was notsensitive enough to detect a low frequency of recombi-nant retroviruses with a reconstituted LTR, we also ana-lyzed the RNA isolated from Rat-2 cells which had beeninfected with v�E/NEO and selected in 500 �g/ml G418.Again, no full length protected fragments were detected

c.*1

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in cells selected for neo expression (Fig. 2, Lanes 6 and7) demonstrating that the G418R Rat-2 cells were not a

result of infection by rare undetected virus particlescontaining an enhancer element. These data were con-firmed by Southern blot analysis of infected Rat-2 cells(data not shown) and infected �I’2 cells (see below).

Rat-2 cells infected with v�E/NEO and selected inG418 contain a neo transcript corresponding in size tothe genomic transcript of vz�E/NEO (2.3 kb; Fig. 3). Thus,transcription of the neo gene is usually initiated at the

28s-

18s- I,

1 2 3 4 5 6 7 8C

NEO -probe

fig. 3. Characterization of retroviral transcripts in cells infected with

v.�E/NEO. RNA isolated from pools of Rat-2 cells infected with v.�E/NEOand selected in G418 (Lanes 1-8) was analyzed by Northern blotting witha neo probe. Different numbers of cells or clones were used for eachpool: the following estimates were based on estimates of the titer ofinfectious virus and the frequency of infected cells: Lane 1, iO� clones;

Lane 2, i0� clones; Lanes 3-6, 5-10 clones; Lanes 7 and 8, 1-5 clones.Lane C contains RNA from noninfected Rat-2 cells.

Cell Growth & Differentiation 453

LTR promoter. One pool of cells, however, contained alonger transcript (Fig. 3, Lane 6), suggesting that transcrip-tion can occasionally initiate at endogenous promoterelements. The rapidly migrating bands shown in Fig. 3likely represent some degradation or nonspecific bindingto ribosomal RNA resulting from the large amounts oftotal RNA used in this experiment.

Dependence of neo Expression on a Functional Enhan-cer. The basic assumption in our plan to use enhancertrap retroviruses to identify developmentally regulated

genes is that the LTR promoter alone will not producesufficient neo transcripts to make cells resistant to G418.Although others have shown the strong dependence ofthe LTR promoter on a functional enhancer (19), it isnecessary to confirm in this system the dependence ofneo expression on an enhancer. For this purpose, weconstructed three plasmids containing the neo genedriven by the LTR enhancer and promoter (pEPNeo), thepromoter alone (pPNeo), or lacking both an enhancerand promoter (p#{248}Neo). To test neo expression fromthese plasmids, 1 or 0.1 �g of plasmid together with 1 �zgof pBABepuro plasmid, containing the gene for puro-mycin resistance, were transfected into NIH 3T3 cells.Forty-eight h after transfection, cells were plated in 4 zg/ml puromycin or in 500 zg/ml of G41 8. Surviving colonieswere counted after 14 days. The number of coloniesgrowing in puromycin was used as an index of thetransfection frequency to ensure roughly equal levels oftransfection in all groups in the experiment. The coloniesgrowing in G418 indicated the level of expression of theneo gene.

Table 1 summarizes the data obtained from four ex-periments. In the last three experiments, the number ofpuromycin-resistant colonies was similar in all groups,indicating that the transfection efficiency was similar inall groups; the puromycin vector was not evaluated inthe first experiment. Examination of the groups trans-fected with the various plasmids clearly shows that effi-cient expression of neo depends strongly on the pres-ence of an active enhancer. There was little, if any,expression from the vector lacking both enhancer andpromoter elements. Although the enhancerless vectorundoubtedly expressed the neo gene at a low level,leading to resistance to G418 in a few cells, the vectorwith an enhancer and promoter always gave substantiallyhigher expression than the vector with the promoteralone. This difference was most marked when the cellswere transfected with small amounts of DNA and pre-sumably contained few integrated plasmids. This condi-tion is similar to that obtained with retroviruses wheneach integration site contains only a single copy of theneo gene. On the basis of these results, we concludethat random integration of a plasmid with a reporter gene

Table 1 Neo expression of enhancer trap plasmids

Plasmid

lMg O.l�zg

G418#{176} PuroRRelative

expression I Ia)G418#{176} Puro’

Relative

expression ( /o(

p#{248}NeopPNeo

pEPNeo

15575

NDbNDND

1

141

ND

NDND

p#{248}NeopPNeo

pEPNeo

149

112

348224

148

0.429

100

11

8

165320

400

30.315.6

100

p#{248}NeopPNeo

pEPNeo

040

384

5090

63

07

100

01

43

7557

84

0.03.4

100

p#{248}Neo

pPNeopEPNeo

3

6178

6280

32

0.91.3

100

00

23

385238

0.00.0

100

‘ To calculate relative expression, we took the ratio of G418R colonies to PuroR colonies, and then within each group, set the pEPNeo value to 100% and

expressed the others as a proportion of the pEPNeo value.b ND, not done.

100

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454 Enhancer Trap Retroviral Expression

0 200 400 600 800

(;418 ((.tg/ml)

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ii ire nu’ tt’(f is tb �..�E/NE() ,intl solo t(’(I in various ( Ofl( t’ntr,itions ofC4 18. The ( Ol(irl’, nunll)e’r of riO1ilIlf(’( (‘(1 (do(U’d linc) and infected I e)lldI,,),, � ‘I’, � ills surviving (Iitie’r(’nt ( ()i1( (‘oiraiions 01 C4 I 8 iv.is (I(’ternlln(’cI.01(1 ti )nll)ar(’(I Ii) the ( oloi’i� nunihor ot iiorisek’ Rd ( &‘Ils. F.u h poIntri’presi’ots I hi’ rili’,in \ aloe’ i t � di 011(5 1r 111 our 1)1,111’s�. The numl)vr of( ( ilinii’i ( )I)tJiiii’(l .it 1) pg/nil Cl I B ii is (lt’iin(’d as 1O0#{176}/osurviial.

lacking an effective enhancer is not sufficient to allow itshigh level expression.

neo Expression in a High Proportion of v�E/NEOIntegration Sites. To determine the frequency of integra-tion events which lead to expression of the neo gene, �I’2

cells were infected with v.XE/NEO in a ratio of about 1cell to 3 virus particles. The resulting survival curve (Fig.4) indicated that at least 80% of the ‘!‘2 cells expressedthe neo gene (compare noninfected with infected ‘J2’2cells at 200 �zg/ml G418 in Fig. 2). The same pool ofinfected “2 cells was plated under limiting dilution con-ditions (0.3 cells/well), and 48 individual clones wereisolated and expanded in the absence of G418. DNAfrom each clone was examined by Southern blotting toenumerate the number of integrated proviruses (Fig. 5).Each clone was also plated in various concentrations ofG418 (0, 200, 400, and 800 �tg/ml), and survival wasscored after 9 days by estimating the confluence (Fig. 6).Since �I�2 cells were only 10-50% confluent in 200 �zg/mlG418, neo expression was defined as growth in concen-trations greater than 200 jzg/ml G418. Two infectedclones (A50 and A9) also failed to grow in 400 zg/ml

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fig 5. Enumeration of v.�E/NEO proviruses in randomly picked, infected 4’� clones. DNA isolated from each clone was cut with IcoRl, separated byelectrophoresis. and probed with the neo gene. The-clones are grouped according to the highest concentration of G418 in which the clone can grow

(see Fig. 6).

00 0 �

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Cell Growth & Differentiation 455

-r

+ 800

+1- 800

+ 400

+1.400

+ 200

+1- 200

0 1 2 3 4 5 6 7

copies / cell

Fig. 6. Correlation of v.�E/NEO integration sites and expression of theneo gene. Squares, clones. Plotted are the number of proviruses per clone

(derived from the data shown in Fig. 5) vs the ability of each clone to

survive in G418. +, confluent growth in the designated concentration of

G418; ±, 10-50% confluence in G418; and -, no growth.

G418, indicating the lack of neo expression. Southernblot analysis (Fig. 5) indicated that clone A50 did notcontain a provirus, whereas clone A9 contained twoproviruses. All other clones (46 of 48) containing one ormultiple copies of the proviral DNA survived G418 Se-lection to various extents, as summarized in Fig. 6.

The average number of v�E/NEO proviral integrationsper cell was 3.4. With this mean value, one can calculate,using the Poisson distribution, the expected number ofproviruses per cell; comparison of the observed andexpected values gives a x2 value of 3.596, indicating ahigh probability that infection and integration occurredat random.

Clone A9, which has two proviral copies of normalsize (see Fig. 7), did not express the neo gene at adetectable level. Since it is unlikely that both proviralcopies acquired mutations altering the function of theneo gene, clone A9 supports the hypothesis that leakytranscription of the enhancerless LTR promoter cannotaccount for the observed expression of the neo gene inthe G418-resistant clones. Rather, clone A9 most likelyrepresents integration of the viral DNA into regions ofthe host genome which did not facilitate the expressionof the neo gene.

All 7 clones containing a single provirus grew to variousextents in the presence of 400 �g/ml G418 (Fig. 6). Nineof 10 clones with two proviral copies survived G418selection in concentrations �400 zg/ml. In the latterclones, at least one of the two proviruses must be tran-scriptionally active. Thus, the frequency of proviral inte-gration sites which led to expression of the neo gene isat least 16 of 27, or 59%. The actual number of expressedproviral copies is probably somewhat higher. Five of theclones containing two proviral copies are resistant tointermediate concentrations of G418, similar to the re-

sistance seen with cells having only a single provirus. Theother four clones resist higher doses of G418, suggestingthat in the former, only one provirus expresses the neogene, and in the latter, both proviruses express. Basedon this analysis, we estimate that a high proportion,between 16 and 20 of 27 proviruses, or 59-74%, of

integrated copies of vi�E/NEO are transcriptionally activein �1�2 cells.

The data presented in Fig. 6 indicate that althoughresistance to G418 increases with increasing numbers ofproviruses, other factors obviously also contribute to neoexpression and resistance to G418. For example, consid-ering only the clones with two integrated proviruses, twoclones, 10 and 33, grow only marginally in 400 zg/mIG418, but two other clones, 30 and 49, are completelyresistant to 800 zg/ml of drug. These observations areconsistent with the hypothesis that neo expression isstrongly influenced by the site of integration in the hostgenome.

Integrated Proviruses Lack LTR Enhancer. To excludethe possibility that recombinations between v�E/NEOand endogenous retroviruses or the helper virus presentin *‘2 cells reconstituted an active enhancer in the LTR,we determined the structure ofthe proviral DNA in thoseclones containing one or two proviruses (Fig. 7). Follow-ing digestion with Nhel, a restriction enzyme which cutsat the 5’ end of the LTR, virus with an enhancer in the5’ LTR will be 2.4 kb, and those without, 2.2 kb (Fig. 7;compare E with z�E). Southern blots probed with the neogene (Fig. 7A) or the 186-bp fragment encompassing theLTR enhancer element (Fig. 7B) show that none of thev�E/NEO proviruses in the infected �4’2 cells contain aviral enhancer element. A small proportion (4 of 27) ofthe proviral DNAs are smaller than expected (Fig. 7; AX),and are likely due to small deletions within the proviralDNA, an event often observed with integrated retrovi-ruses (25).

High Frequency of Expression of vMF/NEO in He-matopoietic Cells. To test further the expression of en-hancer trap proviruses integrated into other cell types,two additional experiments were performed with anotherenhancer trap retrovirus, v�EF/NEO, containing the supi

gene in the LTR (see “Materials and Methods”). In thefirst experiment, Jurkat cells were infected with v�EF/NEO and then grown in culture to produce colonies.Individual clones were picked from plates grown in theabsence of G418, and some of the clones were testedfor the presence of the neo gene using PCR, as describedin “Materials and Methods.” These results indicated that23% of colonies contained the neo gene. Analysis of allclones for growth in G418 showed that 9.9% of theclones were G418R. These data indicate that 43% (9.9 of23) of the Jurkat cells infected with v�EF/NEO expressedthe neo gene.

In the second experiment, three cell lines from devel-opmentally different origins, EL-4 (murine 1-cell line),U937 (human macrophage line), and 313 (murine fibro-blasts, closely related to ‘I’2 cells), were infected withv�EF/NEO, and clones were selected randomly withoutselection as described above for the ‘I’2 clones. Eachclone was examined for the presence of the neo geneand for resistance to G418 (400 jzg/ml). Table 2 summa-rizes the results of this experiment. Because of the rela-tively short interval of growth in G418 used in this ex-periment (5 days), all lines gave some neo clones thatsurvived in G418. Nevertheless, for all three lines, over50% of the clones containing the neo gene expressedthe gene, as indicated by resistance to growth inhibitionby G418.

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Discussion

Several investigators have constructed retroviral vectors

lacking the LTR enhancer and/or promoter elements tocreate self-inactivating vectors. These studies showedthat such vectors integrated efficiently into the host ge-nome and allowed expression of cloned genes from an

�- E‘�AE

internal promoter with reduced interference by the tran-scriptional control regions in the LTR (26-30). We haveexploited this observation to construct a retroviral enhan-cer trap vector by deleting the enhancer but leaving thepromoter, including the CAAT-and TATA-boxes, in-tact (19, 31). It has been shown that an enhancerless LTRpromoter is transcriptionally silent in transient expression

456 Enhancer Trap Retroviral Expression

Fig. 7. Structure of proviral DNA in infectedcells. DNA of the plasmid p.�E/NEO, �2 cells,the virus-producing cell line PA/.�sE/NEO, andthe 1 7 �1’2 clones with one and two proviralcopies was digested with Nhel, which cuts at

the 5’ end of the LTRs, and Southern blots wereprepared. A, the Southern blot was probed withthe neo gene. Fragments containing provirus

without an enhancer should be 2.2 kb (indicatedby arrow .XE(. Recombinant proviruses with an

enhancer in the 5’ LTR will be 2.4 kb (arrow El.

Some samples (10, 31, 32, and 35( containedsmaller than expected proviral DNA (arrow AX).

B, an identical blot was probed with the LTRenhancer element. At the top of both blots isindicated the ability of each clone to grow inG4 18.

Cell Growth & Differentiation 457

Table 2 Expression of neo gene in various cells infected with v�EF/

NEO

CellsPresence ofneo gene

Responseto G418’

R S

% G418R

EL-4 +

-

25 12

3 5968

4.8

U937 +

-

7 3

3 29

70

9.3

3T3 +

-

16 12

3 16

57

16

Presence or absence of the neo gene was determined by PCR usingprimers specific for neo.b G418#{176} was determined by growing each clone in 400 �g/ml of activeG418.

assays (16-20). To test the enhancer dependence of theLTR promoter in our assay, we constructed plasmids inwhich the neo gene was expressed from a wild-type LTRpromoter and from an enhancerless LTR promoter. Wereasoned that these plasmids would insert into cells morerandomly than retroviruses and that, because of theirpresence in a plasmid, the promoters would be separatedfarther than the promoters in a provirus from activecellular enhancers. Transfection of these constructs intoNIH 313 cells showed that neo expression depends onan enhancer (Table 1). Especially when we used lowlevels of DNA (0. 1 �g) for transfection to mimic the singleintegration site for retroviruses, few, if any, of the enhan-cerless promoters gave G418 resistance. On the basis ofthese results, we conclude that transcription of the neogene from the enhancerless promoter in the v�E/NEOvector occurs only when the provirus integrates near anactive cellular enhancer and, less frequently, near anactive promoter.

The results obtained demonstrate that a large propor-tion of cells from various origins, fibroblasts, 1-cells, andmacrophages, infected with v�E/NEO or v�EF/NEO, areresistant to G418. Leaky transcription from the enhan-cerless LTR promoter cannot account for the observedresistance of the infected fibroblast cells. Tests with thedeleted LTR used in the v�E/NEO vector demonstratethat it is inactive in transient expression assays (16-20).The finding that one clone with two integrated provirusesis sensitive to G418 shows that not all proviruses expressthe neo gene. If the results depended on leaky transcrip-tion, we would not expect to find negative clones. Finally,the levels of resistance observed in the infected fibroblastclones illustrate a high level of neo gene transcription,unlikely due to leakiness of the LTR promoter. Therefore,the presented data suggest strongly that expression ofthe neo gene in the infected fibroblast cells results fromintegration near active cellular enhancers which stimulatetranscription of the neo gene from the LTR promoter.

It is important to emphasize that the experimentsdescribed above involved no selection; all clones werepicked at random in the absence of G418. Since theaverage number of integrations per cell in the �I’2 clonesis 3.4, it is difficult to determine accurately the numberof proviruses expressing the neo gene. Nevertheless, byconsidering the 1 7 clones containing one or two proviralcopies, we can estimate the frequency of active provi-ruses. As described in detail above, a very high propor-

tion, between 60 and 74%, of integrated v�E/NEO copiesare transcriptionally active in these clones. In other celltypes, the frequency of transcriptionally active provirusesranged from 43% (Jurkat, human T-cell line) to 70%(U937, human macrophage line). These high frequenciesof expression in the absence of selection are consistentwith the hypothesis that retroviruses prefer to integratenear active enhancers.

As shown recently by Scherdin et a!. (7), a high pro-portion of retroviral integration sites are within transcrip-tionally active regions, and, in fact, approximately 50%of the flanking sequences contain detectable exons. Theyestimated that the probability of integration near an exonby chance alone is only 5%. Presumably, the chance ofintegration near an active enhancer is similar or evenless. This assumption is supported by the finding that lessthan 10% of the integration sites of plasmids used asenhancer trap vectors lead to expression of a reportergene (32, 33). Thus, our observation that between 43 and74% of the integrated enhancer trap proviruses expressthe reporter gene indicates a strong preference for retro-viral integration near transcriptionally active enhancers.

Shih et a!. (8) estimated that there are approximately500-1000 highly preferred targets for retroviral integra-tion. Since the estimated number of expressed genes isat least 10 times this number, one must ask what isunique about the 1000 integration sites detected by Shihet a!. Since approximately 10% genes are thought to bedevelopmentally regulated, whereas the remainder arehousekeeping genes that often lack enhancer elements,we suggest that the preferred integration sites of retroviralDNA are the active enhancer region(s) of develop-mentally regulated genes.

It is worth noting that retrovirus-like elements showsimilar preferences in their integration pattern. Retrotran-sposon 17.6 from Drosophila (34) and Ty from yeast (35)show site specific insertion into promoter sequences.Furthermore, transposable P-elements show a biasedintegration pattern in the genome of Drosophila. Notonly are they generally located outside heterochromatin,but they are found often in the 5’ upstream and untrans-lated regions of genes (36). Using the P-element as anenhancer trap vector, Gehring and his colleagues devel-oped a screening method to identify developmentallyregulated genes in Drosophila embryo genesis (37-39).About 65% of the analyzed integration sites of the P-element showed spatially restricted embryonic expres-sion of the lacZ reporter gene. This remarkably highfrequency indicates that P-elements also integrate pref-erentially close to active enhancer elements important incontrolling developmentally regulated genes.

Although retroviruses can integrate in vitro into nakedDNA (1), integration into a chromosome appears to re-quire both DNA replication and a transcriptionally activeregion (40). As summarized by Svaren and Chalkley (41),it is likely that transcription factors compete with theproteins which build nucleosomes for sites on the nakedDNA. During DNA replication, the nucleosomes fall off,allowing transcription factors to bind to the appropriateregions, thus preventing the creation of nucleosomes inthat region. In addition to the presence of transcriptionfactors binding to this newly replicated DNA, the pres-ence of a growing replication fork with new ends of DNAmay facilitate the integration of a retrovirus and/or retro-transposons into the host genome. Indeed, it is possible

458 Enhancer Trap Retroviral Expression

that retroviral integration is facilitated through some ofthe proteins associated with transcriptional activation.

Materials and MethodsConstruction of Retroviral Enhancer Trap Vectors p�E/NEO and vMF/NEO. The defective retroviral vector p�E/NEO was constructed by inserting a 1 .2-kb Bglll-BamHIfragment of the neomycin gene, which was derived fromMoTN (22), into the BglIl site of the retroviral vector p�E(a gift from I. B. Robson and A. Bernstein, Mount SinaiHospital Research Institute, Toronto, Ontario, Canada).p�E was constructed by replacing the wild-type 3’ LTRof the plasmid pEVX (42) by a defective LTR which has a186-bp deletion 5’ of the Xbal site encompassing theretroviral enhancer element and the GC-rich region. Thedefective LTR was derived from plasmid DpMLV-C/R/B(19).

The p�EF/NEO retroviral vector carries a 202-bp frag-ment of the bacterial suppressor tRNA gene (supF) in its3’ LTR. The supF gene was inserted as a Xbal fragmentinto pz�E, and the generated pz�E/supF fragment was thenligated to a 2.3-kb Apal fragment from MoTN containingthe 5’ LTR and the gag region. The neomycin gene wasinserted as a 1 .2-kb Sail fragment into MoTN/�E/supF.

Test of Dependence of neo Expression on the Enhan-cer. To test the importance of the LTR enhancer in neoexpression, we constructed several plasmid vectors,p#{248}NeopA, pPNeopA, and pEPNeopA. The neo geneincluding the SV4O polyadenylation signal was subclonedinto the BamHl site of pGEM7zf+ (Promega). This vector(p#{248}NeopA), containing no eukaryotic promoter, servedas a negative control for the transfection experiment. The3’ LTR of p�E/Neo, which contains the retroviral pro-moter but lacks the enhancer element, was subcloned5’ of the neo gene cassette of p#{248}NeopA, resulting inplasmid pPNeopA. Similarly, the 5’ LTR of p�E/Neo,

containing both enhancer and promoter, was insertedinto p#{248}NeopA to obtain pEPNeopA. This vector wasused as a positive control in the transfection experiments.

To test the expression of these vectors, NIH 313 (5 x10�) cells were plated on 60-mm dishes in Iscove’s me-dium with 10% calf bovine serum, and 24 h later, thecells were transfected using the calcium phosphatemethod. Each transfection mixture contained 1 �zg of thepBABepuro plasmid with the gene for puromycin resist-ance and 1 �g or 0.1 zg neo construct. An RSV-f3galplasmid was used to bring the total amount of transfectedDNA to 10 �zg. Twenty-four h after transfection, cellswere washed three times in phosphate-buffered salineand then incubated in Iscove’s medium with 10% calfbovine serum for an additional 24 h, when the cells weretrypsinized and replated on 100-mm dishes in mediacontaining either 500 �zg/ml G418 (active) or 4 �zg/mlpuromycin. Colonies were counted after 14 days.

Isolation of the Virus-producing Cell Lines PA/MINEO and PEF1O9. Plasmid DNA, pz�E/NEO (5 �g) orp�\EF/NEO (10 izg), was transfected by the calcium phos-phate method (43) into the helper cell line PA317 (21).Infected cells were selected in G418 (400 �zg/ml), andculture supernatant from individual clones was used toinfect Rat-2 fibroblast cells (22). The highest virus titerobtained for v�E/NEO was 2 x 10� G418R (500 zg/mI)Rat-2 cells/mI supernatant. The same supernatant con-tamed � virus particles/mI as estimated in a quanti-

tative slot blot assay (44). For the vMF/NEO virus, theclone, PEF1O9, had a titer of 2 x 10� virus particles/mi;this titer was confirmed with a slot blot assay of culturesupernatants.

Infection of 4’2 Cells with vM/NEO. Supernatant ofthe virus-producing cell line PA/Z�E/NEO was used toinfect �1’2 cells in a ratio of 3 infectious particles/cell. After24 h, the infected cells were subcultured. To determinea survival curve for the infected cells, 500 infected cellswere plated in varying concentrations of G418 (0, 100,200, 400, 600, and 800 �g/ml active compound). Surviv-ing colonies were counted 8- 10 days later, and the meanof 4 plates was calculated for each G41 8 concentration.

Infection of Various Cell Lines with vMF/NEO. Su-pernatants from PEF1O9 cells were collected after 24 hfrom semiconfluent plates, filtered through a 0.45-zmfilter (Nalgene), and used to infect three different celllines: Jurkat, a human 1-cell line; EL-4, a murine 1-cellline; U937, a human macrophage line; and NIH 313murine fibroblasts, at a ratio of approximately 2 viralparticles/cell in RPMI 1640 media containing 5% fetalcalf serum and 4 zg/ml Polybrene. After 24 h, cells werewashed twice and subcultured under limiting dilutionconditions (1 cell/well in 96-well plates) without G418selection. Cultures were fed every fourth day with freshmedia supplemented with 10% fetal calf serum. Underthese conditions, the cloning efficiency was 42% for EL-4 cells, 24% for 313, and 49% for U937. Individual cloneswere maintained by weekly passage to new 96-wellplates. In some experiments, cell lines were analyzedrandomly for survival in the presence of G418 at aconcentration of 400 �g/ml active G418. This concentra-tion was sufficient to kill >95% of noninfected cells inthe three cell lines used; clones were examined after 5days to determine sensitivity or resistance to G418.

Identification of Provirus in Clones Derived fromvMF/NEO-infeded Cells. To determine whether theclones derived from vMF/NEO-infected cells containedprovirus, all clones were analyzed for the presence ofthe neo gene by PCR amplification. Approximately 1000cells from each cell line were washed twice in phosphate-buffered saline and pelleted in Eppendorf tubes. Gen-omic DNA was isolated by lysis in 50 zl of sterile distilledH2O at 95#{176}Cfor 10 mm, followed by incubation with250 jzg/ml proteinase K at 55#{176}Cfor 90 mm. Proteinase Kwas heat inactivated at 95#{176}Cfor 10 mm, and cell debriswas removed by centrifugation. The DNA solution wasmixed with 50 pmol of each primer and 200 zM concen-trations of each desoxyribonucleotide triphosphate in atotal volume of 100 MI containing 16.7 mt�i (NH4)2SO4, 10mM 2-mercaptoethanol, 3 m�i MgCl2, and 67 mi�.i Iris-HCI, pH 8.8. Each reaction mixture was overlaid with 60�l of paraffin oil. Amplification was performed usingdenaturation (30 s, 94#{176}C),annealing (30 s, 69#{176}C),andextension steps (1 mm, 72#{176}C)for 35 cycles. Primers usedwere N 1 , 5’-ATGGCTGATGCAATGCGGCGGCTGCAT-3 ,, and N2, 5’-ATGCTCTTCGTCCAGATCATCCTG-3’.

RNase Protection Assay. A Sau3A-KpnI fragment of thewild-type LTR encompassing the viral enhancer wascloned into Bluescript SK (Stratagene), and antisense RNAprobes complementary to the Pvu2-Kpnl fragment of theLTR were generated in vitro (24). Radiolabeled RNA wasseparated on a 5% polyacrylamide gel, and full lengthRNA probes were isolated (44). One to 20 �zg of viralRNA or cytoplasmic RNA isolated from infected fibroblast

Cell Growth & Differentiation 459

cells were hybridized overnight at 50#{176}Cto the RNAprobe (10� cpm/reaction). Single-stranded RNA was di-gested with 20 �zg/ml RNase A (24), and the protectedfragments were separated on 6% polyacrylamide gels.

Northern and Southern Analysis. Isolation of RNA andDNA and subsequent Northern and Southern hybridiza-tions were performed according to standard procedures(44).

Acknowledgments

We thank I. B. Robson and A. Bernstein for generously providing the

DNA of the retroviral vectors MoTN and p.�E. We are grateful to M.Ward, S. Chance. and C. Spencer for expert technical help. and to 1.

Dunn, A. Gossler, and A. Cumano for helpful discussions.

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