Proc. Nati. Acad. Sci. USAVol. 88, pp. 478-482, January 1991Genetics

Heterologous introns can enhance expression of transgenes in mice(gene expression/transgenic mice)

RICHARD D. PALMITER*, ERIC P. SANDGRENt, MARY R. AVARBOCKt, D. DIANE ALLEN*,AND RALPH L. BRINSTERt*Howard Hughes Medical Institute, Department of Biochemistry, University of Washington, Seattle, WA 98195; and tLaboratory of Reproductive Physiology,School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104

Contributed by Richard D. Palmiter, October 11, 1990

ABSTRACT In a previous study we showed that genomicconstructs were expressed more efficiently in transgenic micethan constructs that were identical except for the lack ofintrons. Using the mouse metallothionein promoter-rat growthhormone gene construct as a model, we show that the rrstintron ofthe rat growth hormone gene is essential for high-levelexpression, whereas the other three introns are less effective.Several heterologous introns placed 3' of the coding region ofan intronless rat growth hormone gene are also ineffective.However, insertion of some heterologous introns between themetallothionein promoter and the growth hormone gene im-proves expression. To determine whether addition of heterol-ogous introns would provide a general strategy for improvingexpression, we have tested them in conjunction with otherintronless genes and with different promoters.

In principle, it is possible to direct the expression of any geneto any specific cell type of an animal by using establishedtransgenic methodology. This approach relies upon the factthat it is possible to combine the regulatory region(s) ofa genethat is expressed in a cell-specific manner with any mRNA-encoding structural gene. The structural gene should then betranscribed according to the specificity dictated by the reg-ulatory elements. This strategy has been used to explore awide variety of fascinating developmental and physiologicalproblems (see refs. 1-3 for reviews). However, not all geneconstructs work well in transgenic mice. The two mostcommon problems are inappropriate expression patterns andfailure to achieve adequate expression levels.

Inadequate expression is often associated with attempts toexpress cDNA constructs. The most baffling aspect of thisphenomenon is that cDNA constructs that fail to work intransgenic mice are often expressed efficiently when trans-fected into tissue culture cells. These observations suggestedthat introns may be of special importance for achieving geneexpression in transgenic mice. Because most of the earlyexperiments that led to this suspicion were not well con-trolled, we set out to test it directly by comparing constructsthat were identical except for the presence or absence ofintrons. With each ofthree test genes, mouse metallothioneinI (mMT-I), mMT-rat growth hormone (rGH), and human,3-globin (hf3G), we observed a striking improvement both inthe number of transgenic mice that expressed the gene and inthe average level of expression when the natural introns wereincluded (4). Furthermore, nuclear run-on assays indicatedthat the introns had a major effect on transcription rather thanmRNA processing or stability. Because the mMT and mMT-rGH constructs showed no dependence on introns for expres-sion after stable transfection into BHK (baby hamster kid-ney) cells, we postulated that introns may contain sequencesthat are recognized during embryogenesis.

Introns could improve expression of transgenes by a num-ber of different mechanisms. For example, some introns maycontain enhancers or other cis-acting elements which bindproteins that influence transcriptional initiation or elongation(5). Alternatively, the process of mRNA splicing mightenhance mRNA stability in the nucleus, which could lead toaccumulation of more mature mRNA in the cytoplasm (6).Another possibility is that introns contain sequences thatfacilitate opening of chromosomal domains, perhaps by af-fecting nucleosome composition, position, or higher-orderpacking (7).Because many genes that one might like to express in

transgenic animals have not been isolated or are too large tomanipulate easily, we set out to devise a general method toachieve expression of cDNA constructs. We chose themMT-rGH gene as a test gene and tried to improve expres-sion by adding either endogenous or heterologous introns invarious positions. In those cases where expression wasimproved, we tried to generalize by testing the effects ofintrons with other structural genes and other promoters.These studies provide the foundation for future analysis ofthe underlying mechanisms(s) involved.

RESULTSEffect of Natural rGH Introns. In our previous experi-

ments, we compared a mMT-rGH gene containing all four ofits natural introns with an identical construct in which theintrons were precisely removed. When transgenic mice wereproduced and total rGH mRNA was measured in liver of16-day fetuses, we observed a 10-fold difference in theaverage level ofmRNA (Fig. 1 a and b). Individual introns orcombinations of introns were added to the intronless rGHgene by use of convenient restriction sites within the exons.Adding intron A alone improved expression to about half ofthe value obtained with all introns, introns C and D had asmaller effect, and the combination of A, C, and D wasintermediate (Fig. 1 b-f). Surprisingly, intron B squelchedthe positive effect of intron A but this effect was overcomeby introns C and D. The variation in expression obtained withthese constructs is summarized in the figure legend.

Effect of Heterologous Introns Placed 3' of the IntronlessrGH Gene. In this set of experiments, we added an intron andpolyadenylylation sequence to the 3' end of the intronlessrGH gene at the Nsi I site that lies 10 base pairs (bp) upstreamof the polyadenylylation site of rGH. The mouse protamineI (mPrm-I) intron, h(3G intron B, and simian virus 40 (SV40)small tumor antigen (t antigen) intron along with their poly-adenylylation regions did not rescue expression (Fig. 2 b-d).

Abbreviations: hAF, human angiogenesis factor; h$G, human(-globin; hGH, human growth hormone; hNGF, human nervegrowth factor; mAlb, mouse albumin; mMT, mouse metallothionein;mPrm, mouse protamine; rE, rat elastase; rGH, rat growth hormone;rIns, rat insulin; rTGFa, rat transforming growth factor a; SV40,simian virus 40.

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

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mMT-I Exp/Tg Mol/Cella A 2 B _ 3C 4 D 5 - 9/1 1 300

mMTI .rGH rGH 7/15 29

mMT-I rGH. rGHCA3-'4.5 r 6/8 155mMT-I,_ rGH rGH

e A-'22h BC34_34 5 : 11/15 19

mMT-I GH..GH

mMT-1 rGH r3f-i A,2 3;C 4D5$ 12/14 128

FIG. 1. Effect of rGH introns in their natural location. Theconstruction of the mMT-rGH gene (a) and its intronless version (b)was described previously (4). For constructs in c-f, introns wereinserted into the intronless gene by substituting regions from thegenomic copy for comparable regions of the intronless gene throughthe use of convenient restriction sites. The mMT-I promoter extendsto the EcoRI site at nucleotide -1774, and the rGH gene extendsabout 2.75 kilobases (kb) downstream of the polyadenylylation site.Exons are indicated by hatched boxes and numbers, while introns areindicated by open boxes and letters. Total nucleic acid was isolatedfrom 16- to 17-day fetal livers by the SDS/proteinase K method, andrGH mRNA was measured by solution hybridization using a 30-meroligonucleotide complementary to the sequence extending from+328 to +369 that was end-labeled with 32p (4). Exp/Tg, fraction oftotal transgenic mice produced that gave measurable expression ofmRNA; Mol/Cell, average amount ofmRNA (calculated as mRNAmolecules per cell) in all the transgenic samples. The data in a andb were taken from our previous paper (4). The mRNA values(molecules per cell, mean ± SEM) ofjust the expressor mice are asfollows: a, 365 + 66; b, 63 ± 11; c, 207 ± 62; d, 26 ± 5; e, 98 ± 20;f, 150 ± 48.

Substituting the SV40 polyadenylylation sequence alone wasalso ineffective (Fig. 2e).

Effect of Heterologous Introns Placed 5' of the IntronlessrGH Gene. When heterologous introns were placed betweenthe mMT-I promoter and intronless rGH gene, there wassubstantial rescue of expression. In each of these constructsthe introns and flanking exonic sequences were chosen sothat they would not carry translational initiation codons that

mMT-I _rGH_ H52--4

mMT-l siHrGH mPrm-lb 2 --;3?4 --.5.l

mMl-I _rGH hBGc- 327 4 5T*----mMT-l _rGH SV40-td 2 3. 4 5 -

mMT-I rGH SV40-pAe 2 3 4 5

Exp/Tg Mol/Cell

7/15 29

7/8

6/9

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6

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FIG. 2. Effect of heterologous introns placed 3' of the intronlessrGH gene. Construct in a is included for comparison; it is the sameas Fig. lb. To make constructs shown in b-e, the construct in a wascut with Nsi I, which cuts about 10 bp 5' ofthe polyadenylylation site,and DNA containing an intron and polyadenylylation site was addedto constructs in b-d whereas DNA containing only a polyadenyly-lation site was added to the construct in e. For b, a 530-bp Nco I-BglII fragment of the mPrm-I gene (8) was used; for c, a 2810-bpBamHI-Xba I fragment of the hI3G gene (9) was used; for d, a 450-bpHindIII-BamHI fragment from pKOneo was used, which includedthe SV40 small t intron (nucleotides 4710-4550; New EnglandBiolabs numbering) followed by the large tumor antigen (T-antigen)polyadenylylation region (nucleotides 2825-2533); the construct in econtained only the Bcl I-BamHI fragment of SV40 (nucleotides2770-2533). Expression was measured as in Fig. 1. The mRNAvalues (mean ± SEM) ofjust the expressors are as follows: a, 6311; b, 4.5 ± 1.6; c, 31.5 ± 8.5; d, 6.4 ± 2.0; e, 41 ± 12.

would prevent translation of rGH; however, we did not testfor rGH protein production. Intron A of the rat insulin II(rIns-Il) gene, intron B of the h,3G gene, and the SV40 smallt intron resulted in expression (Fig. 3 b-e) comparable to thatachieved with the natural first intron ofrGH. A tandem arrayof two rIns introns was ineffective, however (Fig. 3c). SincerGH intron A was beneficial (Fig. 1c), we tested the analo-gous region of the hGH gene by substituting the region 5' ofthe conserved Pvu II site that lies in exon 2. The hGH intronand flanking exonic sequences did not rescue expression ofthe intronless rGH gene (Fig. 3f). The first introns of hGHand rGH have about 50% nucleotide identity and are 254 and185 nucleotides long, respectively.

amMT-I-_- rGH rGHa, 2 3. 4 58

mMT-r rins-Il rGH Hb |1A1 2 3 4 5.r

mMT rrknsj1rins 11 rGHC inE4AJ A|112 3 4 '}z=_

hiiSGO rGH

mMT-I hG rGH rGHd 234 -

mMT-ISV40-t rGH mMT-H

mPAT-II rGH mM-

e It t2 3 4? S -

mnMT hN _GrFHf1 A 2Z3 ------

Vi~ol PYAJII

mM-l rGH MmMT-ll-- A -1 2 :3: 4 -5 } ....

mMTT-11 rGH MmMT-IIh.-.A '2 -.3 4 5 B

mMT-1 hNGF rG:H

nMTI rins-ll hNGF_:5A[ -: ....-

Exp[Tg Mol/Cell

7/15 29

8/8 179

5/6 39

4/7 102

4/7 111

9/24 8

0/9 0

2/9 2

1/13 0.2

8/11 15

FIG. 3. Effect of introns placed between MT promoters andintronless genes. a is included for comparison; it is duplicated fromFig. lb and Fig. 2a. For constructs in b-e, heterologous introns werecloned into the Xho I site (+6) that joins the mMT-1 promoter to therGH gene. For b, a 171-bp fragment extending from +6 to +177 ofthe rIns-II gene (10) that includes intron A was amplified by poly-merase chain reaction with primers that included Xho I and Sal Irestriction sites. For c, two copies of this Xho I-Sal I fragment werecloned into the Xho I site in the sense orientation. For d, the 920-bpBamHI-EcoRI fragment of the hBG gene that includes intron B wasblunt-end-ligated into the Xho I site. For e, a 127-bp fragment[nucleotides 554-681 (11)] that includes the SV40 small t intron wasamplified by polymerase chain reaction with primers that includedXho I and Sal I restriction sites and was inserted into the Xho I site.Forf, the Xho I-Pvu 1I (+6 to +386) region of the rGH gene wasreplaced with the comparable BamHI-Pvu II (+2 to +459) region ofthe human GH (hGH) gene. For g, the intronless rGH gene (XhoI-Nsi I fragment) was cloned into the BamHI site in exon 2 of themMT-II gene (12) in which the initiation codon in exon 1 had beendestroyed and intron B of mMT-II had been removed by deleting the280-bp BamHI-Pvu II fragment. The construct in h is the same as ing except that intron B ofMT-II was retained; thus, the rGH gene wasbetween two introns. The construct in i had the 2.5-kb Sca I-EcoRIfragment that contains the entire open reading frame ofhuman nervegrowth factor (hNGF; ref. 13) inserted between the Xho I and secondPvu II site of the mMT-rGH gene (see Fig. 4a). Construct inj is thesame as in i but with the rIns-II intron inserted into the Xho I site.For a-h, expression was measured as in Fig. 1. For i andj, fetal liverhNGF mRNA was measured by solution hybridization with anoligonucleotide (5'-GACTTGGGGGATGGTGTGTCCTG-3') com-plementary to hNGF mRNA; for quantitation ofmRNA per cell, weassumed that this oligonucleotide was of the same specific activityand hybridized with similar kinetics as the rGH oligonucleotide. ThemRNA values (mean + SEM) ofjust the expressors are as follows:a, 63 + 11; b, 179 + 80; c, 47 + 15; d, 179 + 56; e, 195 + 109;, 21.3+ 5.0; g, 0; h, 8.4 ± 0; i, 2.6;j, 20.7 ± 5.6.

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The endogenous mMT-II gene, which has a convenientBamHI site in its second intron, is regulated coordinatelywith mMT-I and its expression is about 70%o that of mMT-I(14). When the intronless rGH gene was inserted into exon 2of the mMT-II gene and tested in transgenic mice, there wasno expression of rGH (Fig. 3h). When transfected into BHKcells, there was a high level of mRNA produced from thisconstruct but the rGH sequences were deleted from themRNA, presumably by splicing from exon 1 to 3 (data notshown). Aberrant splicing can also be a problem associatedwith the use of the SV40 small t intron (15). To try toovercome this problem, intron B of mMT-II was removed,but that did not rescue expression (Fig. 3g).To ascertain whether heterologous introns could rescue the

expression of other intronless genes, we compared the effectof the rIns-TI intron on expression of the hNGF gene drivenby the mMT-I promoter. This heterologous intron increasedexpression of this construct about 75-fold (Fig. 3 i and j),although the absolute level of expression was low comparedto rGH.

Effect of Introns Placed Within (or Downstream of) theIntronless rGH Gene. To explore further whether the locationof the intron is important, the rIns-II intron was inserted intoeach of the Pvu II sites within the coding region ofrGH (Fig.4). Although these insertions would not have generated afunctional rGH mRNA after splicing, we neverthelesswanted to determine whether they would allow efficientmRNA production. Both were ineffective (Fig. 4 b and c).Likewise, insertion of the rGH intron A sequence 1.5 kbdownstream of the intronless rGH gene had no effect (Fig.4d). These experiments indicate that the position ofthe intronis important and they argue against an enhancer-like functionof these introns.

Effect of Using Heterologous Introns with Other Promoters.The previous results indicated that heterologous intronscould enhance the expression of two different intronlessgenes driven by the mMT-I promoter. We next wanted toascertain whether they would also work with other promoter/enhancers. Therefore, the rIns-II intron was inserted be-tween the rat elastase I (rE-I) promoter/enhancer and theintronless rGH gene and expression was measured in pan-creas of 15-day-old mice. This intron was ineffective (Fig. Sc)compared to the controls (Fig. 5 a and b). We also tested therIns-II intron with the naturally intronless hAF gene drivenby the rE-I promoter/enhancer (Fig. 5 d and e). None of themice without the intron expressed hAF and only one of ninetransgenic mice with the intron expressed it; thus, this introndoes not appear to work well with the rE-I promoter/enhancer.

Effect ofInserting Intronless Genes (or cDNAs) into the hGHGene. Another strategy that allows expression of some

mMT-I rGH rC-Ha- - 2 3 4 5

rnMT-I rins 11 rGH GH

b _-42l 3: 4 5

Mr Ii..rA . rins-.l

mI12'3 4

mMT- rH rGH rGIH-- 1 2 3 4 5 A

Exp/ilg Mol/Cell7/15 29

0/4 0

7/9 64

3/6 21

FIG. 4. Effect of insertion of introns at alternative sites within therGH gene. In b and c, the rIns-lI intron (see Fig. 3b) was inserted intoeither the Pvu II site in exon 2 or the Pvu II site in exon 5. In constructd, the 176-bp Sph I-Xba I fragment that includes most of the 185-bpintron A of rGH was inserted into the HindIlI site about 1.5 kbdownstream of the rGH gene. Expression was measured as in Fig. 1.The mRNA values (mean ± SEM) of just the expressors are as

follows: a, 63 ± 11; b, 0; c, 82 ± 17; d, 42 ± 13.

rr rC2I- 1 A 2

rr. Gt,ti-. 1 2 3 4

Ci

4.3C 4 [

CI.5

,

F-] rlris-11 'rG I-1 Gi A 2.CA

I- Al ?. 3 4 fj (

Lxpil a Mol/Cel!

5/6 595

215 44

5/9 24

MhF

0/9 0

F-rI rIr's-I1e I Al

MIf;

1/9 81

FIG. 5. Effect of the rIns-II intron on expression with the rE-Ipromoter. Constructs in a and b are controls and the data are fromour previous publication (4). In c, the rIns-II intron (see Fig. 3) wasinserted into the Xho I site that joins the 4.5-kb rE-I promoter/enhancer to the intronless rGH gene. In d, the 0.2-kb rE-I promoterwas fused to the intronless human angiogenesis factor (hAF) gene atan Xho I linker that was introduced at position +63 of the hAF gene(16); the hAF gene extends to the EcoRI site at +3 kb and includesits own polyadenylylation signal. In e, rIns-II intron A was insertedinto the Xho I site thatjoins the rE-I promoter to the hAF gene. rGHmRNA was assayed as in Fig. 1 in total nucleic acids isolated from15- to 16-day postnatal pancreas. [Note that in our previous publi-cation (4) we incorrectly indicated that the mRNA was measured inembryonic day 15-16 pancreas.] hAF mRNA was assayed in totalnucleic acid isolated from adult pancreas by solution hybridizationwith an oligonucleotide (5'-GTGTGTGTACCTGGAGITATCCTG-3') complementary to hAF mRNA. The mRNA values (mean ±SEM) ofjust the expressors are as follows: a, 715 ± 537; b, 110 ±33; c, 44 ± 16; d, 0; e, 730.

cDNAs involves inserting them into the first exon of aheterologous gene, between the promoter and initiationcodon, such that they represent the first open reading frame.This strategy was used, for example, to achieve expression ofdiphtheria toxin (17). We have inserted a variety of cDNAsbetween the mMT-I promoter and the hGH gene shown inFig. 6a. Expression of rGH was less than that achievedwithout the hGH gene (compare Fig. 6b with Fig. lb). Wealso could not achieve significant expression of insulin-likegrowth factor I, heart gap junction, MyoD, or rat proteinkinase C cDNAs (data not shown). Expression ofrTGFa wasachieved (Fig. 6c), whereas the control that had only the hGHpolyadenylylation region gave no expression (Fig. 6d). Note,however, that Jhappan et al. (20) achieved expression of ahuman TGFa cDNA in three or four transgenic mice with aconstruct very similar to that shown in Fig. 6d. This samestrategy has been tried with other promoter/enhancers.About 10 constructs in which cDNAs have been insertedbetween the p56 ck proximal promoter and the hGH gene havebeen expressed successfully, although unusual splicing pat-terns were observed in some instances (ref. 21 and R.Perlmutter, personal communication). Likewise, rTGFa wasexpressed reasonably well in the liver and pancreas by usingthe mAlb and rE-I promoter/enhancers (Fig. 6 f and g).However, the naturally intronless hAF, ,B-adrenergic recep-tor, and influenza virus hemagglutinin genes were not ex-pressed from the mAlb promoter/enhancer using this ap-proach (data not shown). Furthermore, hemagglutinin andseveral different rat protein kinase C constructs were notexpressed in the pancreas by using the rE-I-hGH construct(data not shown).An alternative means of achieving rTGFa expression in-

volved construction of a minigene that incorporated twointrons of rTGFa into the cDNA. This construct was alsoexpressed (Fig. 6e).

DISCUSSIONIn our previous paper (4) we showed that genomic constructswith introns were expressed at a higher level in transgenic

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Exp/Tg Mol/CellmMT-1 hGH hGH

a pI' A 2 BC4 D 5 -D 23/33 610BamHl

mMT-I rGH hGH hGb _T ___ IA 2j B -3 C 4 D -5 r 4/13 5

mMT-1 rTGF hGH , ,hGH._........ _. __A 2 B 3 C 4 D_ 5 10/13 463

mMT-I_ rTGF hGH

BgIlI SmalmMT-I rTGF -D. -- .. hGH

e _~ D _ E

0/6 0

4/11 172

mAlb rTGF hGH hGHf--K_._.._.-'- 1T A 2 _3 C: D 5 11/161.

98

rEl _ rTGF __hGH khGH0 - ~_= = 1 A 2-CB3-C 4 D 5 16/19 363

FIG. 6. Expression ofthe rGH and rat transforming growth factor a (rTGFa) genes inserted between a promoter and the hGH gene. Constructin a had the mMT-1 promoter fused at the BamHI site (+2) of hGH as described (18). The data for both construct 111 and construct 112 of ref.18 are summarized here; the average mRNA levels obtained from eight adult mice that had been induced with either zinc or cadmium wasmultiplied by the Exp/Tg value to obtain the value shown here. Construct in b represents the insertion of the intronless rGH gene (Xho I-NsiI fragment) into the BamHI site; expression was determined in fetal liver as in Fig. 1. In c, the 700-bp rTGFa cDNA Sma I fragment that includesthe entire open reading frame was inserted into the BamHI site of construct in a; mRNA was measured in adult liver of mice maintained ona diet supplemented with zinc (19). In d, as a control, the rTGFa coding region was inserted between the BamHI site and the Sma I site ofhGHthat lies in exon 5 near the termination codon. Construct in e was derived from that in d by substituting a genomic 2.3-kb BgI II-Sma I fragmentthat includes the fourth and fifth introns for the comparable region of the cDNA. Constructs infand g are comparable to that in c, except thatthe rTGFa cDNA fragment was inserted between the mouse albumin (mAlb) promoter or the rE-I promoter and the hGH gene; mRNA wasassayed in adult liver or pancreas, respectively. rTGFa mRNA was measured with an oligonucleotide complementary to the 3' untranslatedregion of hGH. The mRNA values (mean ± SEM) ofjust the expressions are as follows: a, 873 ± 302 (n = 8); b, 15.3 ± 3.7; c, 602 ± 132; d,0; e, 472 ± 175;f, 142 ± 20; g, 432 ± 77.

mice than intronless constructs. Here we have attempted todecipher which of the natural introns are responsible forenhanced expression. We then asked whether heterologousintrons could rescue expression and, if so, whether theirlocation was important. Finally, we inserted intronless genesinto exons of otherwise intact genes that are expressed wellin transgenic mice. Our data indicate that the natural firstintron of rGH is sufficient to achieve most of the expressionof the intact rGH gene. Some (three of five tested) heterol-ogous introns placed between the mMT-I promoter andintronless rGH gene improved expression in fetal liver oftransgenic mice. One of the heterologous introns was alsoeffective when placed between the mMT-I promoter and thehNGF gene. In contrast, rIns-II intron A was ineffectivewhen inserted at two other locations within the intronlessrGH gene and rGH intron A did not function when inserted1.5 kb downstream of the gene. Likewise, three differentheterologous introns were ineffective when inserted down-stream ofthe rGH coding region. Insertion ofcDNAs into thefirst exon ofhGH gave mixed results: a few (e.g., rTGFa andref. 21) worked quite well whereas most did not work at all.The same result was obtained with several different promot-er/enhancers. Thus, from a practical point of view, thegeneral outcome from this and previous work supports theuse genomic constructs whenever possible for transgenicexperiments. The next best strategy may be to use a minigenein which as much genomic sequence as possible is retainedbut large intronic regions are removed. Ifgenomic sequencesare not available or the gene of interest lacks introns, theninsertion of a heterologous intron between the promoter andcoding region may improve expression. If a cDNA is usedwith this strategy, then a functional polyadenylylation regionshould also be appended. Alternatively, a cDNA can beinserted into the first exon of a gene that is known to beexpressed well, so that the cDNA contains the first openreading frame. Although there are numerous reports ofexpression in transgenic mice of constructs lacking introns(1-3), most negative experiments remain unpublished and the

level and/or frequency of expression could probably beimproved substantially by including introns.The mechanism(s) underlying the apparent benefits of

including introns is not easy to discern. The position depen-dence of the functional introns appears to rule out splicingperse or strong enhancer-like elements within those introns asbeing important determinants for expression. Weak enhanc-ers or position-dependent cis-acting elements (22-24) mightreside within those introns that are naturally located near thepromoter. The position dependence of the rGH and rIns-IIintrons might be due to this sort of mechanism. In fact, rGHintron A has been reported to contain a DNase I-hypersen-sitive site that may be a binding site for thyroid hormonereceptor (25). For the introns to be effective in our assay theywould have to bind general transcription factors, because thetransgenes were tested with a heterologous promoter inhepatocytes rather than in cells where they normally func-tion.A general idea that might bear on this issue is that se-

quences around promoters may have evolved in a way thatfacilitates transcription factor assembly. These sequenceswould obviously include transcription factor binding sites,but there might be more subtle effects ofDNA sequence onnucleosomal positioning with respect to important promoterelements (ref. 26; for review see ref. 27). Nucleosomalpositioning might be influenced directly by the DNA se-quence so that nucleosomes are preferentially bound orexcluded. Alternatively, protein-DNA interactions may cre-ate boundary conditions which alter nucleosomal position-ing. In our study, all the gene constructs were made by fusingthe 5' flanking region of one gene with the coding region ofanother gene, and the junctions were made in the 5' untrans-lated region of each gene. As a consequence, if there wereany evolutionary selection for optimal sequences that spanthis junction, then these sequences would most likely bedisrupted in construction of the chimeric genes. Further-more, when the first intron is close to the transcription startsite, the optimal sequence for promoter function might ex-

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tend into the intron, suggesting that this intron may beparticularly important. Regardless of the underlying mecha-nism, it is becoming increasingly clear that there are inter-actions between promoters and structural genes that influ-ence transcription. For example, not all structural genesdriven by the same promoter are transcribed at the same rate(28), and when an inducible promoter such as the mMT-Ipromoter is combined with different structural genes, thedegree of induction varies tremendously (unpublished data).Both of these observations suggest that there are sequencesdownstream of the promoter that influence transcriptionalinitiation or elongation.Another consideration is that introns may enhance nuclear

mRNA stability or transport by the assembly of a spliceo-some complex (6). Nuclear run-on assays revealed that theintronless mMT-rGH construct was not transcribed effi-ciently compared with the genomic construct (4); thus, webelieve that most of the effects studied here are at thetranscriptional level. However, in all of the experimentspresented, the inserted heterologous introns would leavesome flanking ex~onic sequences in the rGH transcript, andthe influence of these sequences on niRNA stability was notassessed.

In this study, we have tried to improve the expression ofintronless transgenes by adding sequences containing intronsat various positions within the test gene. Although we tend tothink of intronis in the context of splicing, it is possible thatmany of the effects are mediated by changes in transcriptionfactor assembly or nucleosomal phasing. Furthermore, al-though effects of introns on gene expression in cultured cellshave been described (22-24, 29, 30), an important aspect ofour previous study (4) is that introns affected expression intransgenic mice but not after transfection into cultured cells.For example, introns are not important for expression of thebovine GH gene in cultured cells (31). Thus, it appears thatgenes are subjected to events during development that-are notrealized in cultured cells, and any general hypothesis mustaccount for this result. In the transfection experiments, aselection process was used to establish stable clones,whereas there was no selection involved before assay of thetransgenic mice. However, even if one compares only theaverage level of expression in those mice that expressed thegene at some level and hence presumably integrated thetransgene into an active chromatin domain, there is still asignificant difference between transgenes with or withoutintrons. It is clear that position-independent transgeneexpression depends upon other sequences, called locus con-trol regions, that may help establish chromosomal domains(32). Perhaps these control elements, together with enhancerelements and sequences contained within introns, alter nu-cleosome-DNA interactions and determine whether func-tional transcription complexes can be assembled at the pro-moter.

We thank our colleagues for their suggestions and support duringthe progress of this study. This work was supported in part byNational Institutes of Health Grants CA38635, HD09172, HD19018,and HD23657.

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