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Biochemical and Biophysical Research Communications 287, 1099–1104 (2001)

doi:10.1006/bbrc.2001.5707, available online at http://www.idealibrary.com on

NAi in Mouse Oocytes and Preimplantation Embryos:ffectiveness of Hairpin dsRNA

etr Svoboda, Paula Stein, and Richard M. Schultz1

epartment of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6018

eceived September 4, 2001

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RNA interference (RNAi), the targeted mRNA degra-ation by double-stranded RNA (dsRNA), is a usefulool for studying gene function in several organisms.ere we report results of experiments with mamma-

ian dsRNA expression vectors that are suitable totudy gene function in mouse oocytes and preimplan-ation embryos. The plasmid vectors were constructedo contain the SV40 small intron, EGFP coding se-uence to permit detection of expression, and an in-erted repeat to mos mRNA that would form a hairpinsRNA. Results of the experiments indicated that (i)airpin dsRNA was just as effective as dsRNA (i.e.,nnealed sense and antisense RNA) in promoting theestruction of targeted mRNA, (ii) the EGFP markerould be expressed from the construct, and (iii) theistance of the SV40 intron from the inverted repeatas critical for the transcribed RNA to function inNAi. © 2001 Academic Press

RNA interference (RNAi) is a recent approach basedn the original observation that double-stranded RNAdsRNA) can efficiently ablate gene function by pro-oting selective mRNA degradation (1). RNAi has

een documented in many species, and in Caenorhab-itis elegans and Drosophila melanogaster it has be-ome a widely used method to study gene function. Theolecular mechanisms of dsRNA-mediated degrada-

ion of the targeted mRNA and the linkage betweenNAi, posttranscriptional gene silencing (PTGS) anduelling (two related gene silencing mechanisms thatccur in plants and Neurospora, respectively) are rap-dly being elucidated (reviewed in 2, 3).

Sequence-specific interference mediated by dsRNAas been recently described in the mouse oocyte andreimplantation embryo (4, 5). We demonstrated thatsRNA directed against Mos and Plat mRNAs in

1 To whom correspondence should be addressed at Department ofiology, University of Pennsylvania, 415 South University Ave-ue, Philadelphia, PA 19104-6018. Fax: 215 898-8780. E-mail:[email protected].

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truction of the targeted mRNA in both a time- andoncentration-dependent manner and that dsRNA cane used to block gene function in mouse oocytes. Thisnd our recent unpublished data which demonstratehat mouse oocyte and preimplantation embryo ex-racts can process dsRNA into the 22-mer that areesponsible for degrading the targeted mRNA (6–8)onfirm that the mechanism interfering with geneunction in mouse oocytes and early embryos is veryimilar, if not identical, to the RNAi mechanism de-cribed in Caenorhabditis and Drosophila.Here we report the design and functional analysis of

lasmids that direct the expression of dsRNA and en-anced green fluorescence protein (EGFP) from eitherhe oocyte-specific ZP3 promoter or the ubiquitouslyxpressed cytomegalovirus (CMV) promoter. Theselasmids can be used not only for microinjection but, inrinciple, also for the generation of transgenic mice.he transgenic RNAi approach has been described inther model systems, namely Caenorhabditis (9), Dro-ophila (10), and Arabidopsis (11). Our work extendshese previous studies by providing new and valuablenformation about the design of constructs expressingsRNA in mammalian cells.

ATERIALS AND METHODS

Plasmids. An inverted repeat (IR) from the mos gene was createdy self-ligating the previously described 535 bp mos PCR product (5)igested 20 bp from the 59 end with Apa LI (New England Biolabs,A). The band of the correct size was extracted from a 1% agarose

el using the Gel Extraction Kit (Qiagen, CA) and cloned into theCRII vector using the TOPO-TA cloning kit (Invitrogen, CA). Ex-ensive screening yielded only one clone that was composed of twoead-to-head oriented Apa LI-digested and undigested PCR prod-cts. The excess sequence from the undigested product formed ahort 20-bp spacer in the middle of the inverted repeat (IR). Thislone was sequenced and used for subsequent experiments (Fig. 1A,onstruct A).The construct A was further modified to generate two additional

emplates (Fig. 1A, constructs B and C) for in vitro transcription. The.8 kb NheI/XbaI fragment from the pEGFP-N2 plasmid carrying theGFP coding sequence (cds) was inserted into a unique XbaI site inonstruct A. We obtained clones with inserts in both orientations

0006-291X/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

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NheI and XbaI produce the same cohesive ends) and used them toenerate in vitro RNA molecules in which EGFP is either upstreamr downstream of the IR (Fig. 1A, constructs B and C).For in vivo expression experiments we modified two plasmids:

MoZP3-EGFP and pEGFP-N2 (Clontech, CA). The pMoZP3-EGFPlasmid (generously provided by Jurrien Dean, NIH) contains ahortened ZP3 promoter inserted in the pEGFP-1 plasmid (Clontech,A). Generally, pMoZP3-EGFP differs from pEGFP-N2 only in theromoter sequence and the multiple cloning site upstream of theGFP cds. These two plasmids were ancestors of several different

onstructs, described as D, E (Fig. 2A), F, G, H (Fig. 3A), and I (Fig.). We inserted the Mos IR upstream (construct E, CMV/ZP3 39GFP) or downstream (construct D, CMV/ZP3 59 EGFP) of EGFP;

he 59 or 39 in the name of the plasmid designates the location ofGFP with respect to the hairpin (as it generates 59 or 39 single-tranded overhangs). These plasmids (D, E) were used for analysis ofoth the effect of location of the IR on EGFP fluorescence and theffect of the location of the EGFP cds in the plasmid on specificRNA degradation.Plasmids F, G and H (Fig. 3A) were used to evaluate the effect of

he SV40 intron location on RNAi. Construct G was generated onlyith the ZP3 promoter, whereas all other constructs were preparedith both promoters. The SV40 small intron was isolated as a 310-bpCR product from the pGL2 plasmid (Promega, WI). For PCR wesed the following primers: upstream primer 59-GCCTGGTG-TACGCCTGAATAA-39, downstream primer 59-GTCAGCAGTA-CCTCATCATC-39. PCR cycle conditions were as follows: 36 cycles

FIG. 1. (A) Schematic diagrams of pCRII-derived plasmid constlasmid is shown. T7, T7 promoter, SP6, SP6 promoter, IR, inverteditro. Ethidium bromide stained nondenaturing 1.2% agarose gel wi, and C from panel A); each lane contains about 400 ng of RNA. M, RNase T1 treatment; lane 3, RNase A treatment. The arrowheouble-sized band in nontreated samples (most apparent in lane A1) irom multiple cloning site pCRII sequence) that persists in the ntraditional” and hairpin dsRNAs on the MAP kinase and H1 kinaseethods. The relative MAP and H1 kinase activities compared to w

tandard deviations, and 12 microinjected oocytes were analyzed iningle oocytes. An autoradiogram illustrating the effect of dsRNA onkinase substrate; MBP, myelin basic protein, a MAP kinase substra

y annealing single-stranded sense and antisense RNAs as describehose shown in lanes 1 of the panel B.

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f 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s. The PCR productas cloned into the pCRII plasmid by TA cloning and reinserted intoMV- or ZP3-driven D constructs (Fig. 1) at three different positions:pstream of EGFP (construct F), between EGFP and the IR (con-truct G), and downstream of the IR (construct H).Finally, we created two plasmids for cloning the IR of interest into

he XbaI site downstream of the SV40 intron and EGFP (construct I,ig. 4). These plasmids were generated by inserting the SV40 intron

nto the multiple cloning sites of pEGFP-N2 and pMoZP3-EGFPlasmids. The entire expression cassette can be removed by digestingith AseI and AflII (CMV-driven construct) or with BglII and AflII

ZP3-driven construct). The XbaI insertion site should providenough versatility for cloning an inverted repeat because NheI andpeI enzymes produce compatible cohesive ends with XbaI.

dsRNA preparation and analysis. Annealed Mos (“traditional”)sRNA was generated as previously described (5). Mos dsRNA hair-ins were generated in an in vitro transcription reaction where ainearized plasmid containing an inverted loop was transcribed withP6 or T7 polymerase (Promega, WI). Transcribed RNAs were phe-ol extracted and diluted in water to obtain a concentration of 106

olecules/5 pl for microinjection. These RNAs were not exposed toemperatures over 37°C. Formation of dsRNA was analyzed byNase T1 (Roche, IN, USA) treatment. About 300 ng of RNA wasigested with RNase T1 in buffer containing 1 mM Tris–HCl (pH.9), 1.5 mM NaCl and 1.5 mM MgCl2 for one1 hour at 37°C. DigestedNA was analyzed electrophoretically (Fig. 1B).

ts used for in vitro transcription. Only the transcribed part of theeat. The diagram is not drawn to scale. (B) Formation of dsRNA inarious treatments of in vitro transcribed RNAs (from templates A,

0 bp ladder (New England Biolabs, MA), lane 1, no treatment; lanedepicts the position of the dsRNA fragment. The presence of theost likely caused by annealing of single-stranded RNA ends (deriveddenaturing gel. (C) Comparison of the effect of microinjection oftivities. The assay was performed as described under Materials andr-injected oocytes (control) is shown. The error bars represent twoh group. (D) A typical result of a double kinase assay performed onand MAP kinase activities is shown. H1, histone H1, a p34cdc2/cyclinEach lane represents one oocyte. “Traditional” dsRNA was obtainedreviously (5). The remaining three hairpin RNAs were the same as

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RNA isolation and RT-PCR. RNA was isolated from oocytes ei-her as previously described (12) or using magnetic beads (Dynaliotech, NY) according to the microscale protocol. Typically, RNA

rom 15 to 20 oocytes per sample was isolated. For reverse transcrip-ion, total RNA or poly(A) mRNA (eluted from the magnetic beads)as reverse transcribed with Superscript II (Life Technologies, MA)sing the manufacturer’s protocol. Prior to RNA isolation, 0.125 pgabbit b-globin mRNA/oocyte was added to the samples as an exter-al standard. Semiquantitative PCR and analysis of globin, Mos andlat PCR products were performed as described previously (5). ForCR analysis of splicing of the SV40 intron, a reverse transcribedRNA equivalent to a single microinjected oocyte was amplified in a

0-ml reaction with the following primers: forward primer: 59-GC-TGGTGCTACGCCTGAATAA-39, reverse primer 59-GTCAGCAGT-GCCTCATCATC-39. PCR conditions were the same as for the clon-

ng of SV40 intron fragment from pGL2 plasmid (see above).

Oocyte and embryo collection, microinjection, culture and microscopy.ully-grown, germinal vesicle (GV) intact oocytes were isolated, micro-

njected and cultured as described previously (5). Meiotically incompe-ent oocytes were obtained from 13-day-old female CF-1 mice (Harlan,ndianapolis, IN) by incubating pieces of ovarian tissue in Ca21-, Mg21-ree CZB medium (13) containing 1 mg/ml collagenase and 0.2 mg/mlNase I at 37°C for up to 60 min. The oocytes released from the growing

ollicles following gentle pipeting with a mouth-operated micropipetere transferred to CZB medium and placed in an incubator at 37°C in

FIG. 2. (A) Schematic diagrams of pEGFP-N2 or pMoZP3-EGFP-erived plasmids carrying a Mos IR and EGFP; only the expressedart of the plasmid relevant to this study is shown. CMV/ZP3, CMVr ZP3 promoter; green arrow, EGFP cds; black arrows, Mos IR; A,olyadenylation site. The diagrams are not drawn to scale. (B) EGFPuorescence in incompetent oocytes microinjected with ZP3-drivenonstructs D and E. Bright-field images are on the left, and fluores-ence images are on the right. Control oocytes were microinjectedith pMoZP3-EGFP plasmid without the hairpin. All oocytes micro-

njected from one representative experiment are shown. The vari-bility in the level of fluorescence among oocytes is probably due toifferences inherent in the microinjection process. This experimentas also performed several times with the CMV-driven constructs in

ncompetent oocytes and early embryos, and similar results werebtained. (C) Autoradiogram showing semiquantitative RT-PCR as-ay of incompetent oocytes injected with pMoZP3-EGFP (control)nd ZP3-driven constructs D and E. This experiment was conductedwice and similar results were obtained in each case. The differencen the effect of constructs D and E on the Mos mRNA level was notignificant due to the sensitivity of the RT-PCR assay and was notbserved in other experiments that are not shown.

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as microinjected with 1 pl of plasmid solution containing 10,000 copiesf the plasmid; microinjection, which was conducted in bicarbonate-freehitten’s medium (14) containing 15 mM Hepes, pH 7.2, and 0.01%

olyvinyl alcohol, was performed as previously described (15). Microin-ected oocytes were cultured in an atmosphere of 5% CO2 in air at 37°C for8 h in CZB. Fluorescence was examined under a fluorescence stereomi-roscope (Leica MZ FLIII, Leica, Wetzlar, Germany) equipped with GFPxcitation sources and appropriate filters (Leica GFP fluorescence filteret). Photographs were taken at 1003 magnification with a CoolSNAPigital camera and CoolSNAP color capture software (Roper Scientific,renton, NJ). Image processing was done using Adobe Photoshop.

Histone H1 and MAP kinase assay. The histone H1 and MAP ki-ase assay on single oocytes was performed as described previously (5).he mobilization of the Mos mRNA during oocyte maturation results inhe ultimate activation of MAP kinase, whose activity is required toaintain arrest at metaphase II (16, 17); oocytes lacking the Mos geneature to metaphase II but then undergo spontaneous activation, i.e.,

hey emit the second polar body and form a pronucleus (18, 19). Inddition, H1 kinase activity, a marker for p34cdc2/cyclin B activity, alsoncreases during oocyte maturation. In the double kinase assay, MAPinase activity serves as an indirect measure of the amount of MosRNA and the H1 kinase activity serves as a biochemical marker for

gg activation, since it decreases following egg activation.

ESULTS

loning of an Inverted Repeat

Our results confirmed the previously published ob-ervation that direct cloning of an inverted repeat is

FIG. 3. (A) Schematic diagrams of pEGFP-N2 or pMoZP3-EGFP-erived plasmids carrying Mos IR, EGFP, and SV40 intron. ZP3, ZP3romoter; CMV/ZP3, CMV or ZP3 promoter; green arrow, EGFP cds;lack arrows, Mos IR; A, polyadenylation site. Construct G was pre-ared only with the ZP3 promoter. The diagrams are not drawn to scale.B) EGFP fluorescence in meiotically incompetent oocytes microinjectedith ZP3-driven constructs F, G, and H. Bright-field images are on the

eft, and fluorescence images are on the right. Uninj, uninjected oocytes.he experiment was performed two times with similar results; allocytes microinjected from one representative experiment are shown.C) Autoradiogram showing a semiquantitative RT-PCR assay of in-ompetent oocytes microinjected with constructs F, G, and H. Theelative decrease of Mos mRNA level in the second lane is about 80%fter normalization to the nontargeted Plat mRNA.

difficult (9). We failed to clone several different IRsti2a(nowwttm

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hat lacked a spacer; at least 36 colonies were screenedn each experiment. When we inserted a short spacer of0–50 bp we were successful. For example, in one case50-bp spacer increased the cloning efficiency to 25%

3/12 screened clones were positive). In addition, weoticed that antibiotic selection effected the efficiencyf isolating an inverted repeat in the pCRII plasmid;e had a higher success rate with ampicilin resistancehen compared to kanamycin (the pCRII plasmid con-

ains genes for both ampicilin and kanamycin resis-ance). The molecular basis for this observation re-ains unresolved.

nalysis of dsRNA with EGFP Single-StrandedOverhang

We initially tested if in vitro transcription of an IRielded detectable dsRNA and if attachment of theGFP cds to the hairpin interfered with formation of

he dsRNA in vitro and with RNAi in vivo. Treatmentf in vitro transcripts with RNase T1 revealed thatGFP single-stranded overhang did not interfere withos dsRNA formation in vitro in either the 39 or the 59

nd of the hairpin (Fig. 1B). Using our established Mosodel system (5), we then tested if there was any

ifference in efficiency of RNAi between “traditional”sRNA (annealed sense and antisense strands) andairpin dsRNA.Microinjection of “traditional” and hairpin Mos

sRNA molecules (106 molecules per oocyte) revealedhat the hairpin RNA (Fig. 1A, construct A) was ateast as effective as the traditional dsRNA in inhibitinghe maturation-associated increase in MAP kinase ac-ivity, i.e., in targeting the destruction of Mos mRNAFigs. 1C and 1D). In another set of experiments weompared the effect of “traditional” dsRNA, hairpinsRNA (Fig. 1A, construct A), and hairpin dsRNAsith 59 or 39 EGFP single-stranded overhangs (Fig. 1A,

onstructs B and C, respectively). We found that allour forms of dsRNA efficiently interfered with theeneration of MAP kinase activity (Fig. 1D) and whenll the experiments were analyzed, there was no sta-istically significant difference in the RNAi effect ofarious hairpin forms with respect to either MAP or H1inase activities (data not shown). It should be notedhat based on insensitivity to RNase T1 digestion, theresence of the EGFP single-strand RNA overhang inhe Mos dsRNA had no inhibitory effect on eithersRNA formation in vitro or on the RNAi effect.

xpression of EGFP and dsRNA from a Plasmid

We were further interested whether we could ex-ress dsRNA with the EGFP marker from the plasmidnd if this expression would produce green fluores-ence and a detectable RNAi effect. Expression of theairpin RNA was driven by either the CMV promoter

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he Mos inverted repeat was placed upstream or down-tream of EGFP (Fig. 2, constructs D and E). Theerminal vesicle of incompetent oocytes was injectedith constructs employing the ZP3 promoter andGFP fluorescence and Mos mRNA level were ana-

yzed 48 h later. Little, if any, fluorescence was de-ected when the IR was placed upstream of the EGFPds, whereas a low, but detectable, level of fluorescenceas detected when the IR was located downstream ofGFP (Fig. 2B). Similar results were obtained whenMV constructs were injected into 1-cell embryos or to

ncompetent oocytes (data not shown). RT-PCR analy-is revealed that the Mos mRNA was greatly reducedn oocytes microinjected with constructs expressing the

os hairpin dsRNA but the nontargeted Plat mRNAemained unchanged (Fig. 2C). The efficiency of theNAi-mediated destruction of the targeted mRNA was

ndependent of the position of the IR with respect toGFP cds. Thus, the expression from plasmids with

he EGFP cds placed upstream of Mos IR producedoth detectable levels of fluorescence, and efficient andpecific Mos mRNA degradation.Last, we analyzed the effect of inserting an intron

nto the expression cassette. We were concerned that ifGFP fluorescence wasn’t detected we would need aonvenient marker to detect expression from the plas-id and detecting the spliced RT-PCR product would

rovide such an alternative marker. Second, the long-erm objective was to develop these plasmids for aransgenic RNAi approach and there is a body of evi-ence that indicates that the presence of an intronnhances expression of a transgene. Therefore, we in-erted an SV40 intron downstream of the IR cloningite (Fig. 3A, construct H), which is where this intron isypically placed. The ability to detect the presence ofhe spliced RT-PCR product following injection of theifferent constructs demonstrated that splicing of thentron could also be used to monitor expression of theonstruct (data not shown). Nevertheless, placing thentron immediately downstream of the hairpin inter-ered with RNAi, since the mRNA level of the targeted

essage did not decrease (Fig. 3C). Therefore, weested two other plasmids in which the SV40 intronas inserted at different positions relative to the IR

Fig. 3A, constructs F and G). Inserting the SV40 in-ron adjacent to the IR did increase the level of fluo-escence when compared to the intron inserted up-tream of the EGFP (Fig. 3B). This placement adjacento the IR, however, inhibited RNAi, whereas when thentron was located upstream of EGFP RNAi was ob-erved, i.e., the destruction of the targeted mos mRNAFig. 3C). Semiquantitative RT-PCR revealed about0% decrease in mos mRNA level in incompetent oo-ytes microinjected with construct F (Fig. 3C). Basedn all of these results, we finally generated ZP3 andMV driven plasmids where the SV40 intron was in-

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ISCUSSION

The RNAi approach is very attractive for the analy-is of gene expression in mammalian systems and inhis report we describe the design and analysis of suchn approach based on plasmid-borne expression of annverted repeat and two markers, i.e., the presence ofreen fluorescence and a spliced RT-PCR product. Thistudy confirms some previous observations as well asrovides novel findings that should be taken into ac-ount when making similar constructs.

First, a spacer as short as 20–50 bp inserted betweenhe sense and antisense sequences greatly enhanceshe efficiency of IR cloning. It has been reported thatloning an inverted repeat is difficult (9), and one so-ution used in several RNAi studies in Drosophila andrabidopsis (10, 11, 20) is to insert a spacer thatanges from hundreds to thousands of base pairs. Al-hough others have suggested that the spacer sizeeeds to be in the range of hundreds of base pairs (20),ur data, which show that a spacer as short as 20–50p is sufficient for relatively high IR cloning efficiency25%), suggest that there is no minimal spacer size forncreased IR cloning efficiency.

Second, we were concerned about the effect of single-tranded overhangs on RNAi because it has been re-orted that longer 39 overhangs on dsRNA inhibitsNAi in vitro (8). In our model system, however, aingle-stranded RNA overhang at 39 and/or 59 end ofhe dsRNA loop does not block the RNAi effect in vivohen the RNA is either microinjected or expressed

rom a plasmid. The possibility that the overhang isroblematic in vivo is minimized by the observationhat transgenic RNAi (where single-stranded 39 over-angs do occur in vivo) works efficiently in severalodel organisms.Third, if EGFP is expressed as a single-stranded

verhang at the 59 end of the hairpin (upstream), aetectable amount of EGFP protein is translated (butignificantly lower when compared to non-IR controls).

FIG. 4. Final construct for insertion of an IR for RNAi experi-ents. The expression is driven by the ZP3 or CMV promoter. Re-

triction sites for cloning into both vectors (XbaI) and sites for re-oval of the expression cassette from the vector (BglII and AflII in

he ZP3-driven version and AseI and AflII in the CMV-driven ver-ion) are shown.

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tream) the fluorescence is extremely low or not detect-ble. A likely explanation for this observation is thathe EGFP located upstream can be translated for aimited period of time despite the processing of dsRNAnd subsequent degradation of the single-strandedGFP mRNA that would lack a poly(A) tail. In con-

rast, EGFP located downstream of the dsRNA hairpins not accessible to the translation machinery andsRNA processing leaves behind an uncapped single-tranded mRNA fragment that is rapidly degraded. Weried to circumvent this problem by introducing annternal ribosome entry site (IRES) between thesRNA hairpin and downstream EGFP cds using theIRES-EGFP2 plasmid (Clontech, CA). This approachailed as we observed that the IRES itself inhibitedGFP fluorescence following microinjection of the plas-id into the GV of either incompetent or fully-grown

ocytes, as well as in the male pronucleus of 1-cellmbryos (unpublished observations).Last, we note the presence of an intron enhances

xpression, as detected by fluorescence of the EGFPeporter. In the event that fluorescence is still very lowr undetectable, the presence of the spliced RT-PCRroduct can be used to detect the expression. In addi-ion, the presence of an intron enhances the level andtability of expression in transgenic mice (21). Ouresults suggest that the intron should not be placedirectly adjacent to the IR because this inhibits RNAmediated destruction of the targeted mRNA. Althoughhe basis for this inhibition is not known, the intronequence, perhaps by recruiting the splicing machin-ry, may interfere with the dsRNA formation. Ineffi-ient dsRNA formation would explain higher level ofuorescence in constructs with the intron located nexto the inverted repeat (Fig. 3B).

The plasmids described here can be used for severalifferent applications, e.g., microinjection, to studyene function in oocytes and preimplantation embryos.he most attractive use, however, will likely lie inransgenic technologies. In principle, the RNAi trans-enic approach using the oocyte-specific ZP3 transgenerovides a general tool to study gene function in theocyte and preimplantation embryo. The ZP3 promoters activated at the onset of oocyte growth and hence thexpressed hairpin RNA should direct the degradationf the targeted mRNA, which in turn should result inliminating or markedly reducing the size of the ma-ernal pool of the targeted protein. The presence ofaternally-derived protein in the oocytes in conven-

ional knockout animals, which typically are heterozy-ous, delays when a phenotypic change is first detectednd hence masks the time when the function of theene is first required. We believe that this approachas a high likelihood of success since a similar ap-roach was used successfully to drive the expression oftransgene encoding an antisense Plat RNA (22). The

expression of this antisense-Plat RNA was restricted tottpgs(lbtdrmaw

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he growing oocytes and decreased the level of theargeted Plat mRNA about 60–80%. Our proposed ap-roach differs from this proven model in that the trans-ene expresses a hairpin dsRNA, which, we havehown, is far more effective than the antisense RNA5). If expression of dsRNA in somatic tissues does notead to cell death, the transgenic RNAi approach coulde used to target genes in somatic tissues by usingissue-specific promoters. In the event that the dsRNAoes trigger cell death, the PKR-mediated apoptoticesponse to dsRNA could be avoided by using PKR 2/2ice (23). The transgenic RNAi would be a simple, fast

nd inexpensive method to analyze the gene functionhen compared to the current Cre-lox approach.In contrast to a gene knockout, RNAi may not totally

liminate the function of the targeted gene, i.e., itesults in a knockdown. This, in fact, can be an advan-age in certain cases because RNAi could generate aange of informative phenotypes that are missed whenhe “classical” gene knockout approach is used, sinceypomorph phenotypes frequently provide critical in-ights into the function of a gene. Moreover, this knock-own approach can unmask the existence of a thresh-ld level of gene function required to observe a mutanthenotype. For example, we demonstrated that a crit-cal amount of Mos activity appears required for theevelopment of MAP kinase activity (5), a result con-istent with a recently proposed switch mechanism forAP kinase activation, as well as other cellular

witches (24).

CKNOWLEDGMENTS

This research was supported by a grant from the NIH (HD 22681o R.M.S.), and portions of this work are being submitted by Petrvoboda in partial fulfillment of the Ph.D. requirements at theniversity of Pennsylvania. The authors thank Scott Poethig for use

f his fluorescence stereo-dissecting microscope.

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