Toward Improving Caenorhabditis elegans Phenome
Mapping With an ORFeome-Based RNAi Library
Jean-François Rual,1,2,6Julian Ceron,3,6John Koreth,4Tong Hao,1Anne-Sophie Nicot,1
Tomoko Hirozane-Kishikawa,1Jean Vandenhaute,2Stuart H. Orkin,5David E. Hill,1
Sander van den Heuvel,3,7and Marc Vidal1,7
1Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute and Department of
Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA;2Faculte ´s Universitaires Notre-Dame de la Paix, 5000
Namur, Belgium;3Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, Massachusetts 02129;
USA;4Department of Medical Oncology,5Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical
School, Boston, Massachusetts 02115, USA
The recently completed Caenorhabditis elegans genome sequence allows application of high-throughput (HT) approaches
for phenotypic analyses using RNA interference (RNAi). As large phenotypic data sets become available,
“phenoclustering” strategies can be used to begin understanding the complex molecular networks involved in
development and other biological processes. The current HT-RNAi resources represent a great asset for phenotypic
profiling but are limited by lack of flexibility. For instance, existing resources do not take advantage of the latest
improvements in RNAi technology, such as inducible hairpin RNAi. Here we show that a C. elegans ORFeome
resource, generated with the Gateway cloning system, can be used as a starting point to generate alternative
HT-RNAi resources with enhanced flexibility. The versatility inherent to the Gateway system suggests that additional
HT-RNAi libraries can now be readily generated to perform gene knockdowns under various conditions, increasing
the possibilities for phenome mapping in C. elegans.
[Supplemental material is available online at www.genome.org.]
Thirty years of classical genetics in Caenorhabditis elegans identi-
fied and characterized ∼800 genes (∼4% of all genes; Brenner
1974; Jorgensen and Mango 2002). With the availability of the
worm genome sequence (The C. elegans Sequencing Consortium
1998), it is now possible to apply genome-wide reverse genetic
approaches for functional characterization. One way to uncover
the functions of genes is to study their loss-of-function pheno-
types after deletion (or gene knockout). Given the lack of ho-
mologous recombination in C. elegans, gene deletions are not
easily amenable to high-throughput (HT) generation, although
efforts are currently underway to achieve this goal. Instead, RNA
interference (RNAi; Fire et al. 1998), which relies on the intro-
duction of dsRNA in the worm, readily allows genome-wide loss-
of-function screens by gene “knockdowns” (Fraser et al. 2000;
Gonczy et al. 2000; Piano et al. 2000, 2002; Hanazawa et al. 2001;
Maeda et al. 2001; Zipperlen et al. 2001; Ashrafi et al. 2003; Ka-
math et al. 2003; Lee et al. 2003; Pothof et al. 2003; Simmer et al.
2003; Vastenhouw et al. 2003).
An RNAi-by-feeding (Timmons and Fire 1998) library was
generated by using PCR-amplified genomic DNA fragments as a
template (Kamath et al. 2003). This resource targets ∼78% of the
19,920 genes currently predicted in Worm Sequence 112 (WS112;
WormBase, http://www.wormbase.org; see Methods for calcula-
tions; Kamath et al. 2003; Harris et al. 2004). This library has
been successfully used for several genome-wide RNAi screens, for
example, in wild-type (WT) N2 animals for systemic functional
analysis (Kamath et al. 2003) and for the analysis of genes in-
volved in the regulation of body fat storage (Ashrafi et al. 2003),
as well as in the hypersensitive rrf-3 C. elegans strain (Simmer
et al. 2003). These data represent a significant step forward in the
phenotypic characterization of most C. elegans genes.
Ultimately, the combination of loss-of-function data ob-
tained in numerous, different conditions could lead to the clas-
sification of worm genes by “phenotypic signatures.” Phenoclus-
tering methods have been applied on a moderate scale in
C. elegans (Boulton et al. 2002; Piano et al. 2002) and in Dro-
sophila (Boutros et al. 2004). The C. elegans “phenome” map re-
sulting from such genome-wide analyses would increase our un-
derstanding of a variety of biological processes.
However, several technical points need to be resolved before
a comprehensive phenome map can be generated for C. elegans.
First, such a map relies upon the generation of RNAi data for all
genes. Today, not all worm genes are yet targeted by an RNAi
clone. Second, RNAi screens should be performed in different
environmental conditions or various genetic backgrounds, ap-
plying different procedures. For example, experiments leading to
the loss-of-function of two genes, simultaneously, can be carried
out in the worm (Simmer et al. 2003; Tewari et al. 2004). Fur-
thermore, RNAi can be applied by different methods of delivery,
such as injection (Fire et al. 1998), feeding (Timmons and Fire
1998), or soaking (Tabara et al. 1998), at different times during
the development of the worm such as different larval stages, and
with different DNA templates for the synthesis of dsRNA, such as
genomic DNA fragments, cDNAs, or ORFs. Finally, a new RNAi
technology, referred to as inducible hairpin RNAi (Tavernarakis
et al. 2000; N.M. Johnson, C.A. Behm, and S.C. Trowell, unpubl.),
opens new possibilities for targeting genes. Indeed, genes can be
knocked-down at specific times during the development and spe-
cific cell types such as neurons can now be targeted. Other im-
proved technologies for loss-of-function assays will likely be de-
6These authors contributed equally to this work.
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veloped soon. To accommodate these numerous applications,
RNAi resources should thus be generated by using cloning tools
that are as flexible as possible (Fig. 1).
In the infancy of the genomic era (Collins et al. 2003), re-
sources need to be dynamic and flexible. Ideally, a single, com-
prehensive set of cloned ORFs, that is, an “ORFeome” (Walhout
et al. 2000b; Reboul et al. 2003; Rual et al. 2004), should be usable
for various HT applications. An ORFeome resource should be
updated (ORFs corrected, removed, or added) regularly as new
genome annotations become available. An ORFeome should be
cloned in a format that allows facile transfer of the ORFs to any
desired vector as new technologies become available (Fig. 1). Re-
cently developed recombinational cloning technologies (Liu
et al. 1998; Hartley et al. 2000; Walhout et al. 2000a; Zhang et al.
2002) allow such versatile cloning approaches at HT (Reboul et al.
2003; Dricot et al. 2004).
Upon the release of the C. elegans genome sequence (The
C. elegans Sequencing Consortium 1998), a genome-wide ap-
proach was initiated to clone all predicted ORFs of the worm
ORFeome (Walhout et al. 2000b; Reboul et al. 2001, 2003; Vaglio
et al. 2003; WorfDB, http://worfdb.dfci.harvard.edu) using the
Gateway recombinational cloning system (Hartley et al. 2000;
Walhout et al. 2000a). Version 1.1 of the C. elegans ORFeome
resource contains 11,942 cloned ORFs (Reboul et al. 2003) and
constitutes a platform for proteomic and functional genomic ap-
proaches (Reboul et al. 2003; Rual et al. 2004). The C. elegans
ORFeome resource is dynamic, with new ORFs added or corrected
continuously (Lamesch et al. 2004). Phenome mapping, as well
as other “omic” approaches, should benefit from the flexibility of
By using the C. elegans ORFeome v1.1 resource (Reboul et al.
2003), we began generating a companion resource for RNAi
screening. Herein we describe this ORFeome-RNAi v1.1 library, as
well as the results from using this library for a genome-wide
RNAi-by-feeding screen of WT C. elegans hermaphrodites. We
conclude that the use of an ORFeome approach is feasible for the
generation of an alternative RNAi library, and we suggest that
this cloning strategy could be applied to other functional
Generation of the ORFeome-RNAi v1.1 Library
The C. elegans ORFeome v1.1 library contains 11,942 ORFs
cloned as Gateway Entry clones, comprising 10,623 ORFs cloned
“in frame” plus 1319 ORFs cloned “out of frame” (Reboul et al.
2003). ORFs were cloned out of frame because of mispredictions
of their ATG or STOP codons. Only the in-frame ORFs can be
used for protein expression, but both sets of clones can be used
for RNAi. HT-recombinational cloning protocols were used to
transfer ORFs from the pDONR201 Entry plasmid into the
pL4440-dest-RNAi Destination vector by using the LR Gateway
reaction (see Methods). In all, 11,804 RNAi clones were gen-
erated (Supplemental Table A). These clones were archived as
glycerol stocks of transformed Escherichia coli strain HT115(DE3)
used in RNAi-by-feeding and as miniprep plasmid DNAs (Tim-
mons and Fire 1998). These DNA preps can be used as templates
for in vitro dsRNA synthesis before RNAi application by soaking
or injection. The ORFeome-RNAi v1.1 library is available to the
community upon request (Open Biosystems, http://www.
openbiosystems.com; MRC geneservice, http://www.rfcgr.
mrc.ac.uk/geneservice; WorfDB, http://worfdb.dfci.harvard.edu).
Bioinformatic analysis revealed that the ORFeome-RNAi
v1.1 library contains 11,511 RNAi clones, each expected to target
a single gene (see Methods; Supplemental Table A). Several of
these RNAi clones target the same genes, so the RNAi library in
theory can knockdown the expression of 10,953 (∼55%) of the
19,920 unique protein-encoding genes predicted in WS112
(Supplemental Table B). Of these 10,953 genes, 485 are targeted
by two or more RNAi clones. There are 1736 genes, not previ-
ously targeted by other existing RNAi-by-feeding libraries, that
now have a clone available in the ORFeome-RNAi v1.1 library
(Supplemental Table B). This represents an increase of ∼9% in the
total number of genes that can be targeted by RNAi-by-feeding.
In combination with the previous RNAi-by-feeding library (Ka-
math et al. 2003) a total of 17,201 predicted worm genes (∼86%)
can now be targeted by an RNAi-by-feeding clone.
Genome-Wide Phenotypic Analysis
Using the ORFeome-RNAi v1.1 Library
We first used the ORFeome-RNAi v1.1 library for a synthetic le-
thal screen by conducting a genome-wide RNAi screen simulta-
neously in WT N2 C. elegans hermaphrodites and in a lin-35/Rb
mutant background (see Methods; Fig. 2). The results in the N2
animals are presented here, and the results of the lin-35/Rb syn-
thetic phenotype screen will be reported elsewhere (J. Ceron, J.-F.
Rual, M. Vidal, and S. van den Heuvel, unpubl.). Worms were
subjected to RNAi-by-feeding at the first larval stage (L1). We
identified a variety of phenotypic defects, including lethality and
abnormal body morphology, occurring during the embryonic,
larval, and adult stages.
The results from this phenotypic analysis of 10,953 WS112
predicted genes are described in Table 1 and in Supplemental
Table B. We observed phenotypes different from WT for 1066
WS112 predicted genes, representing ∼10% of the total ORFs
tested. This percentage is similar to that obtained in previous
genome-wide RNAi-by-feeding screens (Ka-
math et al. 2003; Simmer et al. 2003).
We observed an RNAi phenotype for
220 WS112 predicted genes for which no
phenotype was previously reported in
WormBase (Supplemental Table B). For 79
of these 220 genes, no RNAi assay was listed
in WormBase. The other 141 genes, targeted
in previous RNAi screens, had showed a WT
phenotype before. Altogether, our results
increase the total number of genes associ-
ated with any RNAi phenotype by ∼7%,
from 3259 to 3479. Finally, an additional
1145 genes for which no RNAi assay was
reported in WormBase did not show any de-
tectable phenotype and were scored as WT
in our screen.
tions. AntR1 and AntR2 refer to antibiotic resistance markers number 1 and 2.
Generating RNAi resources from flexible Gateway ORFeome and promoterome collec-
C. elegans ORFeome1.1-RNAi Library
A global analysis of the data verifies observations made pre-
viously regarding the distributions of viable postembryonic phe-
notypes (Vpep) and nonviable phenotypes (Nonv) ascribed to
various genes located on either the autosomes or the X chromo-
some (Piano et al. 2000; Maeda et al. 2001; Kamath et al. 2003).
In particular, there appears to be a low incidence of genes located
on the X chromosome that are associated with any phenotype
Gene inactivation by RNAi-by-feeding is often incomplete
and may be too limited to cause fully penetrant loss-of-function
phenotypes, thereby generating false-negative results. For ex-
ample, RNAi is less effective in neurons (Tavernarakis et al. 2000),
and genes encoding proteins with long half-lives are more resis-
tant to RNAi (Hannon 2002). Previous genome-wide RNAi
screens had various rates of false negatives (Kamath et al. 2003;
Simmer et al. 2003), based on comparison of the RNAi results to
the loss-of-function phenotypes caused by genetic mutations.
Nearly 45% of the genes with a reported mutant phenotype did
not show any phenotype in each of these previous studies. More
precisely, rates of ∼25% and ∼55% of false negatives were ob-
served for Nonv mutant phenotypes and for Vpep mutant phe-
notypes, respectively. Matching our RNAi data to the phenotypes
described for 269 genetic mutants (resembling the control set of
genetic mutants used in Kamath et al.  and Simmer et al.
), we observed an overall false-negative rate of ∼59% (∼43%
for Nonv mutant phenotypes and ∼83% for Vpep mutant phe-
notypes; Supplemental Table C). This somewhat higher fre-
quency of false negatives can be explained mostly by our HT
RNAi protocol, in which animals were followed only for one
generation and not transferred to a second RNAi plate (see Dis-
Low rates of false positives were previously described in ge-
nome-wide RNAi-by-feeding screen (Kamath et al. 2003; Simmer
et al. 2003). In these studies, a false positive was scored when the
RNAi phenotype was lethal, whereas the reported genetic mutant
shows a nonlethal phenotype. Published
false-positive rates vary from ∼0% to 0.4%
(Kamath et al. 2003; Simmer et al. 2003). By
applying the same strategy to a similar set
of 103 loci, we counted four Nonv pheno-
types in our RNAi experiment (Supplemen-
tal Table D). After sequence verification, we
found one obvious cross-contamination.
The remaining three clones have the cor-
rect sequence. Two of the three correspond-
ing genes are reported as Nonv by RNAi in
WormBase. Overall, our rate of false-
positive results is low and in part can be due
The ORFeome-RNAi v1.1 library contains
430 pairs in which both clones target the
same gene and should give rise to the same
phenotype. These clones were processed in-
dependently and therefore constitute a
blind control for the reproducibility of scor-
ing. In 78% (337/430), we obtained a WT
phenotype for both clones of the pair. In
10% (45/430), we observed a phenotype for
both clones. Thus, the results are in agree-
ment regarding the scoring of WT versus
phenotype for ∼89% of the pairs of clones
(382/430; Supplemental Table E). In ∼11%
(48/430), there is disagreement. Variability
in the results from repeated RNAi-by-feed-
ing assays was observed previously (Simmer et al. 2003). Variabil-
ity ranges from ∼10% to 30% between experiments and is likely
due to lack of reproducibility from one RNAi assay to another
(Simmer et al. 2003). To verify this phenomenon, we performed
a second RNAi assay for the 48 pairs of RNAi clones in disagree-
ment. In the second assay, we observed similar phenotypes for
both clones ∼52% of the time (25/48; Supplemental Table E).
Thirteen pairs of clones now gave rise to a WT phenotype for
both clones. Ten pairs of clones were still not in agreement; their
correct identities were verified by DNA sequence analysis.
Interestingly, of the 10 pairs of clones in disagreement in
the second RNAi assay, six showed large differences in size of the
cloned ORF. For these six pairs, the longer ORF clone was the one
giving rise to the observed phenotype. These numbers are not
statistically significant but suggest that effectiveness of RNAi-by-
feeding may be somewhat dependent on the length of the clone,
the longer clones being more effective in RNAi. We went on to
perform RNAi assay by injection for three of these six pairs of
clones and obtained a phenotype in all six (three by two) RNAi-
by-injection assays. Thus, the output of an RNAi assay depends
on the method of delivery of dsRNA.
The generation of complete resources of cloned ORFs represents
an important and challenging step in the development of pro-
teomics and functional genomics (Reboul et al. 2003; Rual et al.
2004). Within months after the generation of the C. elegans
ORFeome v1.1 (Reboul et al. 2003), we transferred >11,000 ORFs
into the pL4440-dest RNAi vector, generating a new RNAi re-
source. The technology that allowed this genome-wide cloning
effort is now being used for the production of additional re-
sources, such as Activation Domain and DNA Binding Domain
fusion collections for yeast two-hybrid screens.
The C. elegans ORFeome-RNAi v1.1 library contains 11,511
are inoculated in sixteen 24-wells plates (six clones per plate and four wells per clone). After
overnight induction of the dsRNA synthesis, three to 10 worms synchronized at the L1-stage
(N2 or lin-35 strain) were deposited into the wells. (B) RNAi experiments were performed at 20°C.
We observed a wide range of phenotypes across development.
Overview of the RNAi screening procedure. (A) Overnight cultures of 96 bacterial clones
Rual et al.
2164 Genome Research
Summary of RNAi Data
Kamath et al. (2003)
Viable phenotypes only
Kamath et al. (2003)
Kamath et al. (2003)
Kamath et al. (2003)
Viable phenotypes only
Kamath et al. (2003)
Kamath et al. (2003)
Kamath et al. (2003)
Viable phenotypes only
aThe total number of predicted genes in WS112 is 19,920; 2850 on chromosome I; 3457 on chromosome II; 2639 on chromosome III; 3242 on chromosome IV; 4970 on chromosome V; and 2762
on chromosome X.
bThe total number of predicted genes in WS61 is 19,427; the number of predicted genes for each chromosome could not be found; ND means not determined.
C. elegans ORFeome1.1-RNAi Library
RNAi clones, each expected to target a single gene (Supplemental
Table A), and includes 1736 C. elegans genes not targeted previ-
ously by any RNAi-by-feeding clones (Supplemental Table B). We
used the C. elegans ORFeome-RNAi v1.1 library to perform a ge-
nome-wide RNAi screen in WT C. elegans hermaphrodites (Fig. 2).
We observed a phenotype for ∼10% of the WS112 genes studied
(Supplemental Table A; Table 1). Our RNAi results increase by 220
the number of genes for which an RNAi phenotype has been
described. We confirmed phenotypes for numerous genes.
As previously observed (Simmer et al. 2003), we found that
the RNAi effect varies from one RNAi-by-feeding screen to an-
other. This lack of reproducibility is likely due to the inability to
control the amount of dsRNA animals are exposed to, resulting in
a high rate of false negatives. We also suggest that the RNAi effect
could depend on the method of dsRNA delivery and on the size
of the RNAi clone, with longer clones generally more effective, as
described previously for RNAi-by-injection (Parrish et al. 2000).
As for most large-scale approaches, results obtained from ge-
nome-wide RNAi screens should be interpreted carefully, espe-
cially negative results. Several issues need to be addressed before
a comprehensive phenome map of C. elegans can be generated,
for example, by generating genome-wide RNAi data in different
conditions and by developing new RNAi protocols. In this regard,
ORFeome-based RNAi resources should help improve phenome
mapping by taking advantage of the versatility of recombina-
tional cloning to apply new RNAi technologies to genome scale
Cloned ORFs versus cloned genomic DNA fragments, as
template DNA could improve the efficiency of RNAi. Cloned
ORFs are free of introns and thus provide more template for
siRNAs. Moreover, a study based on the RNAi results obtained
with the genomic RNAi library (Kamath et al. 2003) showed a
lower incidence of RNAi phenotype among loci with a small
portion of coding sequence and large introns. This observation
suggested that genomic DNA as RNAi template reduces RNAi ef-
ficiency (Cutter et al. 2003). Nevertheless, our proportion of hits
(Table 1) and rate of false-negative results are comparable to pre-
vious results (Kamath et al. 2003; Simmer et al. 2003). Further-
more, an analysis using the current version of the genome an-
notation demonstrates that the fraction of coding gene sequence
exerts a weaker negative effect than initially reported (A.D. Cut-
ter, pers. comm.). Overall, it seems that the presence of introns in
the DNA template does not have a dramatic negative impact on
the efficiency of RNAi.
In our screen, animals were exposed to RNAi-by-feeding dur-
ing the L1 stage (Fig. 2), as opposed to the L4 stage in previous
screens (Kamath et al. 2003; Simmer et al. 2003). Consequently,
we observed phenotypes linked to sterility (sterile, sterile prog-
eny, and reduced brood size) for a larger number of genes (∼5.5%
of those analyzed) than reported before (∼2.8%; Table 1; Kamath
et al. 2003). On the other hand, Kamath et al. (2003) observed an
embryonic lethal phenotype (Emb) for ∼5.5% of assayed genes,
whereas we observed 3.1%. This difference might arise because
many genes are essential for both the development of the adult
germ line and the embryos. For these genes, scoring Emb versus
sterile phenotypes could depend on the time of initiation of the
RNAi assay, larval stage L1 versus L4.
Several factors are likely to contribute to a reduced fre-
quency of phenotypes as well as to phenotypes that differ from
those listed in WormBase for a given gene. First, we used a dif-
ferent RNAi protocol, and the criteria for scoring phenotypes
were slightly more stringent than those used previously. To in-
crease the throughput of the screening, we initiated the screen-
ing on L1 stage worms, we followed the animals for one genera-
tion and did not transfer them to a second RNAi plate. Pheno-
types occurring late in the screen were difficult to score because
food was limited. Subtle phenotypes may have been overlooked
because we were scoring for visually obvious phenotypes.
Despite the higher rate of false negatives, we observe a com-
parable rate, ∼10%, of hits for all genes targeted (Table 1). This is
probably due to higher representation in the ORFeome v1.1 re-
source of genes that can give rise to a phenotype. Highly ex-
pressed genes that are more represented in the ORFeome1.1 re-
source (Reboul et al. 2003) have more chance to be essential
(Cutter et al. 2003).
The C. elegans ORFeome v1.1 and ORFeome-RNAi v1.1 li-
braries represent ∼55% of the predicted worm ORFeome. Impor-
tantly, these collections should be considered as dynamic re-
sources. New Gateway Entry clones, generated in the ongoing
ORFeome project (Lamesch et al. 2004), will be subsequently
transferred into the RNAi vector in the near future. If a new
improved RNAi vector is made available to the community, the
Gateway cloning system will allow quick transfer of all cloned
ORFs into it. For example, a hairpin RNAi method (Tavernarakis
et al. 2000) was developed by using the ORFeome v1.1 resource
(Reboul et al. 2003) to generate hairpin RNAi constructs in the
pWormgate system (N.M. Johnson, C.A. Behm, and S.C. Trowell,
unpubl.). In addition, such hairpin constructs could be fused to
any specific promoter cloned by MultiSite Gateway (Fig. 1; Du-
puy et al. 2004).
The complete ORFeome-RNAi v1.1 library (Supplemental
Table A) is available upon request from Open Biosystems (http://
www.openbiosystems.com) and MRC geneservice (http://www.
rfcgr.mrc.ac.uk/geneservice; WorfDB, http://worfdb.dfci.
harvard.edu). The data generated in the WT RNAi screen (Supple-
mental Table B) will be available in WormBase shortly (http://
www.wormbase.org; Harris et al. 2004) and in RNAiDB (http://
nematoda.bio.nyu.edu; Gunsalus et al. 2004). Integrated with
other functional genomic data, these RNAi resources should con-
tribute to a deeper understanding of the biology of C. elegans.
In summary, we demonstrated the value of an ORFeome-
based approach for the generation of alternative HT-RNAi re-
sources and ultimately, for the improvement of phenome map-
ping in C. elegans.
Generation of the ORFeome-RNAi v1.1 Library
ORFs were transferred into pL4440-dest-RNAi, a Gateway-com-
patible RNAi vector adapted from the original pL4440-RNAi vec-
tor (Timmons and Fire 1998). Gateway recombinational cloning
reactions were performed as described by the manufacturer (In-
vitrogen) with minor changes. The pL4440-dest-RNAi vector was
digested with EcoRI and NcoI before the LR reaction, which sig-
nificantly improved (>10-fold) the efficiency of the LR reaction.
LR reaction plates were incubated overnight at 25°C. By using 96
well plates and HT-liquid handling systems, up to 1500 LR reac-
tions were carried out by one person per day.
It is not possible to transform LR products directly into the
RNAi-by-feeding bacterial strain HT115 (DE3), an RNase III–
deficient strain of E. coli. Indeed, HT115 (DE3) bacteria are resis-
tant to the ccdB toxic gene used in the Gateway cloning system.
Therefore, LR products were first transformed into E. coli DH5?
strain. Bacterial transformations were performed with 100 µg/mL
ampicillin selection in liquid cultures. Plasmid DNA minipreps
were prepared in a 96-well format for each pool of transformants
by using a Qiagen 9600 Robot. DNA preparations were subse-
quently transformed into the RNAi-by-feeding bacterial strain
HT115 (DE3). Out of the 11,942 ORFs cloned as Entry clones in
the C. elegans ORFeome v1.1 resource, we obtained HT115 (DE3)
transformants for 11,804 ORFs (Supplemental Table A).
All the Entry clones used in the LR reaction were verified by
DNA sequencing analysis (Reboul et al. 2003). We also verified
Rual et al.
2166 Genome Research
the identity of 96 RNAi clones picked at random from the library
by sequencing. The universal primers used for PCR amplification
and sequencing are pL4440-dest-RNAi-FOR (5?GTTTTCCCAGT
CACGACGTT3?) and pL4440-dest-RNAi-REV (5?TGGATAACCG
TATTACCGCC3?). We observed the expected sequences in 96%
of the time. Three of the clones contained the wrong ORFs, and
one clone did not contain any insert. We conclude that a tiny
portion of the phenotypes may have been attributed to the
wrong genes due to cross-contamination. The identity of the 220
RNAi clones corresponding to genes for which no phenotype was
previously reported in WormBase has been verified by DNA se-
For the C. elegans genome sequence and related genome annota-
tion, we used version WS112 available at WormBase (http://
www.wormbase.org; Harris et al. 2004). There are 19,920 protein
encoding predicted genes in WS112 (Supplemental Table B).
To determine which WS112 genes are targeted by the
ORFeome-RNAi v1.1 clones, we first retrieved the sequences from
the primer pairs used in the ORFeome project (Reboul et al. 2003;
Vaglio et al. 2003) and used these sequences to perform BLAST
analysis against version WS112 of the worm genome. For each
RNAi clone, the resulting position was then queried for an over-
lap of >100 nucleotides with the transcript(s) (UTRs plus exonic
sequences) of one or more predicted WS112 genes.
With this strategy, we determined that there are 153 RNAi
clones that target more than one WS112 predicted gene (Supple-
mental Table A). Furthermore, there are 140 RNAi clones that do
not target any WS112 predicted genes (Supplemental Table A).
These results are somewhat surprising because the cloned ORFs
were PCR amplified from a worm cDNA library (Reboul et al.
2003). The information related to these cloned ORFs should be
corrected in forthcoming releases from WormBase. The results
obtained with these 293 RNAi clones were removed from our
analysis. We found 10,953 WS112 genes targeted by the collec-
tion of 11,511 remaining RNAi clones. Out of the 10,953 genes,
485 were targeted by more than one RNAi clone. Given the low
rate of false-positive results and the high rate of false-negative
results in RNAi screens (see Results; Kamath et al. 2003; Simmer
et al. 2003), all the phenotypes different from WT were scored
by default for those 485 genes in the Supplemental Tables B, C,
The data related to the previous RNAi library (Kamath et al.
2003) were retrieved from WormBase WS112 (WormBase, http://
www.wormbase.org; Harris et al. 2004). In WS112, there are 16,537
RNAi clones that target 15,465 WS112 genes. However, 16,757
RNAi clones were described as targeting 16,757 WS61 genes (Ka-
math et al. 2003). The discrepancy is probably caused by altered
gene predictions between WS61 and WS112, and missing data for
220 RNAi clones in WormBase WS112.
We performed the RNAi screen simultaneously in WT N2 C. el-
egans hermaphrodites and in a lin-35/Rb mutant background
(Fig. 2). Standard methods were used for culturing worms on
nematode growth medium (NGM; Brenner 1974). Screening was
performed in 24-well format plates containing NGM supple-
mented with 50 µg/mL ampicillin, 12.5 µg/mL tetracycline, and
1.43 mg/mL IPTG. Unseeded feeding plates were kept in the dark
for less than a week at 4°C. Before bacterial inoculation, the feed-
ing plates were dried for 2 to 3 h in a laminar airflow hood or in
a 37°C incubator. RNAi bacterial clones were grown in 96-well
format for 24 h at 37°C in 600 µL LB with 50 µg/mL carbenicillin
and 12.5 µg/mL tetracycline. Each well of the 24-well format
plates was inoculated with 115 µL of bacterial culture by using
an extendable automated multichannel pipette. The expression
of dsRNA was induced overnight in the presence of IPTG (1.43
mg/mL), at room temperature and in the dark. After dsRNA in-
duction, three to 10 worms synchronized in the L1-stage were
deposited into the wells (Fig. 2).
Most of the previous RNAi studies were done by initiating
RNAi on young adults (Fraser et al. 2000; Gonczy et al. 2000;
Piano et al. 2000, 2002; Maeda et al. 2001; Zipperlen et al. 2001;
Kamath et al. 2003; Simmer et al. 2003). Instead, we fed worms at
the L1 larval stage (Fig. 2). This procedure necessitates fewer
worm manipulations and thus allows a higher throughput. A
disadvantage of this method is food limitation in the F1 genera-
tion. This might explain the high rate of false-negative results in
our assay, particularly for Vpep phenotypes.
Phenotypes were scored visually twice on days 4 and 5 of the
20°C culture. We looked for 18 different Vpep phenotypes: Lva
(larval arrest), Unc (uncoordinated), Prl (paralyzed), Dpy
(dumpy), Bmd (body morphology defect), Sck (sick), Bli (blister),
Mlt (molting defect), Slm (slim), Him (high incidence of male),
Pvl (protruding vulva), Muv (multivulva), Clr (clear), Lon (long),
Sma (small), Gro (growth defect), Egl (egg laying defect), and Soc
(social). We looked for eight different Nonv phenotypes: Ste
(sterile), Rbs (reduced brood size), Let (Lethal), Emb (embryonic
lethal), Ooc (oocytes), Stp (sterile progeny), Rup (ruptured), and
Lvl (larval lethal). When the phenotype occurs in the F1 genera-
tion, F1 is added before the phenotype name (e.g., F1 Lva). Emb
was defined as >20% dead embryos. Phenotypes observed with
partial penetrance are indicated with a “p”, for example, pEmb
for partially penetrant embryonic lethality. A partial phenotype
is scored when the phenotype occurs in <90% of the P0or F1
We thank the C. elegans Sequencing Consortium for the genome
sequence; members of WormBase for discussions and for making
RNAi data available; the participants of the ORFeome meeting for
discussions; the members of the Plasterk laboratory, and F. Sim-
mer in particular; the Ahringer laboratory, and R. Kamath in
particular; the Piano laboratory, and A. Cutter and G. Achaz for
sharing data and discussions; N. Bertin, V. Goidts, and N. Li for
technical assistance; C. Fraughton for laboratory support; T.
Clingingsmith for administrative assistance; M. Cusick for criti-
cal reading of the manuscript; and all the members of the van
den Heuvel and Vidal laboratories, and D. Dupuy and the ORFe-
ome team in particular. This work was supported by National
Institutes of Health grant CA95281 (S.v.d.H.) and National Can-
cer Institute grant 7 R33 CA81658-02 (M.V.). J.C was the recipi-
ent of a Postdoctoral Fellowship from the Secretaría de Estado de
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WEB SITE REFERENCES
http://www.wormbase.org; Source for C. elegans genome and RNAi data.
http://worfdb.dfci.harvard.edu; Source for C. elegans ORFeome data.
http://www.openbiosystems.com; Source for request of ORFeome-RNAi
http://nematoda.bio.nyu.edu; Source for C. elegans RNAi data.
http://www.rfcgr.mrc.ac.uk/geneservice; Source for request of ORFeome-
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Received February 24, 2004; accepted in revised form May 4, 2004.
Rual et al.
2168 Genome Research