mos1 alleles were selected by visual screening in wormbase ( www.wormbase.org ) for appropriately located transposon insertions and provided by the nemagenetag consortium .
mos1 alleles were identified in wormbase ( www.wormbase.org ) and requested from the nemagenetag consortium ( http://elegans.imbb.forth.gr/nemagenetag/ ) .
the presence of the mos1 insertion was verified with gene - specific primers , annealing inside and outside of the mos1 element .
the mos1 element was crossed into the unc-119(ed3 ) background to make an injection strain and verified by pcr analysis .
targeting constructs were designed to contain ( 1 ) a right homology region ( adjacent to the mos1 element , ( 2 ) a left homology region ( distant from the mos1 element ) and ( 3 ) a c. briggsae - unc-119(+ ) rescue region .
right homology regions were comprised of approximately 2.0 kb of homologous dna immediately adjacent to the mos1 insertion site .
left homology regions were comprised of 2 to 3 kb of homologous dna , which specifies the endpoint of the targeted deletion .
these regions were selected to avoid repetitive dna sequences , in particular short inverted repeats , which are likely to anneal and reduce the frequency of correct deletions .
a c. briggsae unc-119(+ ) rescue fragment was chosen because it is smaller than the c. elegans unc-119 gene .
an injection mix was made that contained the targeting plasmid ( 50 ng / ul ) , mos1 transposase helper plasmid pjl43.114 ( 50 ng / ul ) , mcherry co - injection markers pgh810 ( prab-3::mcherry , 10 ng / ul ) , pcfj90 ( pmyo-2::mcherry , 2.5 ng / ul ) , and pcfj10410 ( pmyo-3::mcherry , 5 ng / ul ) . the injection strain was maintained at 15c on hb101 bacteria .
young adult animals were mounted on an agarose pad under halocarbon oil and injected following standard techniques .
injected worms were left to recover at 15c for a couple of hours and then transferred three at a time to hb101 or op50 seeded ngm plates and placed at 25c . in our hands , approximately 70 % of all injected worms resulted in transgenic progeny . we found that growing worms on hb101 bacteria at 15c significantly improved the health of unc-119 animals . after approximately 7 days each plate was screened for deletion mutants .
screening was greatly facilitated by allowing the plate to starve out completely because unc-119 animals can not form dauers and are therefore selected against .
strains with an unc-119(+ ) insertion were identified on a fluorescence dissection microscope as l1 animals that move like wild - type animals but have none of the fluorescent co - injection markers .
a single rescued , non - fluorescent animal was picked to a new plate and allowed to propagate for one generation . in the case of obvious phenotypes ( for example , dpy-13 ) a single mutant animal was picked from the progeny ; in cases where the phenotype was wild - type ten animals were picked to individual plates and tested for homozygosity .
it takes approximately two weeks from injecting the strain to recovering a homozygous deletion animal .
we calculated the distance of every protein coding gene in the ws205 referential release of wormbase ( http://ws205.wormbase.org15 ) to all current mos1 insertion alleles using a state machine algorithm written in perl .
the closest mos1 element was defined as the distance from the insertion site to the atg start codon .
the number of mos1 elements in the vicinity of each gene was determined by extracting a sequence segment upstream and downstream of the atg and summing the number of elements contained within that span .
the number of intervening genes between a given gene and its nearest mos1 element was determined by extracting the segment ranging from the atg to the insertion site and tallying the number of genes present , including genes that partially reside within the interval .
the analysis was repeated against all genes lacking a deletion allele from either the c. elegans gene knockout consortium in the u.s . and
canada ( ok alleles ) and the national bioresource project in japan ( tm alleles ) .
genomic dna from worms was isolated with the gentra puregene tissue kit ( qiagen ) following the manufacturer s supplementary protocol :  purification of archive - quality dna from nematodes or nematode suspensions using the gentra puregene tissue kit  .
dna labeling , sample hybridization , image acquisition , and determination of fluorescence were all performed as previously described12 .
we used a 3x high - density ( hd ) chip divided into three 720 k whole genome sections for all experiments .
all microarrays were manufactured by roche - nimblegen with oligonucleotides synthesized at random positions on the arrays .
for all experiments , normalization of intensity ratios were performed with a loess regression as previously described12 .
three strains , eg5810 ( 25 kb deletion ) , eg5620 ( 15 kb ) and eg5621 ( 15 kb ) , were tested against wild - type dna .
all samples also showed two identical untargeted deletions : an approximately 8 kb deletion of pgp-6 and pgp-7 on chromosome x and an approximately 4 kb deletion of the telomeric region ctelx3.1 at the left end of chromosome v. we verified by cgh that these deletions were present in the parent strain ( eg5003 ) and the deletions therefore do not represent second site mutations caused by the mosdel technique .
targeting vectors typically consist of three distinct fragments : a  right homology region , a positive selection marker ( cb - unc-119(+ ) ) and a  left homology region . in some cases
the positive selection marker was flanked by the fluorescent marker punc-122::gfp , which is dimly expressed in the coelomocytes .
all targeting vectors were made using the multisite gateway three - fragment vector construction kit ( invitrogen , catalog no .
12537 - 023 ) . to verify deletions by pcr we designed oligos that would amplify short genomic dna fragments inside and outside the targeted regions .
these reactions were reproducible and robust ; pcr amplification was successful on crude genomic lysates from 510 worms or from high quality dna samples prepared with a gentra puregene kit ( qiagen ) . dyn-1
heterozygous verification primers : complete dyn-1 deletions were verified at the 5 end by pcr amplification with the three primers : ogh154 , ogh133 and ocf125 .
oligos ogh154 + ogh133 give a 2.7 kb pcr product when the wild - type copy of dyn-1 is present .
oligos ogh154 + ocf125 give a 3.4 kb pcr product when the dyn-1 locus is deleted .
the deletion was verified at the 3 end by pcr with oligos ogh155 , ogh156 and ocf400 .
oligos ogh155 + ogh156 produce a 2.5 kb pcr product when the wild - type dyn-1 locus and the ttti14024 mos1 element are present .
oligos ogh155 + ocf400 produce a 3.0 kb pcr product when the dyn-1 gene is deleted . all molecular biology analysis and design was done with the program ape ( a plasmid editor ) that is freely available at http://www.biology.utah.edu / jorgensen / wayned / ape/. all oligos and plasmids are listed in supplementary table 3 .
mos1 alleles were selected by visual screening in wormbase ( www.wormbase.org ) for appropriately located transposon insertions and provided by the nemagenetag consortium .
mos1 alleles were identified in wormbase ( www.wormbase.org ) and requested from the nemagenetag consortium ( http://elegans.imbb.forth.gr/nemagenetag/ ) .
the presence of the mos1 insertion was verified with gene - specific primers , annealing inside and outside of the mos1 element .
the mos1 element was crossed into the unc-119(ed3 ) background to make an injection strain and verified by pcr analysis .
targeting constructs were designed to contain ( 1 ) a right homology region ( adjacent to the mos1 element , ( 2 ) a left homology region ( distant from the mos1 element ) and ( 3 ) a c. briggsae - unc-119(+ ) rescue region .
right homology regions were comprised of approximately 2.0 kb of homologous dna immediately adjacent to the mos1 insertion site .
left homology regions were comprised of 2 to 3 kb of homologous dna , which specifies the endpoint of the targeted deletion .
these regions were selected to avoid repetitive dna sequences , in particular short inverted repeats , which are likely to anneal and reduce the frequency of correct deletions .
a c. briggsae unc-119(+ ) rescue fragment was chosen because it is smaller than the c. elegans unc-119 gene .
an injection mix was made that contained the targeting plasmid ( 50 ng / ul ) , mos1 transposase helper plasmid pjl43.114 ( 50 ng / ul ) , mcherry co - injection markers pgh810 ( prab-3::mcherry , 10 ng / ul ) , pcfj90 ( pmyo-2::mcherry , 2.5 ng / ul ) , and pcfj10410 ( pmyo-3::mcherry , 5 ng / ul ) . the injection strain was maintained at 15c on hb101 bacteria .
young adult animals were mounted on an agarose pad under halocarbon oil and injected following standard techniques .
injected worms were left to recover at 15c for a couple of hours and then transferred three at a time to hb101 or op50 seeded ngm plates and placed at 25c . in our hands , approximately 70 % of all injected worms resulted in transgenic progeny . we found that growing worms on hb101 bacteria at 15c significantly improved the health of unc-119 animals . after approximately 7 days
screening was greatly facilitated by allowing the plate to starve out completely because unc-119 animals can not form dauers and are therefore selected against .
strains with an unc-119(+ ) insertion were identified on a fluorescence dissection microscope as l1 animals that move like wild - type animals but have none of the fluorescent co - injection markers .
a single rescued , non - fluorescent animal was picked to a new plate and allowed to propagate for one generation . in the case of obvious phenotypes ( for example , dpy-13 ) a single mutant animal was picked from the progeny ; in cases where the phenotype was wild - type ten animals were picked to individual plates and tested for homozygosity .
it takes approximately two weeks from injecting the strain to recovering a homozygous deletion animal .
we calculated the distance of every protein coding gene in the ws205 referential release of wormbase ( http://ws205.wormbase.org15 ) to all current mos1 insertion alleles using a state machine algorithm written in perl .
the closest mos1 element was defined as the distance from the insertion site to the atg start codon .
the number of mos1 elements in the vicinity of each gene was determined by extracting a sequence segment upstream and downstream of the atg and summing the number of elements contained within that span .
the number of intervening genes between a given gene and its nearest mos1 element was determined by extracting the segment ranging from the atg to the insertion site and tallying the number of genes present , including genes that partially reside within the interval .
the analysis was repeated against all genes lacking a deletion allele from either the c. elegans gene knockout consortium in the u.s . and
canada ( ok alleles ) and the national bioresource project in japan ( tm alleles ) .
genomic dna from worms was isolated with the gentra puregene tissue kit ( qiagen ) following the manufacturer s supplementary protocol :  purification of archive - quality dna from nematodes or nematode suspensions using the gentra puregene tissue kit  .
dna labeling , sample hybridization , image acquisition , and determination of fluorescence were all performed as previously described12 .
we used a 3x high - density ( hd ) chip divided into three 720 k whole genome sections for all experiments .
all microarrays were manufactured by roche - nimblegen with oligonucleotides synthesized at random positions on the arrays .
for all experiments , normalization of intensity ratios were performed with a loess regression as previously described12 .
three strains , eg5810 ( 25 kb deletion ) , eg5620 ( 15 kb ) and eg5621 ( 15 kb ) , were tested against wild - type dna .
all samples also showed two identical untargeted deletions : an approximately 8 kb deletion of pgp-6 and pgp-7 on chromosome x and an approximately 4 kb deletion of the telomeric region ctelx3.1 at the left end of chromosome v. we verified by cgh that these deletions were present in the parent strain ( eg5003 ) and the deletions therefore do not represent second site mutations caused by the mosdel technique .
targeting vectors typically consist of three distinct fragments : a  right homology region , a positive selection marker ( cb - unc-119(+ ) ) and a  left homology region . in some cases
the positive selection marker was flanked by the fluorescent marker punc-122::gfp , which is dimly expressed in the coelomocytes .
all targeting vectors were made using the multisite gateway three - fragment vector construction kit ( invitrogen , catalog no .
12537 - 023 ) . to verify deletions by pcr we designed oligos that would amplify short genomic dna fragments inside and outside the targeted regions .
these reactions were reproducible and robust ; pcr amplification was successful on crude genomic lysates from 510 worms or from high quality dna samples prepared with a gentra puregene kit ( qiagen ) . dyn-1
heterozygous verification primers : complete dyn-1 deletions were verified at the 5 end by pcr amplification with the three primers : ogh154 , ogh133 and ocf125 .
oligos ogh154 + ogh133 give a 2.7 kb pcr product when the wild - type copy of dyn-1 is present .
oligos ogh154 + ocf125 give a 3.4 kb pcr product when the dyn-1 locus is deleted .
the deletion was verified at the 3 end by pcr with oligos ogh155 , ogh156 and ocf400 .
oligos ogh155 + ogh156 produce a 2.5 kb pcr product when the wild - type dyn-1 locus and the ttti14024 mos1 element are present .
oligos ogh155 + ocf400 produce a 3.0 kb pcr product when the dyn-1 gene is deleted . all molecular biology analysis and design was done with the program ape ( a plasmid editor ) that is freely available at http://www.biology.utah.edu / jorgensen / wayned / ape/. all oligos and plasmids are listed in supplementary table 3 .
( note : large dataset , too large to include in joined pdf file . )
 we have developed a method , mosdel , to generate targeted knock - outs of genes in c. elegans . 
 we make use of the mos1 transposase to excise a mos1 transposon adjacent to the region to be deleted . the double - strand break is repaired using injected dna as a template . 
 repair can delete up to 25 kb of dna and simultaneously insert a positive selection marker . 
Methods
Nematode strains
Deletion protocol
Analysis of 
Comparative Genome Hybridization
Molecular biology
Supplementary Material