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A genome-wide collection of Mos1 transposon insertion mutants for the C. elegans research community.

Vallin E, Gallagher J, Granger L, Martin E, Belougne J, Maurizio J, Duverger Y, Scaglione S, Borrel C, Cortier E, Abouzid K, Carre-Pierrat M, Gieseler K, Ségalat L, Kuwabara PE, Ewbank JJ - PLoS ONE (2012)

Bottom Line: These strains were then subject to molecular analysis, and more than 13,300 Mos1 insertions characterized.In addition to targeting directly more than 4,700 genes, these alleles represent the potential starting point for the engineered deletion of essentially all C. elegans genes and the modification of more than 40% of them.This collection of mutants, generated under the auspices of the European NEMAGENETAG consortium, is publicly available and represents an important research resource.

View Article: PubMed Central - PubMed

Affiliation: Centre de Génétique et de Physiologie Moléculaires et Cellulaires, CNRS UMR 5534, Campus de la Doua, Villeurbanne, France.

ABSTRACT
Methods that use homologous recombination to engineer the genome of C. elegans commonly use strains carrying specific insertions of the heterologous transposon Mos1. A large collection of known Mos1 insertion alleles would therefore be of general interest to the C. elegans research community. We describe here the optimization of a semi-automated methodology for the construction of a substantial collection of Mos1 insertion mutant strains. At peak production, more than 5,000 strains were generated per month. These strains were then subject to molecular analysis, and more than 13,300 Mos1 insertions characterized. In addition to targeting directly more than 4,700 genes, these alleles represent the potential starting point for the engineered deletion of essentially all C. elegans genes and the modification of more than 40% of them. This collection of mutants, generated under the auspices of the European NEMAGENETAG consortium, is publicly available and represents an important research resource.

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Distribution of Mos1 alleles.(A) Graph showing the relationship between chromosome length (as a percentage of the whole nuclear genome) and the proportion of Mos1 alleles per chromosome reported in a previous study [5], and the 10,858 alleles obtained in the current project (black and red circles, respectively). The outliers, concerning chromosomes I and V, from the previous study are highlighted with lines. (B) Distribution of distances from one Mos1 allele to the next, in a 5′ to 3′ direction along each chromosome. The graph shows the cumulative percentage of alleles that are separated by less than the indicated distance. (C) Concentration of Mos1 alleles at the extreme right end of chromosome I (length 15,072,423 bp). The separation of the allele numbers indicates that almost all the alleles were generated independently, except in two cases (ttTi2276 and ttTi2284; ttTi13453 and ttTi13460), highlighted by an asterisk. This region was also preferentially targeted during the previous study as reflected by the presence of several cxTi alleles.
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pone-0030482-g002: Distribution of Mos1 alleles.(A) Graph showing the relationship between chromosome length (as a percentage of the whole nuclear genome) and the proportion of Mos1 alleles per chromosome reported in a previous study [5], and the 10,858 alleles obtained in the current project (black and red circles, respectively). The outliers, concerning chromosomes I and V, from the previous study are highlighted with lines. (B) Distribution of distances from one Mos1 allele to the next, in a 5′ to 3′ direction along each chromosome. The graph shows the cumulative percentage of alleles that are separated by less than the indicated distance. (C) Concentration of Mos1 alleles at the extreme right end of chromosome I (length 15,072,423 bp). The separation of the allele numbers indicates that almost all the alleles were generated independently, except in two cases (ttTi2276 and ttTi2284; ttTi13453 and ttTi13460), highlighted by an asterisk. This region was also preferentially targeted during the previous study as reflected by the presence of several cxTi alleles.

Mentions: Bioinformatics analyses were conducted to analyze the distribution of the complete collection of Mos1 alleles and to evaluate its potential usefulness. As in some cases multiple redundant insertions were obtained, it was important to try to address whether this reflected a real bias of the Mos1 transposon to insert at specific sites, or whether this was the consequence of experimental artifact. Of the 2,476 redundant alleles, 2,239 were found to have an insertion site that exactly matched that of an allele in the non-redundant set of 10,858 alleles (Table S1). When we looked at the allele numbers of such matching pairs, which reflects allele isolation history, we found that in almost 90% (1,972) of cases, the allele numbers differed by less than 25, indicating that they were derived from the same, or closest, 24-well plates. This strongly suggested, as discussed below, that the great majority of redundant alleles arose from experimental artifact. We therefore limited further analyses to the non-redundant set of 10,858 alleles. In our pilot study with 914 alleles, we reported a bias for insertions on chromosome I and against chromosome V [5]; with our new set of 10,858 alleles, this skewed distribution was not detected and the number of Mos1 insertions found on each chromosome was proportional to the chromosome length (Figure 2A). The previously observed imbalance presumably represents a sampling bias. The average distance between neighboring alleles was 9,230 bp, with 33%, 67% and 95% of gaps being less than 3.2 kb, 10 kb and 30 kb, respectively. The single largest gap between adjacent alleles was less than 100 kb (Figure 2B, Table S1). There were, however, a few local “hot spots” for Mos1 alleles. The extreme right end of chromosome III, and especially of chromosome I, for example, had a dense distribution (Figure 2C, results not shown). But otherwise, on the scale of each individual chromosome, the spread of Mos1 alleles was relatively uniform (Figure 3).


A genome-wide collection of Mos1 transposon insertion mutants for the C. elegans research community.

Vallin E, Gallagher J, Granger L, Martin E, Belougne J, Maurizio J, Duverger Y, Scaglione S, Borrel C, Cortier E, Abouzid K, Carre-Pierrat M, Gieseler K, Ségalat L, Kuwabara PE, Ewbank JJ - PLoS ONE (2012)

Distribution of Mos1 alleles.(A) Graph showing the relationship between chromosome length (as a percentage of the whole nuclear genome) and the proportion of Mos1 alleles per chromosome reported in a previous study [5], and the 10,858 alleles obtained in the current project (black and red circles, respectively). The outliers, concerning chromosomes I and V, from the previous study are highlighted with lines. (B) Distribution of distances from one Mos1 allele to the next, in a 5′ to 3′ direction along each chromosome. The graph shows the cumulative percentage of alleles that are separated by less than the indicated distance. (C) Concentration of Mos1 alleles at the extreme right end of chromosome I (length 15,072,423 bp). The separation of the allele numbers indicates that almost all the alleles were generated independently, except in two cases (ttTi2276 and ttTi2284; ttTi13453 and ttTi13460), highlighted by an asterisk. This region was also preferentially targeted during the previous study as reflected by the presence of several cxTi alleles.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3275553&req=5

pone-0030482-g002: Distribution of Mos1 alleles.(A) Graph showing the relationship between chromosome length (as a percentage of the whole nuclear genome) and the proportion of Mos1 alleles per chromosome reported in a previous study [5], and the 10,858 alleles obtained in the current project (black and red circles, respectively). The outliers, concerning chromosomes I and V, from the previous study are highlighted with lines. (B) Distribution of distances from one Mos1 allele to the next, in a 5′ to 3′ direction along each chromosome. The graph shows the cumulative percentage of alleles that are separated by less than the indicated distance. (C) Concentration of Mos1 alleles at the extreme right end of chromosome I (length 15,072,423 bp). The separation of the allele numbers indicates that almost all the alleles were generated independently, except in two cases (ttTi2276 and ttTi2284; ttTi13453 and ttTi13460), highlighted by an asterisk. This region was also preferentially targeted during the previous study as reflected by the presence of several cxTi alleles.
Mentions: Bioinformatics analyses were conducted to analyze the distribution of the complete collection of Mos1 alleles and to evaluate its potential usefulness. As in some cases multiple redundant insertions were obtained, it was important to try to address whether this reflected a real bias of the Mos1 transposon to insert at specific sites, or whether this was the consequence of experimental artifact. Of the 2,476 redundant alleles, 2,239 were found to have an insertion site that exactly matched that of an allele in the non-redundant set of 10,858 alleles (Table S1). When we looked at the allele numbers of such matching pairs, which reflects allele isolation history, we found that in almost 90% (1,972) of cases, the allele numbers differed by less than 25, indicating that they were derived from the same, or closest, 24-well plates. This strongly suggested, as discussed below, that the great majority of redundant alleles arose from experimental artifact. We therefore limited further analyses to the non-redundant set of 10,858 alleles. In our pilot study with 914 alleles, we reported a bias for insertions on chromosome I and against chromosome V [5]; with our new set of 10,858 alleles, this skewed distribution was not detected and the number of Mos1 insertions found on each chromosome was proportional to the chromosome length (Figure 2A). The previously observed imbalance presumably represents a sampling bias. The average distance between neighboring alleles was 9,230 bp, with 33%, 67% and 95% of gaps being less than 3.2 kb, 10 kb and 30 kb, respectively. The single largest gap between adjacent alleles was less than 100 kb (Figure 2B, Table S1). There were, however, a few local “hot spots” for Mos1 alleles. The extreme right end of chromosome III, and especially of chromosome I, for example, had a dense distribution (Figure 2C, results not shown). But otherwise, on the scale of each individual chromosome, the spread of Mos1 alleles was relatively uniform (Figure 3).

Bottom Line: These strains were then subject to molecular analysis, and more than 13,300 Mos1 insertions characterized.In addition to targeting directly more than 4,700 genes, these alleles represent the potential starting point for the engineered deletion of essentially all C. elegans genes and the modification of more than 40% of them.This collection of mutants, generated under the auspices of the European NEMAGENETAG consortium, is publicly available and represents an important research resource.

View Article: PubMed Central - PubMed

Affiliation: Centre de Génétique et de Physiologie Moléculaires et Cellulaires, CNRS UMR 5534, Campus de la Doua, Villeurbanne, France.

ABSTRACT
Methods that use homologous recombination to engineer the genome of C. elegans commonly use strains carrying specific insertions of the heterologous transposon Mos1. A large collection of known Mos1 insertion alleles would therefore be of general interest to the C. elegans research community. We describe here the optimization of a semi-automated methodology for the construction of a substantial collection of Mos1 insertion mutant strains. At peak production, more than 5,000 strains were generated per month. These strains were then subject to molecular analysis, and more than 13,300 Mos1 insertions characterized. In addition to targeting directly more than 4,700 genes, these alleles represent the potential starting point for the engineered deletion of essentially all C. elegans genes and the modification of more than 40% of them. This collection of mutants, generated under the auspices of the European NEMAGENETAG consortium, is publicly available and represents an important research resource.

Show MeSH
Related in: MedlinePlus