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Random and targeted transgene insertion in Caenorhabditis elegans using a modified Mos1 transposon.

Frøkjær-Jensen C, Davis MW, Sarov M, Taylor J, Flibotte S, LaBella M, Pozniakovsky A, Moerman DG, Jorgensen EM - Nat. Methods (2014)

Bottom Line: Genetic and antibiotic markers can be used for selection, and the transposon is active in C. elegans isolates and Caenorhabditis briggsae.We used the miniMos transposon to generate six universal Mos1-mediated single-copy insertion (mosSCI) landing sites that allow targeted transgene insertion with a single targeting vector into permissive expression sites on all autosomes.We also generated two collections of strains: a set of bright fluorescent insertions that are useful as dominant, genetic balancers and a set of lacO insertions to track genome position.

View Article: PubMed Central - PubMed

Affiliation: 1] Howard Hughes Medical Institute, University of Utah, Salt Lake City, Utah, USA. [2] Department of Biology, University of Utah, Salt Lake City, Utah, USA. [3] Danish National Research Foundation Centre for Cardiac Arrhythmia, University of Copenhagen, Copenhagen, Denmark.

ABSTRACT
We have generated a recombinant Mos1 transposon that can insert up to 45-kb transgenes into the Caenorhabditis elegans genome. The minimal Mos1 transposon (miniMos) is 550 bp long and inserts DNA into the genome at high frequency (~60% of injected animals). Genetic and antibiotic markers can be used for selection, and the transposon is active in C. elegans isolates and Caenorhabditis briggsae. We used the miniMos transposon to generate six universal Mos1-mediated single-copy insertion (mosSCI) landing sites that allow targeted transgene insertion with a single targeting vector into permissive expression sites on all autosomes. We also generated two collections of strains: a set of bright fluorescent insertions that are useful as dominant, genetic balancers and a set of lacO insertions to track genome position.

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A modified Mos1 transposon can carry cargo(a) Schematic of recombinant Mosl insertion protocol. Transposon DNA is co-injected with a helper plasmid expressing the transposase (Peft-3:Mos1 transposase). Negative selection markers (Phsp16.41:peel-1, Pmyo-2:mCherry, Prab-3:mCherry and Pmyo-3:mCherry) were used to select against array-bearing transgenic animals. (b) Genomic locations of insertions identified by cb-unc-119(+) rescue of unc-119 mutants. All insertions rescued unc-119, but not all strains expressed GFP-histone in the germline. Germline fluorescence is indicated with turquoise (GFP-positive) or black (no fluorescence) triangles. (c) Fluorescence image of germline expression. Transposon insertion oxTi38 expressed GFP-histone in the germline (Ppie-1:GFP:H2B). Above, differential interference contrast; below confocal fluorescence image. Scale bar = 100 μm. (d) Schematic of the minimal Mos1 transposon (miniMos). 550 bp was enough to retain full insertion frequency. (e) Bar-graph of insertion frequencies with the genetic marker unc-119(+) and antibiotic selection markers G418 (NeoR), puromycin (PuroR) or hygromycin B (HygroR). Values show the average of two independent injections and error bars show the 95% confidence interval (modified Wald method). (f) Bar-graph of insertion frequency at different temperatures. Values shown are averages of three independent replicates (injections) and error bars represent standard error of mean (SEM). Statistics: repeated measures ANOVA (P = 0.0017). Bonferroni post-hoc comparison. **, P < 0.01.
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Figure 1: A modified Mos1 transposon can carry cargo(a) Schematic of recombinant Mosl insertion protocol. Transposon DNA is co-injected with a helper plasmid expressing the transposase (Peft-3:Mos1 transposase). Negative selection markers (Phsp16.41:peel-1, Pmyo-2:mCherry, Prab-3:mCherry and Pmyo-3:mCherry) were used to select against array-bearing transgenic animals. (b) Genomic locations of insertions identified by cb-unc-119(+) rescue of unc-119 mutants. All insertions rescued unc-119, but not all strains expressed GFP-histone in the germline. Germline fluorescence is indicated with turquoise (GFP-positive) or black (no fluorescence) triangles. (c) Fluorescence image of germline expression. Transposon insertion oxTi38 expressed GFP-histone in the germline (Ppie-1:GFP:H2B). Above, differential interference contrast; below confocal fluorescence image. Scale bar = 100 μm. (d) Schematic of the minimal Mos1 transposon (miniMos). 550 bp was enough to retain full insertion frequency. (e) Bar-graph of insertion frequencies with the genetic marker unc-119(+) and antibiotic selection markers G418 (NeoR), puromycin (PuroR) or hygromycin B (HygroR). Values show the average of two independent injections and error bars show the 95% confidence interval (modified Wald method). (f) Bar-graph of insertion frequency at different temperatures. Values shown are averages of three independent replicates (injections) and error bars represent standard error of mean (SEM). Statistics: repeated measures ANOVA (P = 0.0017). Bonferroni post-hoc comparison. **, P < 0.01.

Mentions: The requirements for transposition of mariner elements (Mos1 and the closely related Peach transposon) vary depending on whether the transposon is embedded in chromatin or is contained within injected plasmid DNA. Mariner transposons within chromosomes require internal sequences to transpose17 and can carry cargo only if the cargo is flanked by intact transposons18. By contrast, transposons injected as plasmids can transpose efficiently even if they contain internal deletions and carry cargo19. Experiments in vitro have further demonstrated that modifications to the inverted terminal repeats improve transposition frequency20. We tested whether modified Mosl elements and plasmid injection protocols11 could overcome previously described limitations for Mosl transposition in C. elegans9. We generated a composite Mosl transposon with a 7.5 kb transgene (containing Ppie-1:GFP:histone and cb-unc-119(+)) and tested transposition by plasmid injection (Fig. 1 and Supplementary Fig. 1). We co-injected the composite Mosl transposon with a helper plasmid expressing the transposase and fluorescent extra-chromosomal array markers. We injected 27 unc-119 animals and identified 17 independent lines with recombinant Mosl insertions (62% P0 insertion frequency). 47% (8/17) of the strains expressed GFP in the germline (Fig. 1). We mapped four GFP expressors and four non-expressors by inverse PCR21 to unique insertion sites. Non-fluorescent insertions were found on autosomal arms, which have high levels of repressive chromatin marks22 or the X-chromosome, which is inactivated in the germline23 (Fig. 1). It is likely that these Ppie-1:GFP:histone insertions are silenced through a combination of small RNAs that detect foreign DNAs and protect endogenous gene expression in the germline24-26 and subsequent modifications to the chromatin environment. We are currently characterizing germline and somatic position effects in detail (unpublished C.F.-J. & E.M.J.).


Random and targeted transgene insertion in Caenorhabditis elegans using a modified Mos1 transposon.

Frøkjær-Jensen C, Davis MW, Sarov M, Taylor J, Flibotte S, LaBella M, Pozniakovsky A, Moerman DG, Jorgensen EM - Nat. Methods (2014)

A modified Mos1 transposon can carry cargo(a) Schematic of recombinant Mosl insertion protocol. Transposon DNA is co-injected with a helper plasmid expressing the transposase (Peft-3:Mos1 transposase). Negative selection markers (Phsp16.41:peel-1, Pmyo-2:mCherry, Prab-3:mCherry and Pmyo-3:mCherry) were used to select against array-bearing transgenic animals. (b) Genomic locations of insertions identified by cb-unc-119(+) rescue of unc-119 mutants. All insertions rescued unc-119, but not all strains expressed GFP-histone in the germline. Germline fluorescence is indicated with turquoise (GFP-positive) or black (no fluorescence) triangles. (c) Fluorescence image of germline expression. Transposon insertion oxTi38 expressed GFP-histone in the germline (Ppie-1:GFP:H2B). Above, differential interference contrast; below confocal fluorescence image. Scale bar = 100 μm. (d) Schematic of the minimal Mos1 transposon (miniMos). 550 bp was enough to retain full insertion frequency. (e) Bar-graph of insertion frequencies with the genetic marker unc-119(+) and antibiotic selection markers G418 (NeoR), puromycin (PuroR) or hygromycin B (HygroR). Values show the average of two independent injections and error bars show the 95% confidence interval (modified Wald method). (f) Bar-graph of insertion frequency at different temperatures. Values shown are averages of three independent replicates (injections) and error bars represent standard error of mean (SEM). Statistics: repeated measures ANOVA (P = 0.0017). Bonferroni post-hoc comparison. **, P < 0.01.
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Related In: Results  -  Collection

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Figure 1: A modified Mos1 transposon can carry cargo(a) Schematic of recombinant Mosl insertion protocol. Transposon DNA is co-injected with a helper plasmid expressing the transposase (Peft-3:Mos1 transposase). Negative selection markers (Phsp16.41:peel-1, Pmyo-2:mCherry, Prab-3:mCherry and Pmyo-3:mCherry) were used to select against array-bearing transgenic animals. (b) Genomic locations of insertions identified by cb-unc-119(+) rescue of unc-119 mutants. All insertions rescued unc-119, but not all strains expressed GFP-histone in the germline. Germline fluorescence is indicated with turquoise (GFP-positive) or black (no fluorescence) triangles. (c) Fluorescence image of germline expression. Transposon insertion oxTi38 expressed GFP-histone in the germline (Ppie-1:GFP:H2B). Above, differential interference contrast; below confocal fluorescence image. Scale bar = 100 μm. (d) Schematic of the minimal Mos1 transposon (miniMos). 550 bp was enough to retain full insertion frequency. (e) Bar-graph of insertion frequencies with the genetic marker unc-119(+) and antibiotic selection markers G418 (NeoR), puromycin (PuroR) or hygromycin B (HygroR). Values show the average of two independent injections and error bars show the 95% confidence interval (modified Wald method). (f) Bar-graph of insertion frequency at different temperatures. Values shown are averages of three independent replicates (injections) and error bars represent standard error of mean (SEM). Statistics: repeated measures ANOVA (P = 0.0017). Bonferroni post-hoc comparison. **, P < 0.01.
Mentions: The requirements for transposition of mariner elements (Mos1 and the closely related Peach transposon) vary depending on whether the transposon is embedded in chromatin or is contained within injected plasmid DNA. Mariner transposons within chromosomes require internal sequences to transpose17 and can carry cargo only if the cargo is flanked by intact transposons18. By contrast, transposons injected as plasmids can transpose efficiently even if they contain internal deletions and carry cargo19. Experiments in vitro have further demonstrated that modifications to the inverted terminal repeats improve transposition frequency20. We tested whether modified Mosl elements and plasmid injection protocols11 could overcome previously described limitations for Mosl transposition in C. elegans9. We generated a composite Mosl transposon with a 7.5 kb transgene (containing Ppie-1:GFP:histone and cb-unc-119(+)) and tested transposition by plasmid injection (Fig. 1 and Supplementary Fig. 1). We co-injected the composite Mosl transposon with a helper plasmid expressing the transposase and fluorescent extra-chromosomal array markers. We injected 27 unc-119 animals and identified 17 independent lines with recombinant Mosl insertions (62% P0 insertion frequency). 47% (8/17) of the strains expressed GFP in the germline (Fig. 1). We mapped four GFP expressors and four non-expressors by inverse PCR21 to unique insertion sites. Non-fluorescent insertions were found on autosomal arms, which have high levels of repressive chromatin marks22 or the X-chromosome, which is inactivated in the germline23 (Fig. 1). It is likely that these Ppie-1:GFP:histone insertions are silenced through a combination of small RNAs that detect foreign DNAs and protect endogenous gene expression in the germline24-26 and subsequent modifications to the chromatin environment. We are currently characterizing germline and somatic position effects in detail (unpublished C.F.-J. & E.M.J.).

Bottom Line: Genetic and antibiotic markers can be used for selection, and the transposon is active in C. elegans isolates and Caenorhabditis briggsae.We used the miniMos transposon to generate six universal Mos1-mediated single-copy insertion (mosSCI) landing sites that allow targeted transgene insertion with a single targeting vector into permissive expression sites on all autosomes.We also generated two collections of strains: a set of bright fluorescent insertions that are useful as dominant, genetic balancers and a set of lacO insertions to track genome position.

View Article: PubMed Central - PubMed

Affiliation: 1] Howard Hughes Medical Institute, University of Utah, Salt Lake City, Utah, USA. [2] Department of Biology, University of Utah, Salt Lake City, Utah, USA. [3] Danish National Research Foundation Centre for Cardiac Arrhythmia, University of Copenhagen, Copenhagen, Denmark.

ABSTRACT
We have generated a recombinant Mos1 transposon that can insert up to 45-kb transgenes into the Caenorhabditis elegans genome. The minimal Mos1 transposon (miniMos) is 550 bp long and inserts DNA into the genome at high frequency (~60% of injected animals). Genetic and antibiotic markers can be used for selection, and the transposon is active in C. elegans isolates and Caenorhabditis briggsae. We used the miniMos transposon to generate six universal Mos1-mediated single-copy insertion (mosSCI) landing sites that allow targeted transgene insertion with a single targeting vector into permissive expression sites on all autosomes. We also generated two collections of strains: a set of bright fluorescent insertions that are useful as dominant, genetic balancers and a set of lacO insertions to track genome position.

Show MeSH