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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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Using miniMos to generate universal mosSCI insertion sites(a) Schematic of method to generate universal mosSCI insertion sites. Step 1: Insert miniMos with the ttTi5605 genomic region (including the native Mos1 element) into unc-18 mutants. Cross inserts to unc-119. Step 2: Inject pCFJ150-based targeting vector to insert transgene by mosSCI. All insertions were verified as functional, single-copy insertions. (b) Genomic location of universal mosSCI insertion sites with verified germline expression. Black arrowhead: NeoR marker. Green arrowhead: Pmyo-2:GFP:H2B marker. NeoR = Neomycin Resistance gene.
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Figure 3: Using miniMos to generate universal mosSCI insertion sites(a) Schematic of method to generate universal mosSCI insertion sites. Step 1: Insert miniMos with the ttTi5605 genomic region (including the native Mos1 element) into unc-18 mutants. Cross inserts to unc-119. Step 2: Inject pCFJ150-based targeting vector to insert transgene by mosSCI. All insertions were verified as functional, single-copy insertions. (b) Genomic location of universal mosSCI insertion sites with verified germline expression. Black arrowhead: NeoR marker. Green arrowhead: Pmyo-2:GFP:H2B marker. NeoR = Neomycin Resistance gene.

Mentions: The PhiC31 recombinase has been used in flies to develop universal insertion sites that are compatible with a single targeting vector4,40. We unsuccessfully attempted to adapt the PhiC31 system for C. elegans (M.S. and C.F-J., unpublished observations). As an alternative, we developed a miniMos system that achieves the same goal. We generated a miniMos element containing the ttTi5605 MosSCI site and flanked it with two selection markers, unc-18 and either NeoR or Pmyo-2:GFP:H2B (Fig. 3). The embedded ttTi5605 Mos element within the miniMos transposon can be used as a landing site for single copy insertion using MosSCI12 and is compatible with previously published targeting vectors (pCFJ150 or pCFJ350) (Fig. 3). Furthermore, mosSCI insertions can be followed in crosses by the adjacent selection marker (NeoR or Pmyo-2:GFP:H2B). We generated a set of validated single-copy, full-length mosSCI universal insertion sites that were permissive for germline expression (Fig. 3). Additionally, we targeted the insertion of a universal landing site into the ttTi25545 Mos1 site at the center of Chr. III by mosSCI because no insertion site on Chr. III was compatible with germline expression (data not shown). All universal landing sites were validated: we could generate single copy inserts at frequencies similar to insertions into the native ttTi5605 site and a Pdpy-30:GFP:H2B transgene was expressed in the germline (Supplementary Table 1).


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)

Using miniMos to generate universal mosSCI insertion sites(a) Schematic of method to generate universal mosSCI insertion sites. Step 1: Insert miniMos with the ttTi5605 genomic region (including the native Mos1 element) into unc-18 mutants. Cross inserts to unc-119. Step 2: Inject pCFJ150-based targeting vector to insert transgene by mosSCI. All insertions were verified as functional, single-copy insertions. (b) Genomic location of universal mosSCI insertion sites with verified germline expression. Black arrowhead: NeoR marker. Green arrowhead: Pmyo-2:GFP:H2B marker. NeoR = Neomycin Resistance gene.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126194&req=5

Figure 3: Using miniMos to generate universal mosSCI insertion sites(a) Schematic of method to generate universal mosSCI insertion sites. Step 1: Insert miniMos with the ttTi5605 genomic region (including the native Mos1 element) into unc-18 mutants. Cross inserts to unc-119. Step 2: Inject pCFJ150-based targeting vector to insert transgene by mosSCI. All insertions were verified as functional, single-copy insertions. (b) Genomic location of universal mosSCI insertion sites with verified germline expression. Black arrowhead: NeoR marker. Green arrowhead: Pmyo-2:GFP:H2B marker. NeoR = Neomycin Resistance gene.
Mentions: The PhiC31 recombinase has been used in flies to develop universal insertion sites that are compatible with a single targeting vector4,40. We unsuccessfully attempted to adapt the PhiC31 system for C. elegans (M.S. and C.F-J., unpublished observations). As an alternative, we developed a miniMos system that achieves the same goal. We generated a miniMos element containing the ttTi5605 MosSCI site and flanked it with two selection markers, unc-18 and either NeoR or Pmyo-2:GFP:H2B (Fig. 3). The embedded ttTi5605 Mos element within the miniMos transposon can be used as a landing site for single copy insertion using MosSCI12 and is compatible with previously published targeting vectors (pCFJ150 or pCFJ350) (Fig. 3). Furthermore, mosSCI insertions can be followed in crosses by the adjacent selection marker (NeoR or Pmyo-2:GFP:H2B). We generated a set of validated single-copy, full-length mosSCI universal insertion sites that were permissive for germline expression (Fig. 3). Additionally, we targeted the insertion of a universal landing site into the ttTi25545 Mos1 site at the center of Chr. III by mosSCI because no insertion site on Chr. III was compatible with germline expression (data not shown). All universal landing sites were validated: we could generate single copy inserts at frequencies similar to insertions into the native ttTi5605 site and a Pdpy-30:GFP:H2B transgene was expressed in the germline (Supplementary Table 1).

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
Related in: MedlinePlus