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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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Fosmid insertions are intact(a) Schematic of Mos1-based fosmids (Mosmids). Mos1 and cb-unc-119(+) selection recombineered into the backbone of a fosmid carrying a GFP-tagged gene. (b) Fluorescence microscopy of Mosmid insertions. Four different Mosmid insertions with GFP show expression from the tagged genes. (c) Comparative genome hybridization (CGH) of genomic DNA from four independent insertions of the Mosmid WRM0615dD02 containing tagged cnd-1. CGH is based on dense oligo arrays tiled against a genome of interest and labeling of sample DNA and control DNA with different fluorophores. Genomic regions that differ between sample and control will show a difference in the ratio between the two color intensities. The Mosmid with cnd-1:eGFP contained an error rendering the fusion protein non-fluorescent.
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Figure 2: Fosmid insertions are intact(a) Schematic of Mos1-based fosmids (Mosmids). Mos1 and cb-unc-119(+) selection recombineered into the backbone of a fosmid carrying a GFP-tagged gene. (b) Fluorescence microscopy of Mosmid insertions. Four different Mosmid insertions with GFP show expression from the tagged genes. (c) Comparative genome hybridization (CGH) of genomic DNA from four independent insertions of the Mosmid WRM0615dD02 containing tagged cnd-1. CGH is based on dense oligo arrays tiled against a genome of interest and labeling of sample DNA and control DNA with different fluorophores. Genomic regions that differ between sample and control will show a difference in the ratio between the two color intensities. The Mosmid with cnd-1:eGFP contained an error rendering the fusion protein non-fluorescent.

Mentions: To determine the maximum cargo capacity of recombinant Mos1 elements we generated Mosl-based fosmids (Mosmids) by recombineering36. We inserted a cassette with a 1 kb recombinant Mosl element and cb-unc-119(+) into the backbone of several fosmids with GFP-tagged genes (Fig. 2). We injected five different Mosmids into unc-119 animals and obtained stable integrated lines at P0 frequencies ranging from 2% to 14% (5% ± 2%) of all constructs. The drop in insertion frequency was likely caused by two effects: larger cargo may inhibit transposition and Mosmid injections only inefficiently form extra-chromosomal arrays. Inserted Mosmids expressed GFP in the expected tissues, including the germline (Fig. 2).


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)

Fosmid insertions are intact(a) Schematic of Mos1-based fosmids (Mosmids). Mos1 and cb-unc-119(+) selection recombineered into the backbone of a fosmid carrying a GFP-tagged gene. (b) Fluorescence microscopy of Mosmid insertions. Four different Mosmid insertions with GFP show expression from the tagged genes. (c) Comparative genome hybridization (CGH) of genomic DNA from four independent insertions of the Mosmid WRM0615dD02 containing tagged cnd-1. CGH is based on dense oligo arrays tiled against a genome of interest and labeling of sample DNA and control DNA with different fluorophores. Genomic regions that differ between sample and control will show a difference in the ratio between the two color intensities. The Mosmid with cnd-1:eGFP contained an error rendering the fusion protein non-fluorescent.
© Copyright Policy
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4126194&req=5

Figure 2: Fosmid insertions are intact(a) Schematic of Mos1-based fosmids (Mosmids). Mos1 and cb-unc-119(+) selection recombineered into the backbone of a fosmid carrying a GFP-tagged gene. (b) Fluorescence microscopy of Mosmid insertions. Four different Mosmid insertions with GFP show expression from the tagged genes. (c) Comparative genome hybridization (CGH) of genomic DNA from four independent insertions of the Mosmid WRM0615dD02 containing tagged cnd-1. CGH is based on dense oligo arrays tiled against a genome of interest and labeling of sample DNA and control DNA with different fluorophores. Genomic regions that differ between sample and control will show a difference in the ratio between the two color intensities. The Mosmid with cnd-1:eGFP contained an error rendering the fusion protein non-fluorescent.
Mentions: To determine the maximum cargo capacity of recombinant Mos1 elements we generated Mosl-based fosmids (Mosmids) by recombineering36. We inserted a cassette with a 1 kb recombinant Mosl element and cb-unc-119(+) into the backbone of several fosmids with GFP-tagged genes (Fig. 2). We injected five different Mosmids into unc-119 animals and obtained stable integrated lines at P0 frequencies ranging from 2% to 14% (5% ± 2%) of all constructs. The drop in insertion frequency was likely caused by two effects: larger cargo may inhibit transposition and Mosmid injections only inefficiently form extra-chromosomal arrays. Inserted Mosmids expressed GFP in the expected tissues, including the germline (Fig. 2).

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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