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. 2014 May;11(5):529-34.
doi: 10.1038/nmeth.2889. Epub 2014 Mar 16.

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

Christian Frøkjær-Jensen  1 , M Wayne Davis  2 , Mihail Sarov  3 , Jon Taylor  4 , Stephane Flibotte  4 , Matthew LaBella  5 , Andrei Pozniakovsky  3 , Donald G Moerman  4 , Erik M Jorgensen  2
Affiliations

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

Christian Frøkjær-Jensen et al. Nat Methods. 2014 May.

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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Figures

Figure 1
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.
Figure 2
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.
Figure 3
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.
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