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Single-copy insertion of transgenes in Caenorhabditis elegans

Abstract

At present, transgenes in Caenorhabditis elegans are generated by injecting DNA into the germline. The DNA assembles into a semistable extrachromosomal array composed of many copies of injected DNA. These transgenes are typically overexpressed in somatic cells and silenced in the germline. We have developed a method that inserts a single copy of a transgene into a defined site. Mobilization of a Mos1 transposon generates a double-strand break in noncoding DNA. The break is repaired by copying DNA from an extrachromosomal template into the chromosomal site. Homozygous single-copy insertions can be obtained in less than 2 weeks by injecting approximately 20 worms. We have successfully inserted transgenes as long as 9 kb and verified that single copies are inserted at the targeted site. Single-copy transgenes are expressed at endogenous levels and can be expressed in the female and male germlines.

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Figure 1: Schematic overview of MosSCI.
Figure 2: Single-copy insertions of transgenes.
Figure 3: Independent unc-18::mCherry transgenes are expressed at near-endogenous levels.
Figure 4: MosSCI inserts are expressed in the female and male germlines.
Figure 5: MosSCI inserts can be generated directly by injection.

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Acknowledgements

We thank L. Segalat and the NemaGENETAG consortium for generating and supplying Mos1 insertion alleles; L. Salkoff for twk-18 cDNA clones; G. Seydoux for promoter plasmids; J. Rand for UNC-18 antibodies; C. Thacker at the Utah worm core facility for generating biolistic transformants; G. Stanfield for help in confirming sperm expression; J.-L. Bessereau and V. Robert for sharing unpublished data; and M. Ailion, G. Hollopeter and the rest of the Jorgensen laboratory for scientific input. E.M.J. is an Investigator of the Howard Hughes Medical Institute. S.-P.O. is an Investigator of the Danish National Research Foundation. C.F.-J. was funded by fellowships from Fonden af 17.12.1981 and the Lundbeck Foundation.

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C.F.-J., M.W.D., C.E.H. and E.M.J. designed the experiments. C.F.-J., M.W.D., C.E.H., B.J.N. and J.M.T. carried out the experiments. E.M.J., M.G. and S.-P.O. supervised and funded the experiments. C.F.-J., M.W.D. and E.M.J. wrote the manuscript.

Corresponding author

Correspondence to Erik M Jorgensen.

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Supplementary Figures 1–7, Supplementary Table 1, Supplementary Methods (PDF 2804 kb)

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Frøkjær-Jensen, C., Wayne Davis, M., Hopkins, C. et al. Single-copy insertion of transgenes in Caenorhabditis elegans. Nat Genet 40, 1375–1383 (2008). https://doi.org/10.1038/ng.248

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