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Nat Genet. 2008 Nov;40(11):1375-83. doi: 10.1038/ng.248. Epub 2008 Oct 26.

Single-copy insertion of transgenes in Caenorhabditis elegans.

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  • 1Department of Biology and Howard Hughes Medical Institute, University of Utah, 257 South 1400 East, Salt Lake City, Utah 84112, USA.

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.

PMID:
18953339
[PubMed - indexed for MEDLINE]
PMCID:
PMC2749959
Free PMC Article

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