Single-copy insertion of transgenes in Caenorhabditis elegans

• DOI: 10.1038/ng.248
• Publication Date: 2008-10-26T18:22:03Z
• Authors: Christian Frøkjær...; M Wayne Davis; Christopher E Hop...; Blake J Newman; Jason M Thummel; Søren-Peter Olesen; Morten Grunnet; Erik M Jorgensen
• Keywords: Male; 0301 basic medicine; Green Fluorescent Proteins; Gene Dosage; Injections; Animals, Genetically Modified; Mutagenesis, Insertional; 03 medical and health sciences; Germ Cells; DNA Transposable Elements; Animals; Female; Transgenes; Caenorhabditis elegans

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.