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.
Frøkjær-Jensen, C., Wayne Davis, M., Hopkins, C. E., Newman, B. J., Thummel, J. M., Olesen, S. P., … Jorgensen, E. M. (2008). Single-copy insertion of transgenes in Caenorhabditis elegans. Nature Genetics, 40(11), 1375–1383. https://doi.org/10.1038/ng.248