Single-copy insertion of transgenes in Caenorhabditis elegans.

These authors describe a very important and useful genetic tool that had been missing so far in the Caenorhabditis elegans genetic toolkit, namely the possibility to integrate a single-copy construct at a predefined locus.

I think the method is very promising because reporter constructs should be expressed at levels very comparable to that of the endogenous gene, and because it will be possible to compare the
effects of different constructs for a given gene. In addition, it bypasses to a large extent the need to use biolistic transformation, since the authors found that germline-expressed genes are not silenced. Its main limitation at this point is that it can accommodate only constructs smaller than 9-10 kb and also that it has been designed so far only for insertion into two loci located on chromosomes II and IV (the latter has not been tested for as many constructs). It will be essential to define additional host loci so as to be able to combine constructs linked to different fluorescent reporters and to be able to test the effect of mutations that happen to be located very close to the two already defined insertion loci. Incidentally, the method has been successfully tested in my lab.

Wow! A method to generate single copy insertions of transgenes in C. elegans at a defined insertion site. This article is a must read for any C. elegans researcher.

C. elegans transgenes generated by microinjection often result in extrachromosomal arrays with many copies of the gene-of-interest. These transgenes are thus over-expressed in somatic tissues. In the germline, because of the highly repetitive nature of the array, they are often silenced. This problem was only partially solved with biolistic transformation. It has long been a desire of C. elegans researchers that they be able to generate transgenic animals with a single copy insertion at a defined site in the genome. The authors of this paper have taken advantage of a library of C. elegans strains {1} with unique Mos1 element (from Drosophila) transposon insertions. Since each strain has a single Mos1 insertion, they have shown that transposase can generate a double-strand break in the chromosome through Mos1 excision.

Using an extrachromosomal transgenic array with homologous DNA sequences from each flanking side of the Mos1 element, they were able to target various genes-of-interest into this unique site and get single copy integrations. This method is fast, easy, and appears to be quite efficient. The advantage for C. elegans researchers is that they can now control for integration site effects since all their transgenes can be put into one site. If one has a deletion of their favorite gene, this is a great tool to perform structure-function studies and assay rescuing ability of their transgene. This method will likely turn out to be used by many in the very near future. I already ordered the strains and plasmids.

Frøkjaer-Jensen and colleagues have developed an approach to introduce transgenes in single-copy into a defined location in the C. elegans genome. This method will facilitate studies that demand low-level or germline expression, or depend on comparisons between different constructs (e.g. structure/function analyses).


Experimental organisms have their pros and cons; for C. elegans, transgenesis has been far from ideal, relying on multiple copies of genes embedded in unusual chromatin contexts. This new approach, which takes advantage of the Mos transposase, allows generation of stable transgenes, equivalent expression levels of different constructs (e.g. for structure/function analysis) and expression in the germ line, which is refractory to high-copy transgenes.

The creation of low-copy transgenic lines in C. elegans has been a tricky business, but this paper reports an elegant solution. Frøkjaer-Jensen and colleagues have developed a method to insert single-copy transgenes at a neutral locus in the C. elegans genome.

Currently, popular methods for creating transgenics in C. elegans do not allow precise control over the number of transgene copies or location of integration in the genome. For germline-expressed transgenes, high transgene copy number usually leads to problematic transgene silencing in the gonad. Frøkjaer-Jensen et al. have taken advantage of the Drosophila mariner element Mos1, which can function as a single-copy transposon in the C. elegans genome. The authors achieved insertion of single-copy transgenes at a genetically neutral, intergenic Mos1 site, a method they call Mos1-mediated single-copy insertion (MosSCI). Worms are microinjected with a cocktail that includes a transgene of interest, a Mos1 transposase gene and selection markers, which together form an extrachromosomal array. The transposase excises the Mos1 transposon, creating a double-strand break, and the break is repaired -- often using the chromosomal array sequence. The selection markers are then used to identify transgenic worms that gained an insertion and then lost the extrachromosomal array. These insertions work for somatically-expressed transgenes, for expression in both oocytes and sperm, and with inserts as big as 9 kilobases. We imagine that, for some applications, there might be a disadvantage in single-copy transgenes producing only endogenous protein expression levels, which might force the use of brighter fluorescent tags. The authors point out that such low-copy insertions will be helpful for minimizing germline silencing, and for facilitating protein structure-function studies as well as rescue with sequences from other species' genomes.

The holy grail of metazoan model organism transgenics is the ability to replace a specific gene at its endogenous chromosomal location with a modified copy. Though the technique described by Jorgensen and colleagues falls short of this lofty goal, it nevertheless represents a fantastic technical advance.

This paper describes a new method to insert single copy transgenes into the C. elegans genome at a defined location. Previous methods to make integrated transgenic lines rely on approaches where the site of integration is essentially random {1, 2}. The chromosomal context of the integration site can have a profound effect on reporter expression, thus confounding analyses that rely on comparisons between reporter strains. Now, a single copy of a transgene can be inserted into a defined location in the genome. This approach will enable experiments designed to evaluate reporter expression level -- not just reporter expression pattern. Moreover, the technique is straightforward and can be performed by most C. elegans labs without a need for new equipment.

This paper introduces a new and highly attractive method for transgenic modification of Caenorhabditis elegans, especially since germline expression of transgenes is often inefficient due to the integration of multiple copies of a gene and subsequent gene silencing. Mos1-mediated single-copy insertion (MosSCI) allows the single integration of a gene of interest at a defined locus, enhancing expression efficiency comparable with endogenous transcript levels.

Since recent times, the authors have provided improved reagents and increased numbers of MosSCI insertion sites, which make this technique even more efficient {1}. Furthermore, Zeiser et al. {2} have presented a toolkit that allows the construction of MosSCI-compatible, germline-specific transgenic vectors by MultiSite Gateway cloning.