A rapid method for generating and finding ΦC31 landing sites (attP) with the desired expression characteristics as well as a new method for creating transgene arrays in vivo was recently described by Knapp et al (2015).
Albeit these methods were developed in and applied to Drosophila melanogaster, but they could be applied to species for which transposon-based technologies are available and this list of species continues to grow.
Site specific recombination systems, ΦC31 in particular, have been very useful because they have permitted transgenes to be integrated into known locations of a genome that are not subject to unwanted influences of endogenous regulatory sequences (‘position effects’). In addition, at least in D. melanogaster, rates of integration into these fixed sites can be quite high, making the creation of genetically modified flies that much more efficient.
Finding ‘good’ landing sites is a challenge, as Knapp et al (2015) recount – ‘leaky’ gene expression from sites, variable expression levels among sites, and the degree of gene expression from a given site can vary depending on the cell or tissue in which the transgene is being expressed.
Knapp et al (2015) describe how they placed a “test gene” that was integrated into an active transposon by site specific recombination and then jumped this ‘test gene’-containing transposon around the genome. They used this as a preliminary means for identifying positions in the genome that allowed their ‘test gene’ to be expressed in its well known cell-type and tissue specific pattern. Notably, of the 172 test sites screened, 80% failed initial screening and only 5 met all of their criteria. So, ‘good sites’ are not common in D. melanogaster.
One feature of ΦC31 recombination is that recombined attP and attB sites are no longer substrates for ΦC31 integrase. However, there are integrase variants that do have excisionase activity. Once a ‘good site’ was found based on ‘test gene’ expression Knapp et al (2015) used these integrase variants to excise the ‘test gene’ to reconstitute functional attP sites that could then be used subsequently as a landing site for any transgene.
These variant integrases with excisionase activity also have integrase activity and can use attP-attB hybrid sites (attL and attR sites). Knapp et al (2015) use these activities to create transgene arrays in vivo – integrating transgenes next to existing transgenes.
The system Knapp et al (2015) describe broadens the utility of the ΦC31 system in D. melanogaster. Is any of this relevant to other insect systems? I think so.
A few ΦC31 landing sites have been incorporated into 3 mosquito species but these have not been extensively characterized with respect to the kinds of variable expression seen in D. melanogaster nor have they provided better integration options than direct transgene integration using transposons. But the ΦC31 system has not been well-exercised in mosquitoes so its potential is definitely unrealized.
Knapp et al (2015) ‘site-finding’ system depends on ‘hopping’ a ‘test gene’ around the genome of the host using transposon remobilization. piggyBac remobilization is well established and efficient in Anopheles stephensi, untested in An. gambiae and non-existent in Aedes aegypti (piggyBac seems incapable of being remobilized in this species for unknown reasons). So, at least in the mosquito An. stephensi, Knapp et al’s (2015) strategy could be immediately applied.
Likewise, in the lady beetle Harmonia axyridis piggyBac can be remobilized, as well as Tribolium castaneum and Bombyx mori. So here too Knapp et al’s (2015) ‘site finding’ system could be used with great effect.
I definitely think Knapp et al’s (2015) strategy is worth being aware of if you work on these and perhaps other insect systems. Certainly their manuscript provides a nice primer on the use of the ΦC31 system and the precautions one needs to take if some of the advantages of using this system are to be realized.
Jon-Michael Knapp,Phuong Chung, and Julie H. Simpson (2015) Generating Customized Transgene Landing Sites and Multi-Transgene Arrays in Drosophila Using phiC31 Integrase
Genetics 114.173187; Early online February 12, 2015, doi:10.1534/genetics.114.173187