In systems other than worms (i.e. and also vertebrate cells) Streptomyces temperate phage phiC31 integrase was reported as an efficient mean of inserting the transgenes into specific sites of the genome. In the summer of 2013 we have made an experimental attempt to evaluate if phiC31 integrase would be of possible use in C. elegans. For this purpose we implemented the unc-119(ed3) mutant rescue (1), by linking of two split segments of the unc-119 rescuing cassette. Split segments of unc-119 are two separate plasmids providing respectively: unc-119 promoter and N-terminal UNC-119 CDS and C-terminal UNC-119 followed by unc-119 3′ utr (split at the third intron and flanked with the appropriate phiC31 recognition sites). In addition the third construct was used, intended to provide the phiC31 integrase into the germline cells (essentially based on pJL43.1 – p-glh-2::MosTase::glh-2-utr, where the Mos1 transposase (2, 3) was replaced by codon optimized phiC31 integrase) to facilitate the transgene engineering.

Using the above constructs, we obtained the following evidence for the phiC31 acting efficiently on the transgenic extrachromosomal arrays. We assumed here, the two separate plasmids containing split of two segments of unc-119 gene will rescue the ed3 allele in DP38 derived strain, only if properly re-assembled into functional unc-119 gene. To test for the above assumption the above plasmids, where co-injected into unc-119(-) hermaphrodites carrying homozygous ed3 allele. In the first experiment, two of the above plasmids containing split of two segments of the unc-119 gene, were injected (along with fluorescent markers*) into gonads of the DP38 hermaphrodites. Based on the segregation of the fluorescent markers in the transgenic progeny in this experiment, we concluded that while the injected split unc-119 segments do efficiently form the heritable transgenic array, however fail to rescue the uncoordinated phenotype (observed at least four 4 independent array forming events in 30 injected hermaphrodites).

We contrast the above observation, with the results of the second experiment, where the above plasmids where co-injected with the phiC31 integrase providing plasmid (again along with fluorescent markers) into gonads of DP38 derived hermaphrodites. We observed at least 4 independent events (in 28 injected hermaphrodites) of formation of the heritable arrays resulting in non-uncoordinated movement in unc-119(-) animals, when the split segments of unc-119 were provided with the third plasmid encoding for phiC31 integrase. Consistently the above four of the unc-119 rescued lines, were proven resistant to G418 indicating for the transmission of the selectable marker on the extrachromosomal array and the restored unc-119 function, depending strictly on the germinal provision of the phiC31 expressing plasmid. Collectively, we think we do have  enough evidence, to conclude the phiC31 is working on the transgenic arrays formed of the split unc-119 segments. We think this demonstration could be possibly useful in for example array-array transgenic engineering technology i.e. phiC31 targeted  linking of the two separate extrachromosomal arrays.

* those lines where propagated under G418 selection, as the intact NeoR gene was included into one of the constructs, providing additional line of the confidence over the selection based on fluorescent markers when maintaining the transgenic arrays in the (slow growing) non-rescued uncoordinated unc-119(-) line.

Acknowledgments: We are grateful to Mihail Sarov (, for providing the p-glh-2 driven phiC31 integrase construct and the friendly and supportive laboratory environment.