* present affiliation: Veterinary Consultancy, Conrada 16/65, 01-922 Warsaw, Poland
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 (www.mpi-cbg.de/services-facilities/core-facilities/genome-engineering-facility/), for providing the p-glh-2 driven phiC31 integrase construct and the friendly and supportive laboratory environment.
Frøkjaer-Jensen C, Davis MW, Hopkins CE, Newman BJ, Thummel JM, Olesen SP, Grunnet M, Jorgensen EM. Single-copy insertion of transgenes in Caenorhabditis elegans
Nat Genet. 2008 Nov;40(11):1375-83. doi: 10.1038/ng.248.