Abstract
Previous studies have shown that the transposase and the inverted terminal repeat (ITR) of the Mos1 mariner elements are suboptimal for transposition; and that hyperactive transposases and transposon with more efficient ITR configurations can be obtained by rational molecular engineering. In an attempt to determine the extent to which this element is suboptimal for transposition, we investigate here the impact of the three main DNA components on its transposition efficiency in bacteria and in vitro. We found that combinations of natural and synthetic ITRs obtained by systematic evolution of ligands by exponential enrichment did increase the transposition rate. We observed that when untranslated terminal regions were associated with their respective natural ITRs, they acted as transposition enhancers, probably via the early transposition steps. Finally, we demonstrated that the integrity of the Mos1 inner region was essential for transposition. These findings allowed us to propose prototypes of optimized Mos1 vectors, and to define the best sequence features of their associated marker cassettes. These vector prototypes were assayed in HeLa cells, in which Mos1 vectors had so far been found to be inactive. The results obtained revealed that using these prototypes does not circumvent this problem. However, such vectors can be expected to provide new tools for the use in genome engineering in systems such as Caenorhabditis elegans in which Mos1 is very active.
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Abbreviations
- HTH:
-
Helix-turn-helix
- ITR:
-
Inverted terminal repeat
- ORF:
-
Open reading frame
- SELEX:
-
Systematic evolution of ligands by exponential enrichment
- TE:
-
Transposable element
- UTR:
-
Untranslated terminal region
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Acknowledgments
This work was supported by the University of François Rabelais of Tours, and funded by grants from the European Commission (Project SyntheGeneDelivery, N°018716), the C.N.R.S., the Ministère de l’Education Nationale, de la Recherche et de la Technologie, the Association Française contre la Myopathie, and the Groupement de Recherche CNRS 2157. The technical assistance of Sylvie Bigot is warmly acknowledged. The English text has been revised by Dr M. Ghosh.
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Communicated by M. Grandbastien.
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Casteret, S., Chbab, N., Cambefort, J. et al. Physical properties of DNA components affecting the transposition efficiency of the mariner Mos1 element. Mol Genet Genomics 282, 531–546 (2009). https://doi.org/10.1007/s00438-009-0484-0
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DOI: https://doi.org/10.1007/s00438-009-0484-0