Friday, November 9, 2012

Tn5 as an Insect Gene Vector

Few non-microbial successes for the use of EZ-Tn5™ transposons have been reported, and it is worth going back into the archives to highlight novel uses of the EZ-Tn5™ system (previously known as EZ::Tn™). David O’Brochta and his research team at the University of Maryland report success on using a custom EZ-Tn5 transposon to transfect and insert a custom transposon into the nucleus of the mosquito Aedes aegypti (strain Orlando). The transposon was constructed using the EZ-Tn5 pMOD-2 vector and was developed to include the following markers: (1) The DsRed gene, a marker that conveys autofluorescence when expressed, and (2) a brain and ganglion-specific promoter to tag the Aedes aegypti germ line. A second round of experiments used the EZ-Tn5 KAN-1 Transposome  (since replaced with the EZ-Tn5 KAN-2 Transposome product) to tag the plasmid pUCSacRB, previously documented as a useful tool in determining resistance to sucrose at concentrations >5%. Insertion of the Transposome into the SacRB gene of the plasmid inactivates it and results in resistance to sucrose-based lysis. The plasmid was transfected into the embryonic cells by microinjection. The embryos were permitted to develop after which the gDNA was isolated and transformed into E. coli DH10B and Kanamycin-resistant clones isolated. Evidence for the physical integration of the Tn5::3xP3DsRed element was obtained from Southern hybridization and transposon display experiments.

The authors found that further optimization of the system for the control of self-integration and transposition efficiency would be useful. The results demonstrate the successful use of the EZ-Tn5 Transposon system for developing transposon-based mutagenesis assays for non-bacterial gene expression studies.

ResearchBlogging.orgRowan KH et al. (2004). Tn5 as an insect gene vector. Insect biochemistry and molecular biology, 34 (7), 695-705 PMID: 15242711

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