Abstract
Cabbage moth cells were transfected with the vector pBac{3xP3-EGFPafm} and helper phsp-pBac. Seventeen percent of the transfected cells showed stable EGFP-expression. This indicates successful and stable transformation of M. brassicae cells with a piggyBac-derived vector. Genomic integration of Bac{3xP3-EGFPafm} in stably transformed cells was confirmed by Southern blots and inverse PCR. Since the integrations are stable, and transfection with pBac{3xP3-EGFPafm} alone did not yield in transformations, no cross-reacting transposase activity seems present in M. brassicae cells. Moreover, Southern blotting with a probe for piggyBac transposase indicated the absence of piggyBac-related elements in the genome of Mamestra brassicae. Due to the tissue specificity of the 3xP3-EGFP marker for eye and nervous tissues, it is intriguing that 3xP3-EGFP can successfully be used to identify stably transformed M. brassicae cells of cell line IZD-MB0503, which is hemocyte-derived. Sequence analysis of the insertion sites showed that piggyBac inverted repeats were adjacent to TTAA sequences on both termini in all the clones. The present results are particularly important as they suggest that piggyBac can be used for transgenesis of cabbage moth cells.
Highlights
Mamestra brassicae is a cosmopolitan and polyphagous insect that lives in temperate regions of Eurasia on cruciferae plants
In view of results obtained with other pest crop insects, genetic engineering techniques will allow the development of new strategies for M. brassicae control in the field
We report that the piggyBac transposable element from the cabbage looper T. ni can be used for the effective and stable transformation of a M. brassicae cell line and that the 3xP3-EGFP marker gene (Berghammer et al, 1999; Horn et al, 2000, 2002) can be used to identify such lines
Summary
Mamestra brassicae is a cosmopolitan and polyphagous insect that lives in temperate regions of Eurasia on cruciferae plants. Mutagenesis by insertion of transposable elements into the genome of such a cell line could be interesting to identify and characterize the genes involved in apoptosis of insect cells. We report that the piggyBac transposable element from the cabbage looper T. ni can be used for the effective and stable transformation of a M. brassicae cell line and that the 3xP3-EGFP marker gene (Berghammer et al, 1999; Horn et al, 2000, 2002) can be used to identify such lines.
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