Stable transformation of a Mamestra brassicae (lepidoptera) cell line with the lepidopteran-derived transposon piggyBac

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. (C) 2002 Elsevier Science Ltd. All rights reserved.