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. 1998 Jun 23;95(13):7520-5.
doi: 10.1073/pnas.95.13.7520.

The lepidopteran transposon vector, piggyBac, mediates germ-line transformation in the Mediterranean fruit fly

A M Handler  1 , S D McCombs, M J Fraser, S H Saul
Affiliations

The lepidopteran transposon vector, piggyBac, mediates germ-line transformation in the Mediterranean fruit fly

A M Handler et al. Proc Natl Acad Sci U S A. .

Abstract

The piggyBac (IFP2) short inverted terminal repeat transposable element from the cabbage looper Trichoplusia ni was tested for gene transfer vector function as part of a bipartite vector-helper system in the Mediterranean fruit fly Ceratitis capitata. A piggyBac vector marked with the medfly white gene was tested with a normally regulated piggyBac transposase helper at two different concentrations in a white eye host strain. Both experiments yielded transformants at an approximate frequency of 3-5%, with a total of six lines isolated having pigmented eyes with various levels of coloration. G1 transformant siblings from each line shared at least one common integration, with several sublines having an additional second integration. For the first transformant line isolated, two integrations were determined to be stable for 15 generations. For five of the lines, a piggyBac-mediated transposition was verified by sequencing the insertion site junctions isolated by inverse PCR that identified a characteristic piggyBac TTAA target site duplication. The efficient and stable transformation of the medfly with a lepidopteran vector represents transposon function over a relatively large evolutionary distance and suggests that the piggyBac system will be functional in a broad range of insects.

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Figures

Figure 1
Figure 1
Eye color phenotypes of the medfly strains we (a), wild-type (b), and the Cc[pBw] transformant sublines 220M1 (c), 69M1 (d), 53F4 (e), 5M2 (f), 134M1 (g), and 41F1 (h). Actual visual descriptions are given in Table 2.
Figure 2
Figure 2
Southern DNA blot hybridization analysis of Cc[pBw] transformant sublines, and we host strain and wild-type (wt) control samples. At the top is a schematic diagram (not to scale) of the pB[Ccw] vector showing the BglII and NsiI restriction sites used to digest the genomic DNA and the 1.4-kb SphI–HpaI and 0.95-kb HpaI–AseI piggyBac vector fragments used as hybridization probes (bars). Above the schematic are distances used to calculate internal restriction fragment sizes and minimum sizes for junction fragments. Vector sequences are shaded, and the hsp70-white marker sequences are open. DNA size markers are shown to the left of the autoradiograms.
Figure 3
Figure 3
Inverse PCR strategy to isolate and sequence the pB[Ccw] vector insertion site in transformant sublines. At the top is a schematic diagram (not to scale) of the vector insertion in the host plasmid showing the approximate location of the restriction sites and primers used for PCR. Forward (F) and reverse (R) primers are numbered according to their nucleotide position in piggyBac. The piggyBac sequence is shown shaded, the medfly white marker gene is open, and chromosomal sequence is hatched. Below are shown the piggyBac insertion site sequence in p3E1.2 and the proximal insertion site sequences from several of the transformant sublines.
See this image and copyright information in PMC

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