piggyBac internal sequences are necessary for efficient transformation of target genomes

Abstract

A previously reported piggyBac minimal sequence cartridge, which is capable of efficient transposition in embryo interplasmid transposition assays, failed to produce transformants at a significant frequency in Drosophila melanogaster compared with full-length or less extensive internal deletion constructs. We have re-examined the importance of these internal domain (ID) sequences for germline transformation using a PCR strategy that effectively adds increasing lengths of ID sequences to each terminus. A series of these piggyBac ID synthetic deletion plasmids containing the 3xP3-ECFP marker gene are compared for germline transformation of D. melanogaster. Our analyses identify a minimal sequence configuration that is sufficient for movement of piggyBac vectored sequences from plasmids into the insect genome. Southern hybridizations confirm the presence of the piggyBac transposon sequences, and insertion site analyses confirm these integrations target TTAA sites. The results verify that ID sequences adjacent to the 5' and 3' terminal repeat domains are crucial for effective germline transformation with piggyBac even though they are not required for excision or interplasmid transposition. Using this information we reconstructed an inverted repeat cartridge, ITR1.1k, and a minimal piggyBac transposon vector, pXL-BacII-ECFP, each of which contains these identified ID sequences in addition to the terminal repeat configuration previously described as essential for mobility. We confirm in independent experiments that these new minimal constructs yield transformation frequencies similar to the control piggyBac vector. Sequencing analyses of our constructs verify the position and the source of a point mutation within the 3' internal repeat sequence of our vectors that has no apparent effect on transformation efficiency.

piggyBac transposon remobilization and enhancer detection in Anopheles mosquitoes

“…In most instances where piggyBac underwent transposition in these experiments, elements integrated into TTAA sites, the preferred target site of piggyBac (Datasets S1 and S2); however, in two cases piggyBac was found in noncanonical target sites (CTAC and CTAA). Overall, these observations are consistent with previous reports on piggyBac's preferred target site in various permissive host genomes (29,30). The absence of a complete genome sequence for A. stephensi prevents analysis of the spatial patterns of transposition within the genome at this time; thus, questions regarding regional insertion preferences, intragenic insertion site preferences, and preferences for integrating into linked chromosomal sites must await the availability of more extensive genomic resources, which are expected to be forthcoming.…”

“…pXL-BacII-ECFP contains a piggyBac vector with functional terminal sequences and a marker gene consisting of the synthetic promoter 3xP3 regulating the expression of ECFP (30,39). The plasmid pMi[3xP3-DsRed]-hsp70-piggyBac contains a functional Minos vector carrying the 3xP3 promoter regulating the expression of DsRed and the piggyBac transposase ORF regulated by the promoter from the hsp70 gene in D. melanogaster (26).…”

The Carnegie Protein Trap Library: A Versatile Tool for Drosophila Developmental Studies

“…piggyBac elements are suitable for widespread mutagenesis of Drosophila genes (Thibault et al 2004) and as gene traps (Bonin and Mann 2004). Minimal sequences for piggyBac transposition were defined recently (Li et al 2005). We obtained several hundred piggyBac protein trap insertions that express EGFP, but the piggyBac gene trap vector appeared to be less efficient, on the basis of EGFP intensity and Western blotting, than P-element gene traps in nearby locations (also see accompanying article by Quiñones-Coello et al 2007, this issue).…”

piggyBac is a flexible and highly active transposon as compared to Sleeping Beauty , Tol2 , and Mos1 in mammalian cells

“…Regarding the GAL4 fusion, a PCR fragment containing GAL4 DBD with a linker (primer pairs gatcgaattcaccATGAC-CCCCCCCAAGAAGAAGC and CTCTAATAGTCCTCTGT-GGC) was cloned into the N terminus of pcDNA3.1⌬neo-piggyBac. The donor plasmid was built by inserting the selection cassette into the SmaI͞EcoRV sites of pXLBacIIPUbnlsEGFP, derived from pBSII-ITR1 (33). This donor plasmid is a minimal piggyBac vector with TR of 308 bp and 238 bp at the 5Ј and 3Ј ends, respectively.…”