The relationship between internal domain sequences of <italic>piggyBac</italic> and its transposition efficiency in BmN cells and <italic>Bombyx mori</italic>

Abstract

The piggyBac transposon, which includes terminal inverted repeat sequences and internal domain (ID) sequences, is widely used as a tool for insect transformation. To optimize this system for transgenic research on Bombyx mori, we examined the effects of the amount of the transposase plasmid and its ID sequences on the expression of green fluorescent protein (GFP). 0 ID sequence were constructed with GFP as the marker. After transfecting these four plasmids into BmN cells, we analyzed the transfecting efficiency by comparing the GFP positive to negative cell ratio. Our results indicated that plasmid pB-4 got the highest ratio on the 22nd day. Moreover, the GFP positive to negative cell ratio increased with higher amount of transposase plasmid without overproduction inhibition. Furthermore, we injected three piggyBac transposon plasmids, pB[A3GFP]-1, pB[33P3GFP]-3, and pB[33P3RFP]-4 harboring different markers into preblastoderm stage eggs of B. mori, and found that the transformation efficiency of pB[33P3RFP]-4 was 3.8 folds higher than pB[A3GFP]-1, whereas pB[33P3GFP]-3 failed to produce transformants. Our results suggested that pB-4 may be one of the best piggyBac transposon plasmids currently available for germline transformation in B. mori.

Insect transformation with piggyBac: getting the number of injections just right

“…The 2472‐bp‐long element is structured with two sets of inverted repeats at both ends and a central transposase‐encoding open reading frame (Fraser, ). The insertion site of piggyBac is quasi‐random, with a cut‐and‐paste insertion at the short genome motif site of TTAA (O'Brochta, ; Wu & Burgess, ; Zhuang et al ., ).…”

Minimal piggyBac vectors for chromatin integration

“…PiggyBac vectors are one of the most active and flexible transposon systems available for the stable transfection of mammalian cells. 6,7 The wild-type piggyBac transposon is 2472 base pairs in length, and is composed of two inverted minimal terminal repeats (minTRs), two internal domain sequences and a transposase-encoding domain 8,9 (Figure 1, wild type). Transposase catalyzes the excision of the transposon from one DNA source (that is, a delivered plasmid) and allows its subsequent re-integration into another DNA source (that is, the host cell genome).…”

Analysis of the activity of virus internal ribosome entry site in silkworm <italic>Bombyx mori</italic>

“…3(A)]. As described in our previous report [11], the plasmid pBTrans-RE-IRES-EGFP-3 Â P3-RFP was constructed on the basis of plasmid structure of pB-4 transposon [ Fig. 3(B)].…”

“…Cell line of Sf9 and dual-fluorescence plasmid pFastBacA3RLuc-FL650FLuc were kindly provided by Prof. Changde Lu (Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Shanghai, China). pB-4 and competent Escherichia coli DH10Bac [EGT gene had been knocked out from Autographa californica nuclear polyhedrosis virus (AcNPV) genome] were also preserved in Silkworm Genetic Laboratory [10,11].…”