Construction and characterization of new piggyBac vectors for constitutive or inducible expression of heterologous gene pairs and the identification of a previously unrecognized activator sequence in piggyBac.
BACKGROUND
We constructed and characterized several new piggyBac vectors to provide transposition of constitutively- or inducibly-expressible heterologous gene pairs. The dual constitutive control element consists of back-to-back copies of a baculovirus immediate early (ie1) promoter separated by a baculovirus enhancer (hr5). The dual inducible control element consists of back-to-back copies of a minimal cytomegalovirus (CMVmin) promoter separated by a synthetic operator (TetO7), which drives transcription in the presence of a mutant transcriptional repressor plus tetracycline.


RESULTS
Characterization of these vectors revealed an unexpected position effect, in which heterologous genes adjacent to the 3'- terminal region ("rightward" genes) were consistently expressed at higher levels than those adjacent to the 5'-terminal region ("leftward" genes) of the piggyBac element. This position effect was observed with all six heterologous genes examined and with both transcriptional control elements. Further analysis demonstrated that this position effect resulted from stimulation of rightward gene expression by the internal domain sequence of the 3'-terminal region of piggyBac. Inserting a copy of this sequence into the 5'- terminal repeat region of our new piggyBac vectors in either orientation stimulated leftward gene expression. Representative piggyBac vectors designed for constitutive or inducible expression of heterologous gene pairs were shown to be functional as insect transformation vectors.


CONCLUSIONS
This study is significant because (a) it demonstrates the utility of a strategy for the construction of piggyBac vectors that can provide constitutive or inducible heterologous gene pair expression and (b) it reveals the presence of a previously unrecognized transcriptional activator in piggyBac, which is an important and increasingly utilized transposable element.
Shi
Xianzong
X
Department of Molecular Biology University of Wyoming 1000 E, University Avenue Laramie, WY 82071, USA. dljarvis@uwyo.edu
Harrison
Robert L
RL
Hollister
Jason R
JR
Mohammed
Ahmed
A
Fraser
Malcolm J
MJ
Jarvis
Donald L
DL
D000818:Animals; D001483:Base Sequence; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D004251:DNA Transposable Elements / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D007915:Lepidoptera / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D015533:Transcriptional Activation / Q000235:genetics*; D014170:Transformation, Genetic / Q000235:genetics
D004251:DNA Transposable Elements