|Fraser Jr., Malcolm|
Submitted to: BioMed Central (BMC)Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/18/2007
Publication Date: 2/2/2007
Citation: Shi, X., Harrison, R.L., Hollister, J.R., Mohammed, A., Fraser Jr., M.J., Jarvis, D.L. 2007. 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. BMC Biotechnology. 7(5). Available: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1783651 Interpretive Summary: Transposable genetic elements are pieces of DNA that can be used to introduce novel genes into organisms for research and industrial purposes. PiggyBac elements are one such group of transposable elements that were first identified when they caused mutations in a group of insect viruses known as baculoviruses. The piggyBac elements can be used to place new genes into a wide variety of organisms. A drawback to the use of current piggyBac elements is that only one gene at a time can be introduced into the organism of interest, when sometimes scientists want to introduce more genes simultaneously. For this study, we constructed and tested new piggyBac elements capable of introducing two genes at a time into an organism. In the process, we discovered that a part of the piggyBac DNA sequence can be manipulated to increase the amount of gene product made from the genes carried by the piggyBac element, thus improving its usefulness. The new elements described in this study will be of interest as tools for researchers with a need to introduce multiple genes into a single target organism.
Technical Abstract: The piggyBac transposable element was first identified when it inserted itself into baculovirus genes during infection, causing a mutant plaque morphology phenotype. Since then, piggyBac has been developed into a germline tranformation vector usable with a wide variety of organisms. In this study, we report the construction of new piggyBac vectors designed for the transposition of constitutively- or inducibly-expressible pairs of heterologous genes. The dual constitutive control element in these vectors consisted of two back-to-back copies of a baculovirus immediate early gene (ie1) promoter separated by a baculovirus enhancer (hr5). The dual inducible control element consisted of two back-to-back copies of a minimal cytomegalovirus immediate early gene (CMVmin) promoter separated by a synthetic operator (TetO7), which drives transcription in the presence of a mutant transcriptional repressor plus tetracycline. Characterization of these vectors revealed an unexpected position effect in which the heterologous genes inserted in the leftward orientation consistently induced higher enzyme activity levels than those inserted in the rightward orientation. An examination of the mechanism underlying this position effect revealed that it reflected stimulation of leftward gene expression by asequence known as the internal domain sequence in the left terminal repeat region of piggyBac. Further analysis revealed that a copy of this sequence stimulated expression, albeit to different levels, in an orientation-independent fashion when inserted downstream of the rightward genes. This study is significant because (a) it yielded a substantial new set of dual piggyBac vectors for constitutive or inducible expression of heterologous gene pairs and (b) it revealed the presence of a previously unrecognized transcriptional activator in piggyBac, an important and increasingly utilized transposable element.