Welcome to the piggyBac website!
The TTAA-specific transposon piggyBac is rapidly becoming a highly useful transposon for genetic engineering of a wide variety of species, particularly insects. The purpose of this website is to provide information related to piggyBac in particular, and its uses for transgenesis in general. I will provide information on constructs available from our lab, as well as those available from other labs as information is made available to me. I will also provide information related to the use of these constructs as it is made available, but my hope is that the forum section will serve as an information sharing tool among those who work with the element to allow everyone’s science to benefit. Please feel free to look over the site, contact me for additional information, or provide information in the forum. Please send me relevant information to include on the site. To a large extent, the success of this site is dependent on everyone’s contributions. Since this is my first website, it’s a bit of an experiment for me. I hope it proves successful! – Mac Fraser (email@example.com)
A Brief History of piggyBac
Origin of piggyBac:
The TTAA-specific, short repeat elements are a group of transposons that share similarity of structure and properties of movement. These elements were originally defined in the order Lepidoptera, but appear to be common among other animals as well. While the importance of these elements for genetic manipulation has been appreciated only recently, experimental evidence is accumulating that suggests they will be extremely useful tools for transformation of animals, particularly insects in the orders Diptera, Lepidoptera, and Coleoptera.
The original identification of these unusual TTAA-specific elements came through a somewhat unconventional route relative to most other Class II mobile elements. Spontaneous plaque morphology mutants of baculoviruses were observed to arise during propagation of these viruses in the TN-368 cell line. Genetic characterization of these mutations often revealed an associated insertion of host-derived DNAs, some of which appeared to be transposons (Fraser et al., 1983).
Several different mobile host DNA insertions have been identified within the FP locus of the baculoviruses AcMNPV and GmMNPV(Fraser et al., 1983; 1985; Cary et al., 1989; Wang et al., 1989; Bauser et al., 1996).The insertions most extensively studied are those now designated as tagalong (formerly TFP3) and piggyBac (formerly IFP2). These insertions exhibit a unique preference for TTAA target sites, whether inserting within the viral FP-locus (Cary et al. 1989; Wang et al. 1989) or at other regions of the viral genome (Wang and Fraser, 1993; Fraser et al. 1995). Both of these elements are part of a larger family of TTAA-target site specific insertion elements that includes the T. ni derived piggyBac and tagalong elements, the Spodoptera frugiperda derived elements IFP1.6 (Beames and Summers, 1988; 1990) and 290 bp insertion of Carstens (1987), and the transposon-like insertion within the EcoRI-J,N region of Autographa californica nuclear polyhedrosis virus (Oellig et al. 1987; Schetter et al. 1990), whose origin is undefined.
Interestingly, transposon-induced mutations are also associated with larval propagated virus and are not simply an artifact of in vitro propagation (Jehle et al. 1995). This emphasizes the fact that the relatively high frequency of transpositional and illegitimate (Xiong et al. 1991) recombination that occurs with these viruses is an important aspect of natural baculovirus evolution (O'Reilly and Miller, 1989; O'Reilly et al. 1992; reviewed in Blissard and Rohrmann, 1990). They may also provide a means for horizontal transmission of certain transposons among diverse species (see Fraser 1986; Fraser 2000, for a review).
More recently, analysis of sequences obtained from the human genome has revealed what appear to be 100 to 500 copies of a fossil element called LOOPER, which has sequence homology to piggyBac, terminates in 5' CCY....GGG 3', and apparently targets TTAA insertion sites (J. Jurka, pers. comm.). The LOOPER consensus sequence is on average 77% similar to individual sequences identified in the human genome, indicating it is at least 60 million years old. There are two other TTAA-specific fossil repeat elements, MER75 and MER85 (estimated at 2000 copies per genome) which appear to target TTAA insertion sites and terminate in 5' CCC....GGG 3'. Evidence is accumulating that suggests a superfamily of TTAA-specific mobile elements exists in a diversity of organisms, and that piggyBac-related sequences may be present in a diversity of species.
Why is it called piggyBac?
I’m sure that question has crossed the minds of everyone who hears the name. Well, here’s the story. When I first identified these elements as insertions in Baculovirus mutants I began by naming them IFPx, for Insertion in FP mutant “x”. Upon sequencing one of these insertions and finding that it had characteristic features of a transposon, I named it TFP3, or Transposon in FP3. This nomenclature was a bit boring, and it was hard to attract the attention of anyone outside of Baculovirology to the significance of these elements. A colleague reviewing one of my papers wrote in his review (yes, he did reveal himself afterwards) that I might get more attention for my work if I named these elements along the lines of Drosophila transposon nomenclature rather than the boring “IFP such and such”. I’d never though to do that before, but I sat down and tried to come up with a logical nomenclature based upon the characteristics of these elements. All of them inserted into infecting Baculovirus genomes, and were then carried around in these viral genomes. So I hit upon “piggyback” as the name for the one element that I was most certain would be intact and interesting. To indicate the relationship to Baculoviruses, I dropped the “k” and capitalized the “B”,hence piggyBac. I proceeded to name additional elements as tagalong, hitchhiker, and even clingon. The only problem here is I’m running out of names. I’ll have to resort to alternative languages to name future elements if they are discovered. I am indebted to my reviewer for making the suggestion. If I had not changed the name, there would be a lot less interest.
Structural features of piggyBac.
The piggyBac element is 2.4 kb in length and terminates in 13 bp perfect inverted repeats, with additional internal 19 bp inverted repeats located asymmetrically with respect to the ends (Cary et al. 1989). The initial sequence analysis of the piggyBac element revealed a potential RNA polymerase II promoter sequence configuration, typical Kozak translational start signal, and two apparently overlapping long open reading frames. Primer extension analysis with polyadenylated mRNA positioned the 5' end of the piggyBac transcript near the identified consensus promoter region (Cary et al.1989). Subsequent Northern analyses, and RT-PCR and sequencing of piggyBac-specific RNA transcripts from TN-368 cells confirmed that the major transcript is unspliced (Elick et al. 1996a). Re-examination of additional piggyBac sequences amplified from the TN-368 cell genome, as well as the plasmid p3E1.2, confirmed an error of a single base in the original sequence, and the corrected sequence could be read as a single open reading frame encoding a polypeptide with a predicted size of 64 Kd.
Later cloning and expression of the full length and truncated versions of the ORF confirmed a single protein product of a size predicted for the corrected sequence (Elick and Fraser, unpublished). However, because the sequence of piggyBac or its encoded polypeptide does not resemble any transposases previously described, there was no assurance initially that this sequence represented a full-length transposon capable of autonomous movement, or that the polypeptide encoded had the properties of a transposase.
The piggyBac ORF encodes a functional transposase.
The mobility of the piggyBac element was first examined by developing an assay based upon the already demonstrated mobilization of the element into the baculovirus genome (Fraser et al. 1995). The piggyBac element was tagged by inserting a polyhedrin-driven lacZ reporter gene (polh/lacZ) at the unique, internal PstI site. This tagged transposon, when mobilized into the target virus genome, would produce a blue plaque phenotype. The experiment was performed in the presence or absence of an unmodified helper transposon carried within the plasmid p3E1.2 (see below) by transfecting the plasmids and target viral DNA into SF21AE cells. The SF21AE cell line lacks any endogenous piggyBac-homologous elements, eliminating the possibility that mobilization could occur in the absence of the helper. Subsequent cloning and sequencing analyses established that movement of the tagged transposon into the virus genome in transfected insect cells occured via transposition only when the helper element was co-transfected (Fraser et al. 1995).
This experiment was seminal because it confirmed several important facts of piggyBac movement. First, and perhaps most importantly, the transposon itself encodes a function that facilitates its own movement, and this function acts in a trans fashion, being supplied on a helper plasmid. Second, piggyBac could transpose into the baculovirus genome in these insect cells while carrying a marker gene, polh/lacZ. Third, movement of the piggyBac element could be demonstrated in cells from a lepidopteran species distantly related to the species from which it orignated. These observations verified that this transposon could be used as a helper-dependent vector for transfer of genes in insect cells, and that piggyBac movement was not restricted to the species of origin. These observations led directly to the current interest in the piggyBac transposon as a tool for genetic engineering in insects.
Plasmid-based excision and transposition assays for piggyBac mobilization.
While the baculovirus-based assay provided a foundation for analysis of the movement of the element, the assay generated a considerable background of illegitimate recombination events between the baculovirus target and the co-transfected plasmid, making characterization of the resulting recombinant virus time consuming and labor intensive. A supF tRNA marker gene within the context of the transposon was used to follow movement of the tagged element from the plasmid as detected by reversion to a white colony phenotype in MBL50 cells (Elick et al. 1996b). In all cases we were able to define excision products as being precise, regenerating the characteristic TTAA target site in the plasmid. A second assay was easily developed from the first by constructing a vector having a duplication of the 3' terminal repeat, and tagged with supF between the 5' terminal repeat and the first 3' repeat domain, and a kanamycin resistance gene inserted between the two 3' terminal repeats (Elick et al. 1997). This construct allowed us to replace the proximal 3' terminus with various mutant repeats and examine the effect of mutations in the target site or 3' terminus on the excision of the element. Mutations in the proximal repeat that were prohibitive would force utilization of the distal terminus for excision (Elick et al. 1997). The assay was dependent on the fact that either the proximal repeat region would be favored in non-mutagenized constructs, or that both distal and proximal repeats would be utilized with equal frequency. The control experiment using wild-type termini demonstrated that both terminal repeats were used with equal frequency (Elick et al. 1997) suggesting that piggyBac transposase does not operate by scanning from an internal binding site towards the termini, but rather seems to identify termini directly.
Substitutions and deletions of bases in the 5' GGGTTAA 3' sequence revealed an essential requirement for the terminal 5' GGG 3' residues, as well as an essential requirement for the TTA of the TTAA target site (Elick et al. 1997). These excision results emphasize the uniqueness of the TTAA-specific transposons, and piggyBac in particular. Our research has established that excision of these TTAA-specific elements such as piggyBac and tagalong must involve direct breakage and then immediate joining of the DNA strands at the excision breakpoint (Elick et al. 1997).
Interplasmid transposition assays demonstrate mobility of piggyBac.
While there is reason to believe that excision and transposition are coupled events for the piggyBac transposon, particularly if a cut-and-paste model is proposed, experimental examination of transpositional movement of piggyBac in a species cannot be verified simply through the use of an excision assay. Thibault et al. (1999) were able to demonstrate interplasmid transposition in the pink bollworm, Pectinophora gossypiella using the phspBac helper construct (Handler and Harrell,1999). Both excision and interplasmid transposition were dependent on the presence of the helper transposon. In their hands, the frequencies of interplasmid transposition were comparable to those obtained for other elements developed as transformation vectors for insects. Distribution of the insertions within the target plasmid was restricted to TTAA target sites, as expected, but there appeared to be some preference for only 9 of the possible 21 TTAA sites that might be recovered. Similar analyses were conductedin embryos of D. melanogaster, Ae. aegypti, and T. ni (Lobo et al.1999), and more recently,Ae. albopictus and Ae. triseriatus. To verify a cut and paste mechanism, we employed a DpnI digestion assay that established transposition of piggyBac from the E. coli plasmid is non-replicative (Lobo et al. 1999).
Transformation of insects with piggyBac.
While assays that provide evidence for the movement of a transposon in embryos of a given species are suggestive of the possibility for transformation of that species, they do not provide conclusive proof that a given transposon will be useful for transformation of that species. Many variables impinge on the relative utility of a given element as a vector for transgenesis. Transposon related variables include the relative efficiency of movement into the genome, stability of the transposon insertion in the genome, and the potential for cross-mobilizing or suppressive effects from similar resident elements. Variables related to the insect being examined include the availability of suitable selectable marker genes, survivorship of microinjected embryos, and the survivorship and fecundity of emerged injected insects. Refinements in technique and identification of suitable marker genes have contributed at least as much as the application of new transposons have to the recent surge in the successful transgenic engineering of key species.
The first definitive demonstration of genetic transformation of an insect by the piggyBac transposon was accomplished using the Mediterranean Fruit Fly as a model system (Handler et al. 1998). This system had the advantage of a recently identified white-eye mutation and complementing gene. Insertion of the C. capitata white-eye marker into the piggyBac transposon at the unique HpaI site had no deleterious effect on the movement of the transposon. Co-injection with the helper allowed rescue of the white-eyed flies to a re-eyed coloration. Subsequent hybridization, inverse PCR amplifiation, and sequencing analyses confirmed the insertion of the entire transposon carrying the white gene into the C. capitata genome, with the duplication of a TTAA target site. In one strain, maintenance of the gene at its original position was documented through 15 generations, indicating the piggyBac can provide stable transformation ofthis species. Since this initital report there have been numerous reports of successful transformations of insects in the orders Coleoptera, Diptera, and Lepidoptera using the piggyBac transposon (see list of piggyBac related publications).
The simplicity of movement of this element, and often high frequency of transformation, make it an attractive tool for genetic manipulation of insects. We believe that these properties can be exploited for an even broader range of invertebrate species, and perhaps even vertebrate species, and are hopeful that more attempts will be made to utilize the element for transgenic studies.