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Published as doi: 10.1096/fj.05-5485fje.
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(The FASEB Journal. 2006;20:1880-1882.)
© 2006 FASEB

Chimeric Mos1 and piggyBac transposases result in site-directed integration

K. J. Maragathavally, J. M. Kaminski* and C. J. Coates1

Department of Entomology, Texas A&M University, College Station, Texas, USA; and
* Department of Radiology, Medical College of Georgia, Augusta, Georgia, USA

1Correspondence: Department of Entomology, Texas A&M University, College Station, Texas, USA. E-mail: ccoates{at}tamu.edu

ABSTRACT

Genetic transformation systems based on Mos1 and piggyBac transposable elements are used to achieve stable chromosomal integration. However, integration sites are randomly distributed in the genome and transgene expression can be influenced by position effects. We developed a novel technology that utilizes chimeric transposases to direct integration into specific sites on a target DNA molecule. The Gal4 DNA binding domain was fused to the NH2 terminus of the Mos1 and piggyBac transposases and a target plasmid was created that contained upstream activating sequences (UAS), to which the Gal4 DBD binds with high affinity. The transpositional activity of the Gal4-Mos1 transposase was 12.7-fold higher compared to controls where the Gal4-UAS interaction was absent and 96% of the recovered transposition products were identical, with integration occurring at the same TA site. In a parallel experiment, a Gal4-piggyBac transposase resulted in an 11.6-fold increase in transpositional activity compared to controls, with 67% of the integrations occurring at a single TTAA site. This technology has the potential to minimize nonspecific integration events that may result in insertional mutagenesis and reduced fitness. Site-directed integration will be advantageous to the manipulation of genomes, study of gene function, and for the development of gene therapy techniques.—Maragathavally, K. J., Kaminski, J. M., Coates, C. J. Chimeric Mos1 and piggyBac transposases result in site-directed integration.


Key Words: mariner • Gal4-UAS • transposition assay

TRANSGENIC TECHNOLOGIES PROVIDE powerful tools for the analysis of gene function and the development of genetically modified insect strains for use in agriculture and to combat disease transmission. Transposable elements have been used to transform a wide range of insect species and serve as versatile insect transformation vectors (1 , 2) . However, increasing the efficiency and utility of genetic transformation for insects remains a pressing research problem. Even with the enormous success of gene transfer technology using transposable elements, a consistent set of problems occur, including low transformation efficiency, low transgene expression levels and unexpected transgene expression patterns due to 1) inefficient delivery of the genetic material to the nucleus; 2) unpredictable integration of transgenes due to random insertion into the genome; and 3) misexpression of the transgene due to proximal genetic control elements such as enhancers, promoters, repressors, or as a result of local chromatin folding (3 4 5) .

Transposons have been used as gene vectors in a variety of species including Drosophila, mosquitoes, silkworm, zebrafish, and mouse (6 7 8 9 10 11) . In this study, we used the Mos1 and piggyBac class II transposable elements. Mos1 was isolated from Drosophila mauritiana and is 1286 bp long with 28 bp inverted terminal repeats (ITRs). It has been used to transform a number of species (12 13 14 15 16 17 18) . Tc1/mariner type transposons utilize TA dinucleotide target sites, which are duplicated on insertion (19) . However, integration does not occur with equal frequency into every TA target. For germline transformation systems, transposition is mediated by the transposase, encoded by a helper plasmid, which interacts with the ITRs of a transposon on a donor plasmid to catalyze the excision and insertion into a target site TA dinucleotide in the chromosomes (20) . The transformation efficiency of Mos1 in insects is reported to be less than 5% (1 , 21) .

The piggyBac transposon from the cabbage looper moth, Trichoplusia ni, is 2472 bp long with 13 bp inverted terminal repeats and inserts into the tetranucleotide, TTAA, which is duplicated on insertion (22 , 23) . piggyBac elements have been successfully used to transform the germline of more than a dozen species of insects (24 , 25) .

Site-selective integrating nonviral vectors

Transposon insertion into a functional gene will inactivate it and insertion near regulatory sequences can alter transgene or endogenous gene activity. Integration at predefined sites would result in the appropriate expression of the transgene, and would thus avoid several potential side effects. Therefore, it is important to develop site-selective integration technologies that are highly efficient. There are several integration systems available to facilitate site selective integration, derived from a wide variety of sources (26 27 28 29) .

Recombinase-based DNA integration systems use 30 to 200 nucleotide recognition sequences with integration occurring by double helices exchange and ligation. Site-specific recombinases recognize and pair specific DNA sequences, snip the strands and reconnect. The site-specific recombinases are better termed sequence-selective because of their ability to also recognize pseudo-sites. For example, there are at least 100 pseudo-sites in the human genome that phiC31 recombinase can recognize (30) . Serine recombinases (Resolvases) are structurally different from Tyrosine recombinases (Integrases), however their biochemical properties overlap. The tyrosine recombinases’ catalytic domain and DNA recognition domain are closely interwoven, such that changes in specificity often result in loss of activity. Serine-recombinases have spatially distinct binding and catalytic domains, so these are likely to be more easily manipulated to achieve site-selective integration (reviewed in 7). The Cre-mediated recombination system is effective in prokaryotic and eukaryotic cells. Cre from Bacteriophage P1 and FLP from Saccharomyces cerevisiae belong to the integrase class of recombinases. Recombination takes place between two target sites, LoxP and FRT, for Cre and FLP respectively. Each consists of 34 bp inverted repeats that are used for site specific integration. However, Cre has higher affinity for its target than FLP (31) . The human and mouse genomes contain potential targets for Cre activity. The site specificity of Cre has been used to achieve the integration of therapeutic genes (32) . Both Cre and FLP can recognize target sites flanking a genomic region of interest and can result in inversions and deletion events.

In contrast to Cre, phage phiC31 integrase recognizes "attB" and "attP" target sites, which are dissimilar in sequence and serve as efficient targets for precise site-specific integration. The phiC31 integrase uses a serine-catalyzed reaction mechanism and offers unidirectional integration. Pseudo attP sites in the human and mouse genome are recognized by the phiC31 integrase with significant efficiency, which can lead to genetic alteration at selective genomic sites (30) . The Tn3 resolvase is an efficient site-selective recombinase enzyme that requires directly repeated recombination sites (res) present in a supercoiled DNA molecule. However, both tyrosine (e.g., Cre) and serine (e.g., phiC31) recombinases are mutagnenic (i.e., deletions and substitutions may occur), resulting from multiple target pseudo-sites in the genome or an imperfect recombination reaction (33) .

Encouraging results have emerged utilizing phiC31 and recombination-mediated cassette exchange (RCME) mediated by either Cre or FLP for the production of transgenic insects (34 35 36) . RCME occurs between DNA segments that are flanked by heterospecific target sites, thereby increasing the probability of intermolecular exchange, while minimizing intramolecular excision from the target DNA. Horn and Handler (35) devised a novel FLP recombinase-mediated cassette exchange strategy with a homing sequence from the Drosophila’s linotte locus, which resulted in recombinant Drosophila with targeted insertions at a frequency of 23%.

The incorporation of exogenous DNA binding domains to the transposition or recombination machinery can potentially increase transformation efficiency and result in site-specific integration. Kaminski et al. (37) first proposed the use of DBD-transposase fusion proteins to direct integration at defined target sites. In this study we utilized the Gal4-UAS system to achieve site-directed integration through a chimeric transposase system. The Gal4-UAS system has been used to study gene function in Drosophila (38) , vertebrates (39 , 40) , and plants (41) . Transcriptional activators such as Gal4 often contain separable DNA binding and transactivation domains. The former allows them to bind to a specific target and the latter to recruit the basal transcription machinery. In the present study, we fused the Gal4 DNA binding domain (DBD) to the Mos1 and piggyBac transposases. The Gal4 DBD thus brings the transposase and associated transgene to the specific UAS target site present on the target plasmid. Here we present the results of plasmid-based transposition assays in Aedes aegypti embryos to analyze the efficiency of Gal4-Mos1 and Gal4-piggyBac chimeric transposases. This technology would be profoundly useful for the functional analysis of target genomes and for gene therapy applications. The potential of transposon-based gene therapy is currently under further consideration (6 , 7 , 42) .

MATERIALS AND METHODS

Aedes aegypti (Liverpool strain) mosquitoes were reared in plastic boxes with sufficient water at 25 ± 1°C and 80 ± 5% relative humidity with 12 h cycles of light and dark. Approximately 300 larvae/box were maintained on Rich Mix Tetramin (Tetra Sales, Blacksburg, VA, USA) until pupation. Female and male pupae were placed into cartons covered with fine mesh until adults emerged. Adult mosquitoes were maintained on 10% sucrose-soaked cotton balls. The sucrose was removed 24 h prior to blood feeding, and the females were fed on female mice.

Plasmid constructs
pGDV1-UAS
A DNA fragment containing five copies of the upstream activating sequence 5'-CGGAGTACTGTCCTCCG-3' (UAS) was isolated from the pUAST plasmid (43) by BamHI and EcoRI digestion and ligated into the pBSKS vector (Stratagene, CA, USA). The DNA sequence was verified and was then polymerase chain reaction (PCR) -amplified using primers incorporating Sac1 recognition sites. The PCR amplification product was digested and cloned into the Sac1 (2015) site of a modified pGDV1 target plasmid (44) that contains an E. coli ori at the unique Hinc1I site. Subsequently, the E. coli ori was removed from pGDV1 by restriction digestion with Sph1 and Xba1 and after blunt-ending and self-ligation, the plasmid was transformed into chemical-competent Bacillus subtilis cells for propagation and DNA preparation.

Mos1-Gal4 DBD transposase fusion helper plasmid
The Gal4 DBD was produced by PCR amplification using primers incorporating Sac1I recognition sites and the pAS1–2 plasmid (Clontech, CA, USA) as a template. A consensus nuclear localization signal (NLS) sequence (TPPKKKRKVED) (45) was incorporated upstream of the Gal4 DBD as part of the PCR forward primer (5'-CCGCGGATGACCCCCCCCAAGAAGAAGCGCAAGGTGGAGGACGGAATGAAAGCGTTAACGGCC-3'). A flexible linker (KLGGGAPAVGGGPK) (45) was inserted between the Gal4 DBD and the transposase by incorporation of the linker sequence in the reverse PCR primer (5'-CCGCGGAGCTTGGGGCCGCCGCCCACGGCGGGGGCGCCGCCGCCCAACTTCAGCCAGTCGCCGTTGCG-3') used to amplify the Gal4 DBD. The PCR product was cloned into the pGemT vector (Promega, Madison WI, USA) and the DNA sequence verified. The DBD was excised as a Sac1I fragment and subcloned into a Mos1 transposase helper plasmid (pIE1-Mos1ORF) at the SacII site between the hr5-IE1 enhancer-promoter and the Mos1 transposase. The resulting vector sequence was verified and named pIE1-Gal4-Mos1 (Fig. 1 .).


Figure 1
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  		Figure 1. Structure of the pIE1-Gal4-Mos helper plasmid. The hr5-IE1 enhancer-promoter regulatory region is used to drive the expression of the transposase. The GAL4 DNA binding domain is fused in frame upstream of the Mos1 transposase gene. A nuclear localization signal (NLS) is placed before the DBD, with a linker sequence (L) used to separate the DBD and transposase. The sizes of each genetic element are represented below each segment (base pairs).

piggyBac-Gal4 DBD transposase fusion helper plasmid
The Gal4 DBD was PCR amplified as above using the same forward primer containing the NLS sequence and Sac1I recognition site. The reverse primer contained the same flexible linker sequence and was modified by a single base (shown in bold) to ensure the maintenance of the correct reading frame with the piggyBac transposase (5'-CCGCGGCCTTGGGGCCGCCGCCCACGGCGGGGGCGCCGCCGCCCAACTTCAGCCAGTCGCCGTTGCG-3'). The amplified Gal4 DBD was cloned into a piggyBac helper plasmid (pIE1–3 pB ORF) at the Sac1I site between the hr5-IE1 enhancer-promoter and the piggyBac transposase to form pIE1-Gal4-pB.

Donor plasmid
The Mos1 donor plasmid (pBSMOSoriKan) with an E. coli origin of replication (ori) and kanamycin resistance gene (kan) has been described previously (46) . The piggyBac donor plasmid pB[KOalpha] with an E. coli ori and kanamycin resistance gene has also been used previously (47) .

Injections and transposition assay
A standard transposition assay was performed with two different helper plasmids, pIE1-Gal4-Mos1 (0.25 µg/ml) or pIE1-Gal4-pB (0.25 µg/ml) in Aedes aegypti (Liverpool strain) embryos in individual experiments. Injections were given with a mixture of pGDV1-UAS target (0.5 µg/ml), the pBSMOSoriKan or pB[KO alpha] donor plasmid (0.25 µg/ml), along with the respective helper plasmid into preblastoderm embryos within 2 h of oviposition (Fig. 2 ). The transposition assay was performed as described previously (48) . Control transposition assays were also performed using the chimeric transposases and an unmodified pGDVI target plasmid that lacks the UAS target.


Figure 2
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  		Figure 2. Schematic of the Interplasmid Transposition Assay. The Gal4-Mos1 helper plasmid is as shown in Fig. 1 . The Gal4 DBD binds to the upstream activating sequences (UAS) located on the pGDV1-UAS target plasmid. The tethered transposase then transposes the marked Mos1 element from the donor plasmid into the target plasmid. Transposition products are then recovered in E. coli as Kanamycin and Chloramphenicol-resistant plasmids.

Candidate transposition product clones were analyzed by DNA sequencing with the ABI Prism Bigdye terminator cycle sequencing ready reaction kit, following the manufacturer’s protocols (Applied Biosystem, Foster City, CA, USA) and previously described primers (47 , 49) . Reaction products were resolved on an Applied BioSystems automated DNA sequencer (model # ABI3100) and sequence reads were analyzed using the Vector NTI suite software (InforMax, North Bethesda, MD, USA).

RESULTS

Plasmid-based transposition assays were performed in Ae. aegypti embryos (Fig. 2) . Potential transposition product clones were subjected to BamHI digestion to identify transposition events. The pGDV1-UAS plasmid is 2.727kb in size and has a unique BamHI restriction site at nucleotide position 2000. The Mos1 donor element is 4.2kb, also with a single BamHI site. Thus the combined molecular weight of any restriction fragments from a transposed element is expected to be ~6.9 kb. Putative transposition products with the expected digestion pattern were selected for DNA sequence analysis. The results revealed the duplication of a TA insertion site, the hallmark of Mos1 transposition, which indicates that transposition of the Mos1 element had occurred. The transposition frequency for three replicate experiments was calculated by dividing the number of transposition events by the total number of recovered donor plasmids. As a control we used a pGDV1 target plasmid lacking a UAS target site, such that the Gal4-UAS interaction was absent. The transposition assay results revealed a 12.7-fold increase in transpositional activity over the controls where the UAS target site is absent (Table 1 ). The transposition frequency is almost 20-fold higher when compared with another control where a regular helper plasmid was used. In addition to the enhanced transposition frequency, the Mos1 chimeric transposase showed a high degree of insertion specificity compared with control experiments.


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  		Table 1. Transposition assay data from the use of chimeric transposases and modified target plasmids

The pGDV1 target plasmid contains 251 potential TA target sites, of which 60 have been previously identified as insertion sites and 191 are unused sites (49) . The Cam resistance gene contains 77 TA sites, thus insertions into these sites are not likely to be recovered due to the disruption of the resistance gene used for colony selection, leaving 114 unused sites. Control experiments utilizing the chimeric transposase and an unmodified pGDVI target plasmid lacking the UAS target revealed that integration of the donor element occurs essentially randomly at multiple TA target sites (Fig. 3 ). However, in the presence of the Mos1 chimeric transposase and the modified pGDV1-UAS target plasmid, transposition primarily occurred at the same TA site, position 1061 of the target plasmid, located 954 bp from the inserted UAS target sequence (Fig. 3) . Remarkably, the chimeric transposase directed integration to this specific site 96% of the time. Among the Gal4-Mos1 mediated transposition events at the 1061 site, 98% are in a 5'-3' orientation with respect to the Cam resistance gene. In the control experiments, no integrations occurred at the 1061 TA site of the target plasmid.


Figure 3
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  		Figure 3. Schematic map of the pGDV1-UAS target plasmid showing integrations of Mos1 donor elements when using the Gal4-Mos1 chimeric transposase. Open circles represent insertions of the Mos1 donor element into the standard pGDV1 target plasmid, which lacks the UAS target. The solid circles represent insertions in the opposite orientation with respect to the Chloramphenicol (Cam) resistance gene. 48 insertions were recovered from these control experiments. The "cloverleaf" ({clubsuit}) and solid arrow represent the predominant site hit by Mos1 donor elements when using the pGDV1-UAS target plasmid. As indicated above the solid arrow, this site was hit 51 times during these assays. Two additional insertions recovered when using the pGDV1-UAS target plasmid are indicated by up and down arrowheads. Both experiments shown here utilized the chimeric Gal4-Mos1 transposase helper plasmid. For clarity, the 68 insertions recovered from the assays using the unmodified Mos helper plasmid are not shown; nevertheless, they show a distribution similar to the open and solid circles. The numbers represent the nucleotide location on the pGDV1 plasmid. The UAS target site is shown as a shaded box, the Chloramphenicol (Cam) resistance gene as an open box. Insertions into the antibiotic resistance gene would not be recovered from this assay due to the selection regime used to remove false positives and recombination events.

A parallel transposition assay was also performed in Ae. aegypti embryos using the pIE1-Gal4-pB helper. Putative transposition products were selected based on BamHI digestion. The piggyBac donor element is 5.5kb, contains a single BamHI site and thus the transposition product is expected to be ~8.22kb. Actual transposition products were confirmed using DNA sequence analysis. The sequence results revealed the duplication of a TTAA insertion site; the hallmark of piggyBac transposition, thus confirming that piggyBac mediated transposition had occurred. The transposition frequency was 11.6-fold higher compared to the controls. Moreover, in the presence of the piggyBac chimeric transposase and the modified pGDV1-UAS target plasmid, 67% of transpositions occurred at position 1103 site of the target plasmid, located 912 bp from the inserted UAS target sequence (Fig. 4 ). In the control experiments, no integrations occurred at position 1103 of the target plasmid.


Figure 4
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  		Figure 4. Schematic map of the pGDV1-UAS target plasmid showing integrations of piggyBac donor elements when using the Gal4-piggyBac chimeric transposase. Open circles represent insertions of the piggyBac donor element into the pGDV1-UAS target when utilizing the Gal4-piggyBac chimeric transposase. The solid circles represent insertions in the opposite orientation with respect to the Chloramphenicol (Cam) resistance gene. The asterisk and solid arrow represent the predominant site hit by piggyBac donor elements when using the modified pGDV1-UAS target plasmid. As indicated above the solid arrow, this site was hit 45 times during these assays, representing 67% of the insertions recovered. The open and solid rectangles represent insertions of the piggyBac donor element into the unmodified pGDV1 target when utilizing the Gal4-piggyBac chimeric transposase. The numbers represent the nucleotide location on the pGDV1 plasmid. The UAS target site is shown as a shaded box, the Chloramphenicol (Cam) resistance gene as an open box. Insertions into the antibiotic resistance gene would not be recovered from this assay due to the selection regime used to remove false positives and recombination events.

The pGDV1 target plasmid contains 29 potential TTAA target sites, of which 8 are in the Cam resistance gene, from which insertions cannot be recovered in this assay (47 , 50 , 51) . Control experiments utilizing the chimeric transposase and an unmodified pGDVI target plasmid lacking the UAS target revealed that integration of the donor element occurs at multiple TTAA target sites (Fig. 4) . These sites have been previously used by this element for insertion (47) . Among the Gal4-piggyBac mediated transposition events at the 1103 site, 80% are in a 3'-5' orientation with respect to the Cam resistance gene.

DISCUSSION

Transgenic technologies enable the expression of an exogenous genetic construct in a living organism. Random integration of the transgene may result in insertional mutagenesis, transgene mis-expression, over-expression, or silencing. One approach to minimize position effects and the effects of endogenous enhancers and silencers is the use of scaffold-associated regions (SARs) or matrix-associated regions (MARs) (52 , 53) . Similarly, position effect variegation (PEV) can also affect transgene expression (54 55 56) . PEV occurs when a transgene is integrated near a euchromatin and heterochromatin boundary. PEV is particularly important in Ae. aegypti since its genome is estimated to be up to 50% heterochromatic (57) . If a transgene integrates into heterochromatin, its expression level is dramatically reduced or silenced (58 , 59) . With available gene integration systems for insects, there are certain limitations that revolve around the degree of control over integration site specificity.

Recent research of DNA mediated transposition has opened new opportunities to improve transgenic technologies (60 , 61) . As a part of an emerging technology, we developed a chimeric transposase based on the Gal4-UAS system and has proven to be an effective tool for mediating site-directed integration. This is the first evidence of site-directed integration utilizing Mos1 and piggyBac chimeric transposases.

Since the normal specific target site sequences are very short, these transposons essentially integrate randomly into host genomes. However all target sites are not used with equal frequency. As an example, the Sleeping Beauty transposase prefers certain TA target sites based on DNA structure surrounding the target (62) . Sleeping beauty-mediated transposition is also strongly enhanced by CpG methylation (63) . Interaction with host cell factors might also influence target site selection for certain transposases.

Retrotransposons integrate nonrandomly into eukaryotic genomes. The Ty1 and Ty3 retrotransposons of Saccharomyces cerevisiae, integrate preferentially upstream of tRNA genes or other genes transcribed by RNA polymerase III. Ty1 displays nonrandom integration at N A/T A/T A/T N sites. Ty1 insertions are generally within 700 bp upstream of pol III transcribed genes, and Ty3 integrates within several bp upstream of the transcription start sites of target genes. Ty5 insertions occur on either side of their targets within 800 bp of consensus sequences. Ty5 interacts with the host protein Sir4C to target heterochromatic sites of the host genome (e.g., telomeres and silent mating loci). Targeting to these locations is determined by interactions between a targeting domain (TD), a 6-aa motif at the C terminus of Ty5 integrase (IN) and the heterochromatin protein Sir4p (64) . The preference for Ty5 to integrate near regions of silent chromatin suggested that silent chromatin directs integration (65) . By replacing the TD of Ty5 integrase, which mediates the interaction with Sir4C, it is possible to modulate site specificity (66) .

The Mos1 transposase was chosen in part on the demonstration of a high degree of stability in the insect genome after integration (67) . The helper plasmid was designed to express the Mos1 transposase under the regulation of a hr5-IE1 enhancer-promoter, along with a NLS and Gal4 DBD (Fig. 1A ). The NLS consists of five contiguous positively charged residues and helps direct proteins into the nucleus (68) . A linker between the DBD and transposase was included to separate the DBD and transposase proteins to ensure they both fold correctly. When the Gal4 binding domain binds to its target site, the tagged transposase is presumably brought into proximity and thereby increases the likelihood of transgene integration nearby. We verified the activity of the Gal4-Mos1 and Gal4-piggyBac chimeric transposases in the presence and absence of a Gal4-UAS interaction.

With the use of the Gal4-Mos1 chimeric transposase the transposition efficiency was increased by 12.7-fold compared to controls where the UAS binding site is absent in the pGDV1 target plasmid (Table 1) and ~20-fold compared to the use of a standard helper plasmid. Presumably these increases in transpositional frequency are a result of increased DNA binding affinity of the Gal4 DBD compared to the endogenous Mos1 DBD, even in the absence of a specific UAS target. Compared with other systems, the addition of an N-terminal fusion has not reduced transpositional activity.

In this system, Gal4-UAS mediated transposition occurred at the 1061 site of the target plasmid. This site is 954 bp away from the UAS target site. Surrounding the 1061 site, from site 873 to 1157, there are 26 TA sites available for insertion, among which 8 (nucleotide positions 873, 922, 928, 932, 993, 1100, 1104, and 1157) have been inserted into in previous assays (49) . It is perhaps surprising then that these sites were not used, suggesting that the Gal4-Mos1 transposase may be structurally limited after Gal4-UAS binding. It is reasonable to propose that the Gal4 DBD is binding to the UAS binding site and that the transposase-donor element complex is located at some distance from this region by the linker peptide, resulting in insertion at the 1061 TA site (Fig. 3) . Furthermore, 1061 is an unused target TA site with respect to previous assays; perhaps emphasizing the involvement of the chimeric transposase for the forcible insertion of the transgene. It is not known if 1061 is a true cold spot, limited by DNA structure, or if by chance it had not previously been used for integration. If there is such a structural limitation, it is possible that a hot spot or more favorable TA target site sequence could be placed at an optimal distance and thus result in an even higher integration frequency. It should be noted that potential insertions into the chloramphenicol resistance gene would not be recovered due to the selection regime used for these assays.

In a parallel experiment using a Gal4-piggyBac chimeric transposase the transposition frequency was 11.6-fold higher compared with controls where the Gal4-UAS interaction is absent. In the presence of the chimeric transposase, 67% of transpositions occurred at the 1103 site of the target plasmid, which is an unused site in earlier reports of piggyBac insertions. There are five insertion sites (TTAA) close to the 1103 site (945, 968, 977, 992, and 1169). Among these, three have previously been used (47 , 51) . Insertions primarily at 1103 suggest that the Gal4-piggyBac transposase might also be structurally limited after binding to the UAS site and is thus forced to make an insertion in the same site. It cannot be determined if the 1103 site is a true cold spot at this time. It is worth noting that the two insertions sites used by the chimeric transposases are in close proximity and thus are at similar distances from the UAS target.

Transposases have a theoretical advantage over recombinases in that they are potentially amenable to target any given region, whereas recombinases are inherently limited to specific or closely related (engineered target or pseudo) sites due to specific, larger sequence requirements of the catalytic domain. In the case of Cre and FLP, engineered target sites must be first integrated into the desired genomic location for subsequent gene insertion. Thus, by utilizing zinc finger binding domains, chimeric transposases could allow the design of vectors for integration into or around any genomic site, assuming that the chromatin in that region is permissive for integration. This represents a significant advantage over recombinases in that a prior germline integration event of an engineered target is not required, thus potentially also allowing chimeric transposases to be utilized for somatic gene therapy.

Zinc finger domains are highly conserved DNA binding motifs that are involved in specific DNA binding and protein-protein interactions. A six-zinc finger domain protein will recognize an 18 bp target, sufficiently long to constitute a rare address in the human genome (69 , 70) . Independently folded domains offer a large number of combinational possibilities for DNA recognition. As an example, modifying the control of gene expression with specifically selected zinc finger combinations was used to inhibit an oncogene in a mouse cancer cell line (71) . The site specificity of the Tn3 resolvase was recently modified by exchange of its natural DNA binding domain with the Zn-finger domain of the human transcription factor, Zif268 (26) . The fusion of modifying enzymes with the DNA binding domain of Zif268 is thus promising since Zif268 can theoretically be evolved to recognize any DNA sequence. Similarly, approaches using zinc finger domains as artificial DBD will allow chimeric transposases to be used for the site-directed integration of therapeutic genes into precise genomic locations. The results presented here establish the potential for achieving site-specific integration of Mos1-and piggyBac elements into insect genomes. Furthermore, the recent demonstration of piggyBac activity in mammalian cells (72) will allow these systems to be utilized in a diverse array of settings.

ACKNOWLEDGMENTS

This work was supported in part by National Institutes of Health grant AI047303 to C.J.C.

Received for publication February 16, 2006. Accepted for publication April 27, 2006.

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