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Generation of an inducible and optimized piggyBac transposon system†

Generation of an inducible and optimized piggyBac transposon system† Published online 18 June 2007 Nucleic Acids Research, 2007, Vol. 35, No. 12 e87 doi:10.1093/nar/gkm446 Generation of an inducible and optimized piggyBac transposon system Juan Cadin˜ anos and Allan Bradley* The Wellcome Trust Sanger Institute. Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, CB10 1SA, UK Received March 22, 2007; Revised May 14, 2007; Accepted May 18, 2007 ABSTRACT locus responsible for the observed phenotype. Due to their ability to mobilize around genomes, DNA transposons Genomic studies in the mouse have been slowed by and retrotransposons have been widely used as tools to the lack of transposon-mediated mutagenesis. generate mutation libraries in a variety of organisms. However, since the resurrection of Sleeping Beauty DNA transposons are genetic elements consisting of (SB), the possibility of performing forward genetics inverted terminal DNA repeats (TRs) which in their in mice has been reinforced. Recently, piggyBac naturally occurring configuration flank a transposase (PB), a functional transposon from insects, was also coding sequence (CDS). This transposase follows a ‘cut described to work in mammals. As the activity of PB and paste’ mechanism to excise the transposon from its is higher than that of SB11 and SB12, two hyper- original genomic location and insert it into a new locus (2). Retrotransposons, however, use an RNA active SB transposases, we have characterized and intermediary molecule and retrotranscription to ‘copy improved the PB system in mouse ES cells. We have and paste’ themselves in different locations of the genome. generated a mouse codon-optimized version of the In mammals, the lack of efficient transposon systems has PB transposase coding sequence (CDS) which largely precluded the application of this type of mutagen- provides transposition levels greater than the esis. However, the awakening of the use of DNA original. We have also found that the promoter transposons and the recent success with synthetic retro- sequence predicted in the 5 -terminal repeat of the transposons in the mouse is opening wide the door of PB transposon is active in the mammalian context. forward genetics in this model organism (3,4). Finally, we have engineered inducible versions of the In 1997, Ivics et al. resurrected a Tc1-like DNA optimized piggyBac transposase fused with ERT2. transposon by comparing the nucleotide sequences of One of them, when induced, provides higher levels of a number of dormant inactive elements in salmonid transposition than the native piggyBac CDS, fishes, predicting the active sequence and repairing the whereas in the absence of induction its activity is inactivating mutations. This elegant and meticulous indistinguishable from background. We expect that approach resulted in an active transposon termed these tools, adaptable to perform mouse-germline Sleeping Beauty (SB). The SB system was soon reported to be functional in human and murine cells (3,5,6). mutagenesis, will facilitate the identification of Moreover, since its resurrection, both the inverted repeats genes involved in pathological and physiological and the transposase coding sequence have been optimized, processes, such as cancer or ES cell differentiation. which has yielded an SB element with increased mobiliza- tion activity (7–11). This has been used in mammals for a wide range of applications, such as gene therapy, germline INTRODUCTION mutagenesis and somatic mutagenesis (8,12–15). The activity of the system needs to be regulated, because Forward genetics, where the observation of a phenotype is excessive and uncontrolled transposition results in genomic followed by the identification of the responsible gene(s), instability such as inversions, deletions and translocations provides a valuable tool to carry out functional genomics. (14). These large-scale genomic rearrangements mask The availability of systems to generate tagged mutations the more subtle and informative singular transposition on a large scale facilitates this kind of approach, events, interfering with the identification of single genes permitting the production of collections of genetically responsible for specific phenotypes. Thus, the availability modified cells or organisms which can then be phenotyp- of a highly active, though regulatable, transposase would ically screened (1). The molecular label conferred by the be desirable. tagging mutagen allows for a ready identification of the *To whom correspondence should be addressed. Tel: +44-1223-494884; Fax: +44-1223-494714; Email: [email protected] GenBank accession numbers: EF587698 and EF587699. 2007 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. e87 Nucleic Acids Research, 2007, Vol. 35, No. 12 PAGE 2 OF 8 Table 1. Sequences and properties of the linkers used in the fusion PB transposases engineered in this work Version Nt Linker Ct pI Charge ERT2-L1-mPB ...PATA- -CEFAAT- -MGSS... 4 mPB-L1-ERT2 ...QSCF- -AAA- -SAGD... 5.57 ERT2-L2-mPB ...PATA- -CELSLGAAPDAAPGSEFAAT- -MGSS... 3.57 mPB-L2-ERT2 ...QSCF- -AAASLGAAPDAAPGSGAA- -SAGD... 3.8 ERT2-L3-mPB ...PATA- -CEFKAKLGGGAPAVGGGPKAADKFAAT- -MGSS... 9.05 + mPB-L3-ERT2 ...QSCF- -AAAKLGGGAPAVGGGPKAADKGAA- -SAGD... 9.53 + Columns from left to right: ‘Version’ indicates the different fusion transposases. ‘Nt’ indicates the aminoacidic sequence of the N-terminal half of the fusion protein immediately proximal to the linker. ‘Linker’ corresponds to the sequence of aminoacids between both halves of fusion protein. Bold aminoacids highlight the core linker sequences. ‘Ct’ indicates the aminoacidic sequence of the C-terminal half of the fusion protein immediately proximal to the linker. ‘pI’ corresponds to the isoelectric point of the linker. ‘Charge’ indicates the predicted sign of the net electric charge of the linker at intracellular pH conditions. In addition to SB, piggyBac (PB), a transposable from T2/Onc, kindly provided by Dr L. Collier, and the element originally isolated from the genome of the EMCV IRES sequence was sub-cloned from pPRIG, cabbage looper moth Trichoplusia ni has also been which was a gift by Dr P. Martin (12,22). The minimal 5 reported to be highly active when introduced into and 3 -PB terminal repeats were custom synthesized and mammalian genomes, including human (16–18) and then combined by sub-cloning to produce the desired mouse cells (16). In a direct comparison experiment configurations. The Luc2 sequence corresponds to a involving in vitro transfection, PB has been reported to HindIII/BamHI fragment from pGL4.13 (Promega). be the most active of four tested transposons, including ERT2 was PCR amplified from a cDNA kindly provided 0 0 PB, SB11 (one of the hyperactive SB transposases), Tol2 by Dr P. Chambon and subcloned at the 5 or at the 3 of and Mos1 (17). In a separate study, PB has also been mPB. L1 linkers were created as a result of the cloning shown to be more active than SB12, another hyperactive process. L2 and L3 linkers were introduced between mPB version of SB (18). Although there are other hyperactive and ERT2 by sub-cloning the pairs of primers shown in SB transposases available, which apparently show higher Supplementary Table 1. The core aminoacidic sequence of activity than SB11 and SB12, they have not been directly the L3 linker was kindly provided by Dr Joseph Kaminski. compared to PB (19,20). Given its good performance The CDSs and protein sequences of mPB (GenBank in mammals we have characterized and optimized the accession number: EF587698) and mPB-L3-ERT2 PB system for its utilization in mouse ES cells and for (GenBank accession number: EF587699) are shown in somatic mutagenesis in vivo. We have generated a mouse Supplementary Figure 1. codon-optimized version of the PB transposase CDS and Cell culture and transfection we have observed that it provides levels of transposition considerably higher than those of the native PB CDS. AB2.2 ES cells were cultured on a layer of mitotically We have analysed the differential properties of the 5 and inactive SNL76/7 feeder cells and transfected by electro- 3 -terminal repeats of the PB transposon, uncovering the poration as described previously (23). Puromycin selection existence of promoter activity in the 5 -terminal repeat. was conducted on SNLP 76/7-4 feeders (a puromycin Finally, we have created a highly active and regulatable resistant derivative of SNL76/7) with 3 mg/ml of puromy- PB transposase, by fusing the optimized PB CDS to the cin. Puromycin resistant ES cell colonies were stained for modified ligand-binding domain of the oestrogen receptor, 15 min with 1% methylene blue in 70% EtOH, washed in ERT2. We expect that this optimized transposon system distilled water over night and air-dried. This protocol will significantly expand the utility of transposon-based produced a very low background which allowed counting mutagenesis for the genetic characterization of the mouse colonies with diameters >0.3 mm. COS-7 cells and genome. HeLaS3 cells were grown in 6-well plates and with DMEM supplemented with 10% foetal bovine serum. Plasmids were transfected with Lipofectamine 2000 MATERIALS AND METHODS (Invitrogen), following manufacturer’s instructions. Plasmids Western blot iPB and mPB were custom synthesized. Besides the PB transposase CDS, both sequences included a Kozak Transfected COS-7 cells were washed with PBS and element around the start ATG at the 5 -end and two each well was harvested with 500 ml2  Laemmli buffer. consecutive stop codons at the 3 -end. EcoRI and NotI Fifteen microlitres of lysate were loaded per lane in a 0 0 and 3 -ends, respectively, and they were used flank the 5 4–12% gradient SDS–PAGE gel (Invitrogen). The gel was TM to clone both CDSs into pcDNA3 or pcDNA3-KzHA. To transferred to a Hybond-ECL (Amersham Biosciences) produce the donor plasmids, the puroDTK fusion gene and membrane, and the membrane was dried over night the bovine growth hormone polyA signal were PCR and blocked with 5% non-fat dry milk dissolved in PT amplified from pFlexible (21). En2SA was PCR amplified (0.1% Tween-20 in 1 PBS) for 1 h at room temperature. PAGE 3 OF 8 Nucleic Acids Research, 2007, Vol. 35, No. 12 e87 Then, it was incubated with 12CA5 anti-HA monoclonal In these experimental conditions we did not observe antibody (Abcam, cat #ab16918) or with anti-actin overproduction inhibition, a phenomenon described in monoclonal antibody (Sigma, cat #A5316), both diluted certain transposon systems in which transposition rates 1:1000 in 3% non-fat milk dissolved in PT for 1 h at room decline when the amount of transposase exceeds certain temperature. The membrane was washed 3 times for 5 min levels (10,14,18). Interestingly, the levels of transposition with PT and incubated with the peroxidase-labelled provided by iPB in these series of experiments were far TM anti-mouse antibody from the ECL Western Blot below those of mPB, especially in Figure 1D, where not Analysis System (GE Healthcare) diluted 1/10000 in even the highest amount of iPB plasmid is able to match 1.5% non-fat dry milk for 1 h at room temperature. The the levels of transposition obtained by transfection of membrane was washed again as before and specific protein the lowest amount of mPB. This is likely due to the TM bands were detected using ECL Western Blot Analysis stringent conditions (5  10 cells per transfection and 1 mg System (GE Healthcare). of donor plasmid) of this experiment, aimed at analysing saturation effects. Additional experiments performed in Luciferase assay less-restrictive conditions showed that increasing amounts of iPB provided levels of transposition in the same order Transfected HeLaS3 cells were washed with PBS and each of magnitude as those of mPB (Supplementary Figure 2), well was lysed for 15 min with 100 mlof1 passive lysis indicating that the differences between the performance buffer from the Dual Luciferase Reporter Assay System of iPB and mPB strongly depend on the experimental (Promega). Lysates were harvested and 20 ml of each settings. Despite this, we must remark that mPB showed sample were analysed for luciferase activity following the higher levels of transposition than iPB in all the manufacture’s instructions in a MicroLumatPlus LB 96V conditions tested. luminometer (Berthold Technologies). To ascertain if the higher transposition frequency provided by the optimized sequence was caused by RESULTS higher production of transposase we subcloned iPB and mPB into pcDNA3-Kz-HA, a pcDNA3-based vector Increased transposition by codon-optimization of PB containing a Kozak sequence plus the nucleotide sequence transposase encoding the hemagglutinin epitope (HA) (Figure 2A), As the PB transposon was originally isolated from insect enabling detection of protein production from HA-iPB cells, we hypothesized that a mouse codon-optimization of and HA-mPB by Western blot analysis using an anti-HA the transposase CDS might produce higher levels of antibody. These two plasmids plus an empty pcDNA3- transposition in the murine ES cell context (24). To Kz-HA vector were transfected into COS-7 cells in address this possibility we synthesized the native PB triplicate. Analysis of protein lysates using an anti-HA transposase CDS (hereafter iPB) and a mouse codon monoclonal antibody revealed a clear band at the expected optimized CDS version (hereafter mPB). iPB corresponds size (68 kDa) in the cells transfected with transposase to the original wild-type coding sequence of the PB expression plasmids, but not in the cells transfected with transposase, whereas mPB is a synthetic sequence coding the empty vector (Figure 2B). The amount of PB for the same polypeptide as iPB, but where each codon has transposase in the cells transfected with HA-mPB was been changed for the preferred codon for translation in considerably and consistently higher than in the cells mouse cells. Both iPB and mPB contain a Kozak sequence transfected with HA-iPB, confirming that higher levels of built around the start methionine codon. Besides this, transposase production are likely responsible for the potential cryptic splice sites generated during the codon greater transposition activity provided by mPB. optimization were detected and avoided in the optimized sequence. Expression vectors were assembled by subcloning both 0 0 Differential features of the minimum 5 and 3 -PB versions of the transposase CDSs into pcDNA3. We terminal repeats compared their ability to promote transposition of a PB The original PB transposon is flanked by 13-bp terminal transposon carrying a promoterless puromycin resistance inverted repeats and has additional inverted repeats cassette from a transfected plasmid vector into the ES cell 19-bp long located asymmetrically with respect to the genome (Figure 1A). iPB or mPB expression plasmids end of the element (24). These inverted flanking sequences, were co-electroporated with a transposon donor plasmid 0 0 although able to permit transposition between different (5 -PTK-3 ) into AB2.2 mouse ES cells and the numbers of plasmids in insect cells, were found to be insufficient to puromycin-resistant colonies were assessed. mPB yielded allow transposition from a donor plasmid to genomic considerably more puromycin-resistant colonies than iPB DNA. In fact, additional transposon DNA sequence was (Figure 1B). found to be necessary to provide this kind of transposition To address saturation effects the amounts of helper or 0 0 (25,26). A 5 -terminal repeat (5 -TR) of 313 bp and donor plasmids were varied while keeping a constant 0 0 a3 -terminal repeat (3 -TR) of 235 bp have been described amount of the respective donor or helper plasmid as the minimum PB terminal repeats (25). Combinatorial (Figure 1C and D). In both series of experiments, mPB experiments performed with the SB transposon have provided higher levels of transposition than iPB through- out the whole range of amounts of transfected plasmids. shown that a configuration containing two ‘left’ terminal e87 Nucleic Acids Research, 2007, Vol. 35, No. 12 PAGE 4 OF 8 Figure 1. Comparison of transposase activity of the native and codon optimized piggyBac.(A) Schematic representation of the plasmids used in this 0 0 0 0 experiment. The donor plasmid, 5 -PTK-3 , contains the minimal PB terminal repeats (5 -TR and 3 -TR) flanking a promoter-less puromycin resistance cassette. CMV, cytomegalovirus promoter; ATG, start codon; pA, bovine growth hormone polyA signal; En2SA, mouse engrailed-2 gene splice acceptor; IRES, internal ribosomal entry site from encephalomyocarditis virus; puTK, synthetic fusion CDS between the puromycin- 0 0 resistance gene and a truncated thymidine kinase gene (21). (B)2 mgof 5 -PTK-3 and 2 mg of the transposase plasmid were co-electroporated into 10 AB2.2 mouse ES cells and, after puromycin selection, the numbers or resistant colonies were counted. (C) A fixed amount (1 mg) of transposase 0 0 plasmid was co-electroporated with increasing amounts (1–40 mg) of 5 -PTK-3 into 5  10 AB2.2 ES cells and puromycin-resistant colonies were 0 0 counted. (D) A fixed amount (1 mg) of 5 -PTK-3 transposon plasmid was co-electroporated with increasing amounts (1–40 mg) of transposase plasmid into 5  10 AB2.2 ES cells. Numbers of puromycin-resistant colonies are indicated. n = 3, error bars: SEM. repeats (or 5 -TRs) provides levels of transposition 3 times assessed luciferase activity in transiently transfected higher than the original ‘left’ + ‘right’ combination (11). HeLaS3 cells. The luciferase activity observed with the 0 0 0 In order to study the properties of the 5 and 3 -TRs of 5 -TR-Luc2 construct was 5 times higher than that the PB element and to explore the possibility of enhancing observed in the cells transfected with the 3 -TR-Luc2 the rates of transposition, we subcloned a promoter-less construct (Figure 3B), indicating that the promoter in the puromycin resistance cassette between different combina- 5 -TR repeat is also active in mammalian cells. 0 0 tions of minimum 5 and 3 -PB terminal repeats 0 0 0 0 0 0 0 0 (Figure 3A) (5 –5 ,5 –3 ,3 –5 and 3 –3 ) and cotransfected Generation of an inducible PB transposase the resulting donor plasmids with the mPB expression The success of cancer-gene identification experiments cassette into AB2.2 cells. Only the cells transfected with 0 0 0 0 performed with the SB transposase in mice has been the 5 –3 and 3 –5 configurations yielded significant strongly determined by the levels of transposase activity numbers of resistant colonies (Figure 3A). These results (12,13). However, an excess of transposition has been indicate that the PB transposon requires one 5 and one reported to create undesired genomic rearrangements in 3 -terminal repeat in order to achieve transposition. 0 0 an SB-based system for mouse germline mutagenesis (14). The number of colonies obtained with the 5 –3 0 0 Thus, the ability to control transposition temporally transposon was 4.6 times higher than with the 3 –5 would provide an additional advantage to the PB configuration (Figure 3A). As we had used a promoter- transposase. To explore the possibility of generating an less selection cassette, one explanation of this observation is that the promoter sequence predicted in the 5 -TR, inducible transposase we created fusion proteins between which is functional in insect cells, is also active in the mPB and the modified oestrogen receptor ligand-binding mammalian genome (24,27). To address this possibility, domain (ERT2), at the N or C terminus of mPB (ERT2- we subcloned a luciferase expression cassette (Luc2) mPB and mPB-ERT2, respectively) (28) (Figure 4A). 0 0 downstream of the 5 and 3 -PB TRs (Figure 3B) and The ERT2 domain provides the possibility of regulating PAGE 5 OF 8 Nucleic Acids Research, 2007, Vol. 35, No. 12 e87 more than 800-fold to background levels. To our knowl- edge, mPB-L3-ERT2 is the first PB transposase inducible by 4-OHT, it provides higher activity than iPB and it shows undetectable levels of leakiness. DISCUSSION The recent availability of DNA transposons which are active in the mammalian genome has widened the set of tools to carry out functional genomics and transgenesis. SB, a transposon reconstructed from inactive sequences present in salmonid fishes, has already shown great utility, having being successfully used in cancer-gene identifica- tion, germline mutagenesis and human cell transgenesis experiments (3,8,12–14). More recently, PB, an active transposon from the cabbage looper moth Trichoplusia ni, has also been shown to work in mammalian cells, providing transposition levels higher than those displayed by certain hyperactive SB forms (16–18). These two transposable elements have distinctive properties, for instance target sequence preference, which suggests that they will be used as complementary tools to modify and study the mammalian genome (8,18,30,31). We have characterized, optimized and further enhanced Figure 2. Levels of protein production provided by the native and the functionality of the PB transposon/transposase codon optimized piggyBac.(A) Schematic representation of the system. We have synthesized a mouse-codon-optimized plasmids used in this experiment. HA, sequence encoding the hemagglutinin epitope. (B) pcDNA3-KzHA, HA-iPB or HA-mPB PB transposase CDS, which provides levels of transposi- were transfected into COS-7 cells and the levels of HA-tagged PB tion significantly higher than the native PB transposase transposase were analysed by Western blot using a specific monoclonal CDS over a wide range of transposon/transposase ratios. antibody raised against the HA epitope. The same membrane was Remarkably, the difference in performance between iPB subsequently incubated with a monoclonal anti-actin antibody, as loading control. Three independent transfections were performed and and mPB strongly varied with the experimental condi- analysed per plasmid. tions, although mPB always provided higher levels of transposition. Notably, overproduction inhibition, the decrease of transposition produced by excessive trans- the activity of a protein by the presence or absence of posase previously observed in SB, was not detected for PB 4-hydroxytamoxifen (4-OHT). In the absence of 4-OHT a in the experimental conditions we used (10,17,18). protein containing the ERT2 domain is sequestered by Intriguingly, two previous reports have tried to address heat-shock proteins, preventing it from functioning (29). the existence of this phenomenon in PB with disparate In the presence of 4-OHT, the fusion protein is released results. On one hand, Wu et al. (17) found overproduction and can then play its role (28). inhibition of PB in HEK293 cells. On the other hand, The two key requirements of inducible proteins are high Wilson et al. (18) concluded that PB lacks overproduction activity under induced conditions and low/no activity in inhibition in HEK293 cells. Interestingly, in the same the absence of induction. To achieve the maximum activity study, Wilson et al. transfected equivalent amounts of a and inducibility we evaluated a series of linkers between pCMV-based SB12 helper and donor plasmids in mPB and ERT2 of different lengths, charges and flexibil- HEK293 cells, and observed the previously reported ities (Table 1). Most of the fusion proteins appeared to overproduction inhibition. Although our results support have reduced activity. The L1 fusions show 100-fold the lack of overproduction inhibition in PB, further work reductions compared with mPB. The L2 fusions also is needed to clarify this issue. exhibited reduced activity although one of them (ERT2- We have also shown that the higher transposition L2-mPB) was not leaky when not induced. However, the provided by mPB correlates with increased protein fusion proteins containing L3 linkers provided high levels of transposition (Figure 4B). The L3 linker was based on a production. This result highlights the usefulness of sequence which has previously been used between the codon optimization to increase protein levels. Besides GAL4-DNA-binding domain and the native PB transpo- this, the availability of two different sequences encoding sase, which resulted in levels of activity of the fusion PB transposase offers the opportunity to obtain different protein close to 100% with respect to the wild-type enzyme levels of protein and transposition activity. (17). The number of colonies obtained in the presence of We have analysed the differential properties of the 5 4-OHT using mPB-L3-ERT2 was 6 times higher than those and 3 -PB terminal repeats. In contrast with the previous obtained with iPB, though it was less efficient than mPB. results obtained with different combinations of the SB Importantly, the activity was highly 4-OHT-dependent, terminal repeats we have only detected transposition since in the absence of 4-OHT transposition was reduced activity when the naturally occurring combination of PB e87 Nucleic Acids Research, 2007, Vol. 35, No. 12 PAGE 6 OF 8 Figure 3. Analysis of the properties of the piggyBac terminal repeats. (A) Left, schematic representation of transposon plasmids used in this experiment. Each plasmid contains the same gene trap puromycin resistance cassette flanked by different combinations of 5 -TR (blue arrows) and 3 -TR (red arrows). Right,2 mg of transposon plasmids were co-electroporated into 10 AB2.2 mouse ES cells with 2 mg of mPB helper plasmid. The numbers of puromycin-resistant colonies were counted. (B) Left, schematic representation of the firefly luciferase expression plasmids used in this 0 0 experiment. 5 -TR-Luc2 contains the minimal left terminal PB repeat followed by the firefly luciferase CDS (Luc2). 3 -TR-Luc2 contains the minimal 0 0 right terminal PB repeat followed by Luc2. pA, SV40 late polyA region. Right,5 -TR-Luc2 or 3 -TR-Luc2 were transfected in HeLaS3 cells together with pGL4.74, a transfection control plasmid containing the renilla luciferase gene under the thymidine kinase promoter. Twenty-four hours after transfection firefly and renilla luciferase activities were measured. n = 3, error bars: SEM. 0 0 5 -TR and 3 -TR was used (11). Interestingly, we obtained combinations of mPB with ERT2 (28). The six versions higher numbers of colonies when the resistance cassette have two different arrangements of ERT2 either at the 0 0 was oriented from the 5 -TR to the 3 -TR than in the C-terminus or at the N-terminus of the PB transposase opposite orientation. In these experiments the transposon coupled with three different linkers (L1, L2 or L3) between contained a gene-trap selection cassette. As the 5 -TR has ERT2 and mPB. Four of these fusion proteins show been described to contain a promoter active in insect cells, extremely reduced levels of transposition when compared we hypothesized that the observed differences in colony to the parental mPB, even in the presence of the inducer, numbers could derive from transcriptional activity 4-OHT. However, the other two fusion proteins, ERT2- from the 5 -TR activating the selection cassette (27). In L3-mPB and mPB-L3-ERT2, produce high levels of a promoter analysis experiment using luciferase as a induced transposition. Both of them contain a linker reporter, we observed that the 5 -TR does act as which was based on an aminoacidic sequence previously a promoter which is 5-fold stronger than the 3 -TR. used to fuse the native PB transposase to the GAL4- This observation should be considered in the design of DNA-binding domain (17). The length and the net PB-based promoter-less transposons, as the expression positive charge of this linker compared with the ones of the promoter-less cassette from the 5 -TR repeat which were less successful are likely to underlie the may produce unwanted results. In our view, the huge differences in activity. Remarkably, mPB-L3-ERT2 simplest solution would be to rearrange the transposon yields levels of transposition 6 times higher than iPB in 0 0 so that the 3 -TR is upstream of the 5 -end of the the presence of 4-OHT, whereas no detectable transposi- promoter-less sequence. Remarkably, recent work has tion was observed without induction. In the absence of detected enhancer activity in the 3 -TR of the piggyBac 4-OHT the transposition activity provided by mPB- transposon (32). In our opinion, the presence of promoter L3-ERT2 is reduced 800-fold, returning back to the 0 0 and enhancer sequences in the piggyBac 5 -TR and 3 -TR, low experimental noise levels. Thus mPB-L3-ERT2 is not respectively should also be considered in gene-therapy only an improved version of the PB transposase, but it applications, so as to avoid the negative experiences of can also be temporally regulated. This newly engineered generating activating mutations previously obtained with enzyme will be useful for diverse applications, ranging retroviruses (33). from gene therapy, where the possibility of switching Finally, we have engineered an inducible, optimized PB on and off the enzyme would contribute to making it safer, transposase. We have made and assessed six different to forward genetics cancer screens in mice, where the PAGE 7 OF 8 Nucleic Acids Research, 2007, Vol. 35, No. 12 e87 ACKNOWLEDGEMENTS We would like to thank R. Rad, A. Rodrı´guez, S. Cowley, H. Prosser, E. Ferna´ ndez, C. Lo´ pez-Otı´n, J.M.P. Freije, D. Adams, G. Vassiliou, W. Wang and P. Liu for helpful comments and discussions, as well as A. Beasley for excellent technical assistance. J. Cadin˜ anos is a recipient of a FEBS Long-Term Fellowship. This work was supported by the Wellcome Trust. Funding to pay the Open Access publication charges for this article were provided by the Wellcome Trust. Conflict of interest statement. None declared. REFERENCES 1. Carlson,C.M. and Largaespada,D.A. (2005) Insertional mutagenesis in mice: new perspectives and tools. Nat. Rev. Genet., 6, 568–580. 2. Fischer,S.E., Wienholds,E. and Plasterk,R.H. (2001) Regulated transposition of a fish transposon in the mouse germ line. Proc. Natl Acad. Sci. 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(2005) Cancer gene discovery in solid tumours control. A representative picture of plates with puromycin-resistant using transposon-based somatic mutagenesis in the mouse. Nature, colonies obtained using mPB-L3-ERT2 as helper plasmid in the 436, 272–276. absence (left plate) or presence (right plate) of 4-OHT is shown. 13. Dupuy,A.J., Akagi,K., Largaespada,D.A., Copeland,N.G. and n = 3, error bars: SEM. Jenkins,N.A. (2005) Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system. Nature, 436, 221–226. 14. Geurts,A.M., Collier,L.S., Geurts,J.L., Oseth,L.L., Bell,M.L., Mu,D., Lucito,R., Godbout,S.A., Green,L.E. et al. (2006) Gene temporal regulation of the transposition activity mutations and genomic rearrangements in the mouse as a result of would facilitate the adjustment of the dynamics of the transposon mobilization from chromosomal concatemers. PLoS tumourigenic processes to prevent undesired genomic Genet., 2, e156. 15. 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Generation of an inducible and optimized piggyBac transposon system†

Cadiñanos, Juan; Bradley, Allan
Nucleic Acids Research , Volume 35 (12) – Jun 18, 2007
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Abstract

Published online 18 June 2007 Nucleic Acids Research, 2007, Vol. 35, No. 12 e87 doi:10.1093/nar/gkm446 Generation of an inducible and optimized piggyBac transposon system Juan Cadin˜ anos and Allan Bradley* The Wellcome Trust Sanger Institute. Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, CB10 1SA, UK Received March 22, 2007; Revised May 14, 2007; Accepted May 18, 2007 ABSTRACT locus responsible for the observed phenotype. Due to their ability to mobilize around genomes, DNA transposons Genomic studies in the mouse have been slowed by and retrotransposons have been widely used as tools to the lack of transposon-mediated mutagenesis. generate mutation libraries in a variety of organisms. However, since the resurrection of Sleeping Beauty DNA transposons are genetic elements consisting of (SB), the possibility of performing forward genetics inverted terminal DNA repeats (TRs) which in their in mice has been reinforced. Recently, piggyBac naturally occurring configuration flank a transposase (PB), a functional transposon from insects, was also coding sequence (CDS). This transposase follows a ‘cut described to work in mammals. As the activity of PB and paste’ mechanism to excise the transposon from its is higher than that of SB11 and SB12, two hyper- original genomic location and insert it into a new locus (2). Retrotransposons, however, use an RNA active SB transposases, we have characterized and intermediary molecule and retrotranscription to ‘copy improved the PB system in mouse ES cells. We have and paste’ themselves in different locations of the genome. generated a mouse codon-optimized version of the In mammals, the lack of efficient transposon systems has PB transposase coding sequence (CDS) which largely precluded the application of this type of mutagen- provides transposition levels greater than the esis. However, the awakening of the use of DNA original. We have also found that the promoter transposons and the recent success with synthetic retro- sequence predicted in the 5 -terminal repeat of the transposons in the mouse is opening wide the door of PB transposon is active in the mammalian context. forward genetics in this model organism (3,4). Finally, we have engineered inducible versions of the In 1997, Ivics et al. resurrected a Tc1-like DNA optimized piggyBac transposase fused with ERT2. transposon by comparing the nucleotide sequences of One of them, when induced, provides higher levels of a number of dormant inactive elements in salmonid transposition than the native piggyBac CDS, fishes, predicting the active sequence and repairing the whereas in the absence of induction its activity is inactivating mutations. This elegant and meticulous indistinguishable from background. We expect that approach resulted in an active transposon termed these tools, adaptable to perform mouse-germline Sleeping Beauty (SB). The SB system was soon reported to be functional in human and murine cells (3,5,6). mutagenesis, will facilitate the identification of Moreover, since its resurrection, both the inverted repeats genes involved in pathological and physiological and the transposase coding sequence have been optimized, processes, such as cancer or ES cell differentiation. which has yielded an SB element with increased mobiliza- tion activity (7–11). This has been used in mammals for a wide range of applications, such as gene therapy, germline INTRODUCTION mutagenesis and somatic mutagenesis (8,12–15). The activity of the system needs to be regulated, because Forward genetics, where the observation of a phenotype is excessive and uncontrolled transposition results in genomic followed by the identification of the responsible gene(s), instability such as inversions, deletions and translocations provides a valuable tool to carry out functional genomics. (14). These large-scale genomic rearrangements mask The availability of systems to generate tagged mutations the more subtle and informative singular transposition on a large scale facilitates this kind of approach, events, interfering with the identification of single genes permitting the production of collections of genetically responsible for specific phenotypes. Thus, the availability modified cells or organisms which can then be phenotyp- of a highly active, though regulatable, transposase would ically screened (1). The molecular label conferred by the be desirable. tagging mutagen allows for a ready identification of the *To whom correspondence should be addressed. Tel: +44-1223-494884; Fax: +44-1223-494714; Email: [email protected] GenBank accession numbers: EF587698 and EF587699. 2007 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. e87 Nucleic Acids Research, 2007, Vol. 35, No. 12 PAGE 2 OF 8 Table 1. Sequences and properties of the linkers used in the fusion PB transposases engineered in this work Version Nt Linker Ct pI Charge ERT2-L1-mPB ...PATA- -CEFAAT- -MGSS... 4 mPB-L1-ERT2 ...QSCF- -AAA- -SAGD... 5.57 ERT2-L2-mPB ...PATA- -CELSLGAAPDAAPGSEFAAT- -MGSS... 3.57 mPB-L2-ERT2 ...QSCF- -AAASLGAAPDAAPGSGAA- -SAGD... 3.8 ERT2-L3-mPB ...PATA- -CEFKAKLGGGAPAVGGGPKAADKFAAT- -MGSS... 9.05 + mPB-L3-ERT2 ...QSCF- -AAAKLGGGAPAVGGGPKAADKGAA- -SAGD... 9.53 + Columns from left to right: ‘Version’ indicates the different fusion transposases. ‘Nt’ indicates the aminoacidic sequence of the N-terminal half of the fusion protein immediately proximal to the linker. ‘Linker’ corresponds to the sequence of aminoacids between both halves of fusion protein. Bold aminoacids highlight the core linker sequences. ‘Ct’ indicates the aminoacidic sequence of the C-terminal half of the fusion protein immediately proximal to the linker. ‘pI’ corresponds to the isoelectric point of the linker. ‘Charge’ indicates the predicted sign of the net electric charge of the linker at intracellular pH conditions. In addition to SB, piggyBac (PB), a transposable from T2/Onc, kindly provided by Dr L. Collier, and the element originally isolated from the genome of the EMCV IRES sequence was sub-cloned from pPRIG, cabbage looper moth Trichoplusia ni has also been which was a gift by Dr P. Martin (12,22). The minimal 5 reported to be highly active when introduced into and 3 -PB terminal repeats were custom synthesized and mammalian genomes, including human (16–18) and then combined by sub-cloning to produce the desired mouse cells (16). In a direct comparison experiment configurations. The Luc2 sequence corresponds to a involving in vitro transfection, PB has been reported to HindIII/BamHI fragment from pGL4.13 (Promega). be the most active of four tested transposons, including ERT2 was PCR amplified from a cDNA kindly provided 0 0 PB, SB11 (one of the hyperactive SB transposases), Tol2 by Dr P. Chambon and subcloned at the 5 or at the 3 of and Mos1 (17). In a separate study, PB has also been mPB. L1 linkers were created as a result of the cloning shown to be more active than SB12, another hyperactive process. L2 and L3 linkers were introduced between mPB version of SB (18). Although there are other hyperactive and ERT2 by sub-cloning the pairs of primers shown in SB transposases available, which apparently show higher Supplementary Table 1. The core aminoacidic sequence of activity than SB11 and SB12, they have not been directly the L3 linker was kindly provided by Dr Joseph Kaminski. compared to PB (19,20). Given its good performance The CDSs and protein sequences of mPB (GenBank in mammals we have characterized and optimized the accession number: EF587698) and mPB-L3-ERT2 PB system for its utilization in mouse ES cells and for (GenBank accession number: EF587699) are shown in somatic mutagenesis in vivo. We have generated a mouse Supplementary Figure 1. codon-optimized version of the PB transposase CDS and Cell culture and transfection we have observed that it provides levels of transposition considerably higher than those of the native PB CDS. AB2.2 ES cells were cultured on a layer of mitotically We have analysed the differential properties of the 5 and inactive SNL76/7 feeder cells and transfected by electro- 3 -terminal repeats of the PB transposon, uncovering the poration as described previously (23). Puromycin selection existence of promoter activity in the 5 -terminal repeat. was conducted on SNLP 76/7-4 feeders (a puromycin Finally, we have created a highly active and regulatable resistant derivative of SNL76/7) with 3 mg/ml of puromy- PB transposase, by fusing the optimized PB CDS to the cin. Puromycin resistant ES cell colonies were stained for modified ligand-binding domain of the oestrogen receptor, 15 min with 1% methylene blue in 70% EtOH, washed in ERT2. We expect that this optimized transposon system distilled water over night and air-dried. This protocol will significantly expand the utility of transposon-based produced a very low background which allowed counting mutagenesis for the genetic characterization of the mouse colonies with diameters >0.3 mm. COS-7 cells and genome. HeLaS3 cells were grown in 6-well plates and with DMEM supplemented with 10% foetal bovine serum. Plasmids were transfected with Lipofectamine 2000 MATERIALS AND METHODS (Invitrogen), following manufacturer’s instructions. Plasmids Western blot iPB and mPB were custom synthesized. Besides the PB transposase CDS, both sequences included a Kozak Transfected COS-7 cells were washed with PBS and element around the start ATG at the 5 -end and two each well was harvested with 500 ml2  Laemmli buffer. consecutive stop codons at the 3 -end. EcoRI and NotI Fifteen microlitres of lysate were loaded per lane in a 0 0 and 3 -ends, respectively, and they were used flank the 5 4–12% gradient SDS–PAGE gel (Invitrogen). The gel was TM to clone both CDSs into pcDNA3 or pcDNA3-KzHA. To transferred to a Hybond-ECL (Amersham Biosciences) produce the donor plasmids, the puroDTK fusion gene and membrane, and the membrane was dried over night the bovine growth hormone polyA signal were PCR and blocked with 5% non-fat dry milk dissolved in PT amplified from pFlexible (21). En2SA was PCR amplified (0.1% Tween-20 in 1 PBS) for 1 h at room temperature. PAGE 3 OF 8 Nucleic Acids Research, 2007, Vol. 35, No. 12 e87 Then, it was incubated with 12CA5 anti-HA monoclonal In these experimental conditions we did not observe antibody (Abcam, cat #ab16918) or with anti-actin overproduction inhibition, a phenomenon described in monoclonal antibody (Sigma, cat #A5316), both diluted certain transposon systems in which transposition rates 1:1000 in 3% non-fat milk dissolved in PT for 1 h at room decline when the amount of transposase exceeds certain temperature. The membrane was washed 3 times for 5 min levels (10,14,18). Interestingly, the levels of transposition with PT and incubated with the peroxidase-labelled provided by iPB in these series of experiments were far TM anti-mouse antibody from the ECL Western Blot below those of mPB, especially in Figure 1D, where not Analysis System (GE Healthcare) diluted 1/10000 in even the highest amount of iPB plasmid is able to match 1.5% non-fat dry milk for 1 h at room temperature. The the levels of transposition obtained by transfection of membrane was washed again as before and specific protein the lowest amount of mPB. This is likely due to the TM bands were detected using ECL Western Blot Analysis stringent conditions (5  10 cells per transfection and 1 mg System (GE Healthcare). of donor plasmid) of this experiment, aimed at analysing saturation effects. Additional experiments performed in Luciferase assay less-restrictive conditions showed that increasing amounts of iPB provided levels of transposition in the same order Transfected HeLaS3 cells were washed with PBS and each of magnitude as those of mPB (Supplementary Figure 2), well was lysed for 15 min with 100 mlof1 passive lysis indicating that the differences between the performance buffer from the Dual Luciferase Reporter Assay System of iPB and mPB strongly depend on the experimental (Promega). Lysates were harvested and 20 ml of each settings. Despite this, we must remark that mPB showed sample were analysed for luciferase activity following the higher levels of transposition than iPB in all the manufacture’s instructions in a MicroLumatPlus LB 96V conditions tested. luminometer (Berthold Technologies). To ascertain if the higher transposition frequency provided by the optimized sequence was caused by RESULTS higher production of transposase we subcloned iPB and mPB into pcDNA3-Kz-HA, a pcDNA3-based vector Increased transposition by codon-optimization of PB containing a Kozak sequence plus the nucleotide sequence transposase encoding the hemagglutinin epitope (HA) (Figure 2A), As the PB transposon was originally isolated from insect enabling detection of protein production from HA-iPB cells, we hypothesized that a mouse codon-optimization of and HA-mPB by Western blot analysis using an anti-HA the transposase CDS might produce higher levels of antibody. These two plasmids plus an empty pcDNA3- transposition in the murine ES cell context (24). To Kz-HA vector were transfected into COS-7 cells in address this possibility we synthesized the native PB triplicate. Analysis of protein lysates using an anti-HA transposase CDS (hereafter iPB) and a mouse codon monoclonal antibody revealed a clear band at the expected optimized CDS version (hereafter mPB). iPB corresponds size (68 kDa) in the cells transfected with transposase to the original wild-type coding sequence of the PB expression plasmids, but not in the cells transfected with transposase, whereas mPB is a synthetic sequence coding the empty vector (Figure 2B). The amount of PB for the same polypeptide as iPB, but where each codon has transposase in the cells transfected with HA-mPB was been changed for the preferred codon for translation in considerably and consistently higher than in the cells mouse cells. Both iPB and mPB contain a Kozak sequence transfected with HA-iPB, confirming that higher levels of built around the start methionine codon. Besides this, transposase production are likely responsible for the potential cryptic splice sites generated during the codon greater transposition activity provided by mPB. optimization were detected and avoided in the optimized sequence. Expression vectors were assembled by subcloning both 0 0 Differential features of the minimum 5 and 3 -PB versions of the transposase CDSs into pcDNA3. We terminal repeats compared their ability to promote transposition of a PB The original PB transposon is flanked by 13-bp terminal transposon carrying a promoterless puromycin resistance inverted repeats and has additional inverted repeats cassette from a transfected plasmid vector into the ES cell 19-bp long located asymmetrically with respect to the genome (Figure 1A). iPB or mPB expression plasmids end of the element (24). These inverted flanking sequences, were co-electroporated with a transposon donor plasmid 0 0 although able to permit transposition between different (5 -PTK-3 ) into AB2.2 mouse ES cells and the numbers of plasmids in insect cells, were found to be insufficient to puromycin-resistant colonies were assessed. mPB yielded allow transposition from a donor plasmid to genomic considerably more puromycin-resistant colonies than iPB DNA. In fact, additional transposon DNA sequence was (Figure 1B). found to be necessary to provide this kind of transposition To address saturation effects the amounts of helper or 0 0 (25,26). A 5 -terminal repeat (5 -TR) of 313 bp and donor plasmids were varied while keeping a constant 0 0 a3 -terminal repeat (3 -TR) of 235 bp have been described amount of the respective donor or helper plasmid as the minimum PB terminal repeats (25). Combinatorial (Figure 1C and D). In both series of experiments, mPB experiments performed with the SB transposon have provided higher levels of transposition than iPB through- out the whole range of amounts of transfected plasmids. shown that a configuration containing two ‘left’ terminal e87 Nucleic Acids Research, 2007, Vol. 35, No. 12 PAGE 4 OF 8 Figure 1. Comparison of transposase activity of the native and codon optimized piggyBac.(A) Schematic representation of the plasmids used in this 0 0 0 0 experiment. The donor plasmid, 5 -PTK-3 , contains the minimal PB terminal repeats (5 -TR and 3 -TR) flanking a promoter-less puromycin resistance cassette. CMV, cytomegalovirus promoter; ATG, start codon; pA, bovine growth hormone polyA signal; En2SA, mouse engrailed-2 gene splice acceptor; IRES, internal ribosomal entry site from encephalomyocarditis virus; puTK, synthetic fusion CDS between the puromycin- 0 0 resistance gene and a truncated thymidine kinase gene (21). (B)2 mgof 5 -PTK-3 and 2 mg of the transposase plasmid were co-electroporated into 10 AB2.2 mouse ES cells and, after puromycin selection, the numbers or resistant colonies were counted. (C) A fixed amount (1 mg) of transposase 0 0 plasmid was co-electroporated with increasing amounts (1–40 mg) of 5 -PTK-3 into 5  10 AB2.2 ES cells and puromycin-resistant colonies were 0 0 counted. (D) A fixed amount (1 mg) of 5 -PTK-3 transposon plasmid was co-electroporated with increasing amounts (1–40 mg) of transposase plasmid into 5  10 AB2.2 ES cells. Numbers of puromycin-resistant colonies are indicated. n = 3, error bars: SEM. repeats (or 5 -TRs) provides levels of transposition 3 times assessed luciferase activity in transiently transfected higher than the original ‘left’ + ‘right’ combination (11). HeLaS3 cells. The luciferase activity observed with the 0 0 0 In order to study the properties of the 5 and 3 -TRs of 5 -TR-Luc2 construct was 5 times higher than that the PB element and to explore the possibility of enhancing observed in the cells transfected with the 3 -TR-Luc2 the rates of transposition, we subcloned a promoter-less construct (Figure 3B), indicating that the promoter in the puromycin resistance cassette between different combina- 5 -TR repeat is also active in mammalian cells. 0 0 tions of minimum 5 and 3 -PB terminal repeats 0 0 0 0 0 0 0 0 (Figure 3A) (5 –5 ,5 –3 ,3 –5 and 3 –3 ) and cotransfected Generation of an inducible PB transposase the resulting donor plasmids with the mPB expression The success of cancer-gene identification experiments cassette into AB2.2 cells. Only the cells transfected with 0 0 0 0 performed with the SB transposase in mice has been the 5 –3 and 3 –5 configurations yielded significant strongly determined by the levels of transposase activity numbers of resistant colonies (Figure 3A). These results (12,13). However, an excess of transposition has been indicate that the PB transposon requires one 5 and one reported to create undesired genomic rearrangements in 3 -terminal repeat in order to achieve transposition. 0 0 an SB-based system for mouse germline mutagenesis (14). The number of colonies obtained with the 5 –3 0 0 Thus, the ability to control transposition temporally transposon was 4.6 times higher than with the 3 –5 would provide an additional advantage to the PB configuration (Figure 3A). As we had used a promoter- transposase. To explore the possibility of generating an less selection cassette, one explanation of this observation is that the promoter sequence predicted in the 5 -TR, inducible transposase we created fusion proteins between which is functional in insect cells, is also active in the mPB and the modified oestrogen receptor ligand-binding mammalian genome (24,27). To address this possibility, domain (ERT2), at the N or C terminus of mPB (ERT2- we subcloned a luciferase expression cassette (Luc2) mPB and mPB-ERT2, respectively) (28) (Figure 4A). 0 0 downstream of the 5 and 3 -PB TRs (Figure 3B) and The ERT2 domain provides the possibility of regulating PAGE 5 OF 8 Nucleic Acids Research, 2007, Vol. 35, No. 12 e87 more than 800-fold to background levels. To our knowl- edge, mPB-L3-ERT2 is the first PB transposase inducible by 4-OHT, it provides higher activity than iPB and it shows undetectable levels of leakiness. DISCUSSION The recent availability of DNA transposons which are active in the mammalian genome has widened the set of tools to carry out functional genomics and transgenesis. SB, a transposon reconstructed from inactive sequences present in salmonid fishes, has already shown great utility, having being successfully used in cancer-gene identifica- tion, germline mutagenesis and human cell transgenesis experiments (3,8,12–14). More recently, PB, an active transposon from the cabbage looper moth Trichoplusia ni, has also been shown to work in mammalian cells, providing transposition levels higher than those displayed by certain hyperactive SB forms (16–18). These two transposable elements have distinctive properties, for instance target sequence preference, which suggests that they will be used as complementary tools to modify and study the mammalian genome (8,18,30,31). We have characterized, optimized and further enhanced Figure 2. Levels of protein production provided by the native and the functionality of the PB transposon/transposase codon optimized piggyBac.(A) Schematic representation of the system. We have synthesized a mouse-codon-optimized plasmids used in this experiment. HA, sequence encoding the hemagglutinin epitope. (B) pcDNA3-KzHA, HA-iPB or HA-mPB PB transposase CDS, which provides levels of transposi- were transfected into COS-7 cells and the levels of HA-tagged PB tion significantly higher than the native PB transposase transposase were analysed by Western blot using a specific monoclonal CDS over a wide range of transposon/transposase ratios. antibody raised against the HA epitope. The same membrane was Remarkably, the difference in performance between iPB subsequently incubated with a monoclonal anti-actin antibody, as loading control. Three independent transfections were performed and and mPB strongly varied with the experimental condi- analysed per plasmid. tions, although mPB always provided higher levels of transposition. Notably, overproduction inhibition, the decrease of transposition produced by excessive trans- the activity of a protein by the presence or absence of posase previously observed in SB, was not detected for PB 4-hydroxytamoxifen (4-OHT). In the absence of 4-OHT a in the experimental conditions we used (10,17,18). protein containing the ERT2 domain is sequestered by Intriguingly, two previous reports have tried to address heat-shock proteins, preventing it from functioning (29). the existence of this phenomenon in PB with disparate In the presence of 4-OHT, the fusion protein is released results. On one hand, Wu et al. (17) found overproduction and can then play its role (28). inhibition of PB in HEK293 cells. On the other hand, The two key requirements of inducible proteins are high Wilson et al. (18) concluded that PB lacks overproduction activity under induced conditions and low/no activity in inhibition in HEK293 cells. Interestingly, in the same the absence of induction. To achieve the maximum activity study, Wilson et al. transfected equivalent amounts of a and inducibility we evaluated a series of linkers between pCMV-based SB12 helper and donor plasmids in mPB and ERT2 of different lengths, charges and flexibil- HEK293 cells, and observed the previously reported ities (Table 1). Most of the fusion proteins appeared to overproduction inhibition. Although our results support have reduced activity. The L1 fusions show 100-fold the lack of overproduction inhibition in PB, further work reductions compared with mPB. The L2 fusions also is needed to clarify this issue. exhibited reduced activity although one of them (ERT2- We have also shown that the higher transposition L2-mPB) was not leaky when not induced. However, the provided by mPB correlates with increased protein fusion proteins containing L3 linkers provided high levels of transposition (Figure 4B). The L3 linker was based on a production. This result highlights the usefulness of sequence which has previously been used between the codon optimization to increase protein levels. Besides GAL4-DNA-binding domain and the native PB transpo- this, the availability of two different sequences encoding sase, which resulted in levels of activity of the fusion PB transposase offers the opportunity to obtain different protein close to 100% with respect to the wild-type enzyme levels of protein and transposition activity. (17). The number of colonies obtained in the presence of We have analysed the differential properties of the 5 4-OHT using mPB-L3-ERT2 was 6 times higher than those and 3 -PB terminal repeats. In contrast with the previous obtained with iPB, though it was less efficient than mPB. results obtained with different combinations of the SB Importantly, the activity was highly 4-OHT-dependent, terminal repeats we have only detected transposition since in the absence of 4-OHT transposition was reduced activity when the naturally occurring combination of PB e87 Nucleic Acids Research, 2007, Vol. 35, No. 12 PAGE 6 OF 8 Figure 3. Analysis of the properties of the piggyBac terminal repeats. (A) Left, schematic representation of transposon plasmids used in this experiment. Each plasmid contains the same gene trap puromycin resistance cassette flanked by different combinations of 5 -TR (blue arrows) and 3 -TR (red arrows). Right,2 mg of transposon plasmids were co-electroporated into 10 AB2.2 mouse ES cells with 2 mg of mPB helper plasmid. The numbers of puromycin-resistant colonies were counted. (B) Left, schematic representation of the firefly luciferase expression plasmids used in this 0 0 experiment. 5 -TR-Luc2 contains the minimal left terminal PB repeat followed by the firefly luciferase CDS (Luc2). 3 -TR-Luc2 contains the minimal 0 0 right terminal PB repeat followed by Luc2. pA, SV40 late polyA region. Right,5 -TR-Luc2 or 3 -TR-Luc2 were transfected in HeLaS3 cells together with pGL4.74, a transfection control plasmid containing the renilla luciferase gene under the thymidine kinase promoter. Twenty-four hours after transfection firefly and renilla luciferase activities were measured. n = 3, error bars: SEM. 0 0 5 -TR and 3 -TR was used (11). Interestingly, we obtained combinations of mPB with ERT2 (28). The six versions higher numbers of colonies when the resistance cassette have two different arrangements of ERT2 either at the 0 0 was oriented from the 5 -TR to the 3 -TR than in the C-terminus or at the N-terminus of the PB transposase opposite orientation. In these experiments the transposon coupled with three different linkers (L1, L2 or L3) between contained a gene-trap selection cassette. As the 5 -TR has ERT2 and mPB. Four of these fusion proteins show been described to contain a promoter active in insect cells, extremely reduced levels of transposition when compared we hypothesized that the observed differences in colony to the parental mPB, even in the presence of the inducer, numbers could derive from transcriptional activity 4-OHT. However, the other two fusion proteins, ERT2- from the 5 -TR activating the selection cassette (27). In L3-mPB and mPB-L3-ERT2, produce high levels of a promoter analysis experiment using luciferase as a induced transposition. Both of them contain a linker reporter, we observed that the 5 -TR does act as which was based on an aminoacidic sequence previously a promoter which is 5-fold stronger than the 3 -TR. used to fuse the native PB transposase to the GAL4- This observation should be considered in the design of DNA-binding domain (17). The length and the net PB-based promoter-less transposons, as the expression positive charge of this linker compared with the ones of the promoter-less cassette from the 5 -TR repeat which were less successful are likely to underlie the may produce unwanted results. In our view, the huge differences in activity. Remarkably, mPB-L3-ERT2 simplest solution would be to rearrange the transposon yields levels of transposition 6 times higher than iPB in 0 0 so that the 3 -TR is upstream of the 5 -end of the the presence of 4-OHT, whereas no detectable transposi- promoter-less sequence. Remarkably, recent work has tion was observed without induction. In the absence of detected enhancer activity in the 3 -TR of the piggyBac 4-OHT the transposition activity provided by mPB- transposon (32). In our opinion, the presence of promoter L3-ERT2 is reduced 800-fold, returning back to the 0 0 and enhancer sequences in the piggyBac 5 -TR and 3 -TR, low experimental noise levels. Thus mPB-L3-ERT2 is not respectively should also be considered in gene-therapy only an improved version of the PB transposase, but it applications, so as to avoid the negative experiences of can also be temporally regulated. This newly engineered generating activating mutations previously obtained with enzyme will be useful for diverse applications, ranging retroviruses (33). from gene therapy, where the possibility of switching Finally, we have engineered an inducible, optimized PB on and off the enzyme would contribute to making it safer, transposase. We have made and assessed six different to forward genetics cancer screens in mice, where the PAGE 7 OF 8 Nucleic Acids Research, 2007, Vol. 35, No. 12 e87 ACKNOWLEDGEMENTS We would like to thank R. Rad, A. Rodrı´guez, S. Cowley, H. Prosser, E. Ferna´ ndez, C. Lo´ pez-Otı´n, J.M.P. Freije, D. Adams, G. Vassiliou, W. Wang and P. Liu for helpful comments and discussions, as well as A. Beasley for excellent technical assistance. J. Cadin˜ anos is a recipient of a FEBS Long-Term Fellowship. This work was supported by the Wellcome Trust. Funding to pay the Open Access publication charges for this article were provided by the Wellcome Trust. Conflict of interest statement. None declared. REFERENCES 1. Carlson,C.M. and Largaespada,D.A. (2005) Insertional mutagenesis in mice: new perspectives and tools. Nat. Rev. Genet., 6, 568–580. 2. Fischer,S.E., Wienholds,E. and Plasterk,R.H. (2001) Regulated transposition of a fish transposon in the mouse germ line. Proc. Natl Acad. Sci. 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Published: Jun 18, 2007

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