Abstract:
The present invention is directed to nucleic acid and amino acid sequences of a novel piggyBac transposase enzymes created by modifying the transposase of  Trichoplusia ni.  The piggyBac transposases of the present invention are functionally active or hyperactive for excision and have decreased integration activity compared to wild type  Trichoplusia ni  piggyBac transposase enzyme. These transposases are ideal for use in methods of transforming cells and organisms. In particular embodiments, the present invention provides methods of transient integration and expression of transgenes.

Description:
CROSS-REFERENCE TO PRIOR APPLICATIONS 
       [0001]    This application claims the benefit of U.S. Provisional Application No. 61/508,386, filed on 15 Jul. 2011, the entire contents of which is incorporated herein by reference to the extent permitted by applicable law and regulation. 
     
    
     U.S. GOVERNMENT SUPPORT 
       [0002]    Not Applicable 
       BACKGROUND OF THE INVENTION 
       [0003]    The ability to generate induced pluripotent stem cells (iPSCs) without a permanent DNA sequence change is extremely important for many subsequent applications. A useful strategy is to integrate the genes necessary for transformation into the target genome using the DNA cut &amp; paste piggyBac transposon (1) as the vector. The piggyBac transposon has a large cargo size and a high integration efficiency. The piggyBac transposase promotes insertion of the piggyBac transposon into TTAA target sites by binding to specific sequences at the transposon ends. Upon integration, the element becomes stably associated with the host genome and can serve as a long-term source of transcription factors required for cell transformation. Once iPS cell transformation has occurred, the piggyBac vector can then be re-exposed to transposase and excised by its natural “precise excision” pathway, in which the insertion site is restored to its pre-transposon TTAA sequence (2, 3). To avoid further genome modification, it is important that the excised transposon not re-integrate. 
         [0004]    We are interested in developing improved piggyBac transposons as more useful tools for generating transgene-free iPSCs. A particularly useful tool for removal of the piggyBac vector after iPS cells transformation would be a piggyBac transposase that can promote excision at high frequency but is defective for re-integration following excision, i.e., an Exc + Int −  transposase. 
         [0005]    Using in vitro and in vivo assays in mammalian cells (4), we have isolated a piggyBac Exc + Int −  transposase. In  Saccharomyces cerevisiae  (4), and confirmed in mammalian cells, we have also isolated a transposase that is hyperactive for excision but still integration negative, Exc +hyper Int − . 
     
    
     
       DESCRIPTION OF THE FIGURES 
         [0006]      FIG. 1  shows sequences of transposases;  FIGS. 1A and 1B  are sequence alignments of the  T. ni  transposase sequence with other transposases in the same protein family; and  FIG. 1C  shows the important mutagenized sites of  T. ni  transposase. 
           [0007]      FIG. 2  shows details of Exc+Int− mutant screening in mammalian cells with  FIG. 2A  showing the excision assay by PCR;  FIG. 2B  showing results of colony counting assays; and  FIG. 2C  showing a table of the excision and integration activities; 
           [0008]      FIG. 3  shows the results of in vitro screening of the Ext+Int− candidates;  FIG. 3A  is a diagram of the steps of the piggyBac transposon operation;  FIG. 3B  shows gels of the purified proteins from the various candidates;  FIG. 3C  shows the results of the hairpin resolving and target joining assay;  FIG. 3D  are charts showing quantification of the tests in  FIG. 3C ;  FIG. 3E  shows gels depicting the results of the strand transfer assay; and  FIG. 3F  shows the results of an assay for in vitro nick (at 87 nt) and double strand breaks (at 70 nt). 
           [0009]      FIG. 4  shows the hyperactive R372A/K375A mutant screening in yeast;  FIG. 4A  is a diagram of the process related to the color from the Cherry gene of the yeast cell; and  FIG. 4B  shows the retest of the relative excision activities of the hyperactive mutants; and  FIG. 4C  shows the location of the 35 best candidates. 
           [0010]      FIG. 5  shows the relative excision activities of the hyper reactive R372A/K375A transposase mutants in mammalian cells;  FIG. 5A  shows a diagram of the test strategy;  FIG. 5B  shows the relative activities of the candidates. 
           [0011]      FIG. 6  shows how M194V and D450N were combined with 7PB, a hyperactive transposase for screening;  FIGS. 6A and 6B  show the activity levels in bar graphs and  FIG. 6C  shows the actual cell colonies on plates. 
           [0012]      FIG. 7  shows integration assays demonstrating that the mutants are still defective in integration;  FIG. 7A  shows the retest in yeast cells and  FIG. 7B  shows mutation combinations. 
           [0013]      FIG. 8  shows retest in mammalian cells of mutant combinations demonstrating hyperactive excision. 
           [0014]      FIG. 9  shows tables of excision activity of EXc+Int− mutation combinations;  FIG. 9A  shows mutation combinations with hyperactive excision;  FIG. 9B  shows mutation combinations with WT-like excision activities; and  FIG. 9C  shows mutation combination that give low excision activities. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0015]    The following description is provided to enable any person skilled in the art to make and use the invention and sets forth the best modes contemplated by the inventor of carrying out his invention. Various modifications, however, will remain readily apparent to those skilled in the art, since the general principles of the present invention have been defined herein specifically to provide unique Ext+Int− transposases. 
         [0016]    Transient integration and expression of transcription factors are important approaches to generate transgene-free iPSCs as well as directed differentiation tissues for both research and clinical use. Removal of the transgenes is critical for the therapeutic applications of the iPSCs. The piggyBac transposon, which was isolated from cabbage loop moth,  Trichoplusia ni,  can efficiently transpose genes of interest into the mammalian genome and more importantly can be precisely removed from the integration sites by piggyBac transposase thereby restoring the original sequence at integration sites. However, reintegration of the excised transposon catalyzed by the transposase is a concern (with piggyBac about 40-50% of the excised transposons reintegrate (6)). A piggyBac transposase mutant, which can only excise without integration would be a useful tool for generating iPSCs without any genetic change. 
         [0017]    To that end, we screened a piggyBac transposase target knockout for mutations, which could only catalyze the excision of the transposon but not integration. We further looked for increased excision activity by screening for hyperactive mutants, while still maintaining the integration negative feature. 
         [0018]    Excision +  Integration −  mutant screening in vivo and in vitro. The alignment of the piggyBac family showed that there are some conserved arginines and lysines around the catalytic sites D268, D346 and D447. We mutated these Arg and Lys to Ala. At the same time, we also mutated the other two amino acids, which are located at the conserved second catalytic D of HIV integrase I and asked whether the excision and integration activities of these mutants are abolished.  FIG. 1  shows the amino acid residues chosen for making excision + /integration −  (Exc + Int − ) mutation, R245A, R275A/R277A, K287A, K287A/K290A, R315A, R341A, S351E, K356E, K375A, R372A/K375A, R388A, K409A/K412A, K432A, R460A/K461A. To test the excision activity in vitro, we co-transfected the transposase plasmids, either wild type (WT) or different mutants made by site-direct mutagenesis, together with the donor plasmid which carries piggyBac transposon into HEK293 cells with FuGENE 6 transfection reagent. 
         [0019]    Three days after transfection, the cells were harvested and plasmids were recovered by Hirt methods as described in (5). PCR was then completed with 2 primers against the flanking donor sites (pbexl2, GGAACAGGAGAGCGCACGAG [SEQ ID NO:1] and pbexr2, GAGAGTGCACCATATATGCGGTG [SEQ ID NO:2) using the following conditions (I× 95° C., 4 min, 40× 94° C., 30 sec, 64° C., 30 sec and 72° C., 30 sec; I× 72° C., 5 min). PCR products were run on 1.8% agarose gel and results were shown by ethidium bromide (EtBr) staining.  FIG. 2A  shows the excision assay strategy and  FIG. 2B  shows the results of PCR products from repaired donor DNA after excision. A shorter (277 bp) DNA fragment is shown on the gel if there is excision and the donor side repaired. As shown in  FIG. 2B , R245A, R275A/R277A, R315A, R341A, S351E, K356E, K375A, R372A/K375A, R388A showed excision activity at similar level to WT. K409A/K412A and K432A showed decreased excision activity. No excision activity was observed with K287A, K287A/K290A, (F349A) and R460A/K461A either due to abolished excision activity or to flanking donor sites that cannot be repaired. We were interested in the mutants with WT excision activity and wanted to know whether they are defective in the integration step. Colony counting assays were used to test the integration activity of the mutants. HeLa cells were co-transfected with donor plasmid containing piggyBac transposon carrying blasticidin drug resistance marker and transposase (both WT and mutants) expression plasmids. 
         [0020]    Two days later, cells were trypsinized, diluted and seeded on plates with DMEM medium containing 3.5 ug/ml blasticidin. Drug selection was continued for 18 to 21 days, surviving cells were stained with 0.2% methylene blue, and the plates were scanned. As shown in  FIG. 2C , R275A/R277A, R372A/K375A and R388A have no integration activity (same level as the negative control without transposase) or very low integration activity. Interestingly, S351E showed very low integration activity. S351 is conserved with the Ser119 of HIV integrase I (7) and in a recently solved PFV integrase structure (8), A188 follows the conserved second catalytic D site. Both amino acids are reported to be important for integrase targeting. K356A, R341A and R245A showed decreased integration activity. R315A and K375A showed little change in integration activity as compared with WT. 
         [0021]    Thus, we have several mutant candidates for further study: namely, R275A/R277A, R372A/K375A, R388A and S351E. We also included two mutants with dramatic decreases in integration activity: R341A and R245A. To better understand the mechanism, we performed in vitro assays with proteins purified from an  Escherichia coli  strain having an over-expressed transposase. 
         [0022]    In vitro characterization of the Exc + /Int −  (or Int decreased) mutants. It has been shown that piggyBac transposase catalyzes the transposition in 4-steps (4) as shown in  FIG. 3A : 1) in vitro nick; 2) hairpin of transposon ends excision; 3) opening of hairpins formed after a double-strand break; and 4) target joining. We know that the excision activity of R275A/R277A, R372A/K375A, R388A, S351E, R341A and R245A mutants is about the same level as WT, this means that nick, hairpin formation and double-strand break steps should be about the same as WT. We started from the hairpin opening/resolution step to see whether they show differences in hairpin opening and/or target joining. To test the kinetics of the hairpin resolution step of different mutants, we used a 5′- 32 P-labeled PGB40, a 35 base pair (bp) hairpin with extra TTAA (74 nucleotide (nt) oligo containing 35 bp of PB L-end and flanking TTAA in the middle (PGB40: CATGCGTCAATTTTACGCAGACTATCTTTCTAGGGTTAACCCTAGAAAGATAGTCTGCGTAAAATTGACGCATG [SEQ ID NO:3]). 
         [0023]      FIG. 3  shows the details of the assays.  FIG. 3B  shows a gel of the Int −  proteins.  FIG. 3C  shows the results of the hairpin resolved and target joining assay. A 60-bp R-end oligo with TTAA hairpin was labeled with  32 P at the 5′-end used as substrate and reactions were performed as previous described (4), reactions were stopped at different time points by adding EDTA. Samples were run on denaturing PAGE gel. If the hairpin is opened, it will give a shorter (35 nt) DNA fragment on a denaturing sequencing gel. From the reaction, we can also observe target joining products if this step is not defective for the mutants. As shown in  FIGS. 3E and 3F , R372A/K375A has better hairpin resolving activity as compared with WT, but is defective in target joining. R275A/R277A and S35 1E showed slightly decreased in hairpin resolving, and were defective in target joining. R388A showed dramatic decrease in hairpin opening/resolving and was therefore defective in target joining. The other two mutants—R245A and R341A showed a slight decrease in both hairpin resolving and target joining, which suggest that R245 and R341 are not involved in targeting step. This in vitro data agrees with the in vivo data shown previously. Based on both in vivo and in vitro data, we chose R372A/K375A further studies as the best candidate for piggyBac transposase mutants with excision plus and integration minus features (Exc + /Int − ). 
         [0024]    To better understand the kinetics of the R372A/K375A mutant, we performed a nick and double-strand break assay with a DNA substrate containing 74 bp flanking sequence with TTAA and 87 bp of piggyBac R-TIR and labeled it at 5′-end of both top and bottom strands ( FIG. 3F ). The results showed that the R372A/K375A mutant has slower nick and double-strand break activity in pre-steady state. However, at steady state, R372A/K375A mutant has comparable double strand break products accumulated but cannot further form the target joining product. In the case of WT, partial double strand break products can transform into target joining product. 
         [0025]    In vivo excision assay in yeast,  Saccharomyces cerevisiae.  To more accurately measure the excision activity in vivo, we set up in vivo excision systems in both yeast  S. cerevisiae  and mammalian HEK293 cells. In yeast system, we cloned the piggyBac transposon carrying a kanamycin marker into the mCherry gene at TTAA site located at the very N-terminus of the mCherry gene (position 80-83) on pGAP/Cherry plasmid (9). This disrupts the expression of red fluorescence protein (RFP) ( FIG. 4A ). The mCherry gene will be fully restored in the presence of piggyBac transposase, which can be precisely excised and repaired. RFP signal can be detected under fluorescence microscope (Leica: M165FC). We transformed both WT and R372A/K375A mutants in a pGals expression vector into yeast strain BY4727 with plasmid pCherry::PBTn, 3 days later, colonies were re-suspended in water and spotted onto both SC (synthetic complete) media Trp-Ura+Glucose and Galactose plates. Re-plating plates were scanned under the fluorescence microscope 1, 2 and 3 days later. From  FIG. 4B , we can observe that the excision activity of R372A/K375A mutant was weaker than WT, which is in agreement with the in vitro results. 
         [0026]    Excision and integration assay in mammalian cells A similar strategy was used to measure the excision activity in HEK293 cells. First, we generated a stable HEK293 cell line by Tol2 transposition, in which a Tol2 transposon carrying the Cycle 3-GFP (C3-GFP) under the control of EF-I α promoter. The C3-GFP gene was first mutated at position 72 from T to C of the C3-GFP gene by site-directed mutagenesis which created a HpaI site for cloning of the piggyBac transposon which carries a blasticidin drug resistance gene. The Tol2 transposon will permanently integrate into the chromosome in the presence of Tol2 transposase. With this strategy, we generated a stable cell line—HEK293/T46—to use in mammalian cell excision. Once the piggyBac transposon is excised by piggyBac transposase, the C3-GFP gene will be restored and the GFP signal can be sorted by FACS (fluorescence activated cell sorter) analysis as shown in  FIG. 5A ). To compare the R372A/K375A mutant with WT in excision activity, we transiently transfected plasmids of R372A/K375A mutant and WT in pcDNA3.1/myc-HisA mammalian expression vector into HEK293/T46 line which carries C3-GFP::PBTn. Four to five days later, cells were trypsinized and washed with PBS. Then FACS analysis was used to measure the GFP cell population. As showed in  FIG. 5B , we also observed the R372A/K375A mutant has about 50-60% of WT excision activity. 
         [0027]    Screening hyperactive piggyBac/Exc + Int −  mutants in  S. cerevisiae.  To make a more efficient tool for piggyBac transposon excision, we set up to screen excision hyperactive and integration negative mutants. First, we generated PB/R372A/K375A mutagenesis libraries with 2 primers: pGals-f for priming the 5′-side of the pGals vector cloning site sequence with ATG start codon of the transposase; the second primer, pGals-r for priming the 3′-side of the cloning site with TGA stop codon of the transposase. We used error-prone PCR in the presence of Mn 2+  to generate three mutagenesis libraries and then co-transformed with linearized pGals vector into  S. cerevisiae  BY4727/Cherry::PBTn. Through homologous recombination, the libraries were constructed in pGals vector in the strain containing Cherry::PBTn. Single colonies were picked and re-suspended in water in 96-well plates and pin replicated to SC-Trp-Ura+Glucose or Galactose omitray plates. Plates were grown at 30° C. for 1, 2, 3 and 4 days and scanned by typhoon scanner to see if any colonies showed more fluorescence at the RFP channel on both Glucose and Galactose plates or any of Glucose or Galactose plates. Plasmids were recovered from the yeast strains with Qiagen mini-prep kit and re-transformed into  E. coli  NEB5α. Colonies were sequenced at MCLAB with primers PGB340 and PGB341. 
         [0028]    We screened a total of 6000 colonies from three individual libraries and got 35 candidates to test for further confirmation in the yeast excision assay system. We also tested the candidates in mammalian cells HEK293/T46. As shown in  FIG. 5B , nine candidates showed higher excision activity than R372A/K375A; they are S103P/R372A/K375A, M194V/R372A/K375A, M194T/R372A/K375A, 1221T/R372A/K375A, T560A/R372A/K375A, S573AJR372A/K375A, M589V/R372A/K375A, S373P/R372AJK375A, D450N/R372A/K375A. As two candidate mutants—M194V/R372A/K375A and D450N/R372A/K375A—showed the highest excision rates, we focused on these two mutants and asked whether we could make them more hyperactive by combining these two mutants into one, or combine them separately or together with 7PB, a combination hyperactive mutant from WT piggyBac transposase screening (10). As shown in  FIG. 6A , when combining M194V and D450N together with R372A/K375A into one ORF, the excision activity went down to about WT PB level. When combining M194V/R372A/K375A or D450N/R372A/K375A with 7PB, much higher (5-6 fold) excision activity as compared with R372A/K375A was observed. However, when we combined 7PB, M194V, D450N and R372A/K375A all in one, the excision activity decreased to WT level. This result suggests that certain combination of hyperactive mutants might not be better than each one alone of them in excision activity. 
         [0029]    We also tested the integration activity of 7PB/R372A/K375A/M194V and 7PB/R372A/K375A/D450N to make sure none of the combination recovered the integration activity. As shown in  FIG. 6B , they did not show increases in integration. 
         [0030]    Shuffle-PCR to screen different mutant combination. Next we performed a shuffling-PCR screening of the nine different amino acids with R372A/K375A to see which one gave the highest activity. To get all possible combinations of 511 mutants, we used a Shuffle-PCR strategy. Briefly, the first 100 cycle is based on limiting amounts of PCR primers versus two templates: WT with R372A/K375A and all nine amino acid mutations with R372A/K375A at ratio 1:1, 2:1 and 1:2. We used a very short extension time, denatured immediately at 95° C., and then annealed to make them switch templates as described below in materials and methods. Homologous recombination in yeast cell with pGals linearized vector and gel purified Shuffle-PCR generated PCR products were transformed into yeast strain BY4727/Cherry::PBTn. Single colonies were picked and resuspended in water and spotted to SC-TRP-URA+Gal plates. One day after 30° C. culture, the plates were scanned for intensity of RFP of the spot. WT PB, PB/R3 72A/K3 7 5A, PB/R372A/K375A/M194V, PB/R3 72A/K3 75A/D45 0N, 7PB/R372A/K375A/M194V+D450N and 7PB, a hyperactive from previous screening in our lab (10) were used as control. Among these controls, PB/R372A/K375A/M194V gives strongest signal. The colonies with increased intensity of RFP signal as compared with PB/R372A/K375A/M194V were chosen and plasmids were recovered, sequenced re-transformed into yeast BY4727/pGAPCherry::PBTn and retested for fluorescence. Because some of the mutants showed fluorescence earlier due to the early excision that occurred in specific colonies, especially with hyperactive mutants, retest can tell the real hyperactive mutants. As shown in  FIG. 9A  and  FIG. 7A , some of the mutant combinations with R372A/K375A show hyperactive excision.  FIG. 9B  and  FIG. 7B  show mutant combinations with R372A/K375A give excision activity at around WT PB level.  FIG. 9C  shows mutant combinations with R372A/K375A that give low excision activity. We then tested the mutant combinations shown in  FIG. 9A  in mammalian cell HEK293/T46. Unfortunately, as shown in  FIG. 8 , none of them showed higher activity than 7PB/R372A/K375A/D450N, but HP-8, HP-31, HP-38 and HP-43 showed moderately higher activity than 7PB/R372A/K375A/M194V and 7PB. Since we did not run the FACS at the optimum filter setting (excitation 395 nm, emission 507 nm) for C3-GFP, the difference between different mutants might be enhanced by changing the filter setting. 
         [0031]    Materials and Methods 
         [0000]    
       
         
               
             
           
               
                 Primers for making Exc + Int −  mutant candidates: 
               
               
                 M1 (R245A) 54 mer, aga to gca, 
               
               
                 [SEQ ID NO: 4] 
               
               
                 5′-gagaaaac gatgtatttactcctgttgcaaaaatatgggatctatt 
               
               
                   
               
               
                 atccatcag-3′ 
               
               
                   
               
               
                 [SEQ ID NO: 5] 
               
               
                 (5′-ctgatggataaagagatcccatatttttgcaacaggagtaaataca 
               
               
                   
               
               
                 tcgttttctc-3′) 
               
               
                   
               
               
                 M2 (R275A/ R277A) 64 mer aga to gca/ cgg to gcc 
               
               
                 [SEQ ID NO: 6] 
               
               
                 5′-gaccatagatgaacagttacttggttttgcaggagcctgtccgttta 
               
               
                   
               
               
                 ggatgtatatccc-3′ 
               
               
                   
               
               
                 [SEQ ID NO: 7] 
               
               
                 (5′-gggatatacatcctaaacggacaggctcctgcaaaaccaagtaact 
               
               
                   
               
               
                 gttcatctatggtc-3′) 
               
               
                   
               
               
                 M3 (K287A) 55 mer aag to gcg 
               
               
                 [SEQ ID NO: 8] 
               
               
                 5′-ccgtttaggatgtatatcccaaacgcgccaagtaagtatggaataaa 
               
               
                   
               
               
                 aatcc-3′ 
               
               
                   
               
               
                 [SEQ ID NO: 9] 
               
               
                 (5′-ggatttttattccatacttacttggccgcgtttgggatatacatcc 
               
               
                   
               
               
                 taaacgg-3′) 
               
               
                   
               
               
                 M3+ (K290A) 55 mer aag to gcg/ aag to gcg 
               
               
                 [SEQ ID NO: 10] 
               
               
                 5-′gtatatcccaaacgcgccaagtgcgtatggaataaaaatcctcatga 
               
               
                   
               
               
                 tgtg-3′ 
               
               
                   
               
               
                 [SEQ ID NO: 11] 
               
               
                 (5′-cacatcatgaggatttttattccataggcacttggcttgtttggga 
               
               
                   
               
               
                 tatac-3′) 
               
               
                   
               
               
                 M4 (R315A) 47 mer aga to gca 
               
               
                 [SEQ ID No: 12] 
               
               
                 5′-gataaatggaatgccttatttgggagcaggaacacagaccaacgga 
               
               
                   
               
               
                 g-3′ 
               
               
                   
               
               
                 [SEQ ID NO: 13] 
               
               
                 (5 gttggtctgtgttcctgctccc aaataaggcattccatttatcata 
               
               
                   
               
               
                 t-3′) 
               
               
                   
               
               
                 M5 (R341A) 40 mer cgt to gct 
               
               
                 [SEQ ID NO: 14] 
               
               
                 5′-gcctgtgcacggtagttgtgctaatattacgtgtgacaattggttca 
               
               
                   
               
               
                 c-3′ 
               
               
                   
               
               
                 [SEQ ID NO: 15] 
               
               
                 (5′-gtgaaccaattgtcacacgtaatattagcacaactaccgtgcacag 
               
               
                   
               
               
                 gc-3′) 
               
               
                   
               
               
                 M6 (R372A) 35 mer cga to gca/ aaa to gca 
               
               
                 [SEQ ID NO: 16] 
               
               
                 5′-gtgggaaccgtgcgatcaaacgcacgcgagataccggaagtac-3′ 
               
               
                   
               
               
                 [SEQ ID NO: 17] 
               
               
                 (5′-gtacttccggtatctcgcgtgcgtttgatcgcacggttccca 
               
               
                   
               
               
                 c-3′) 
               
               
                   
               
               
                 M6+ (R372A/ K375A) 36 mer 
               
               
                 [SEQ ID NO: 18] 
               
               
                 5′-gttaaccattgtgggaaccgtggcatcaaacaaacgcgagatacctt 
               
               
                   
               
               
                 aac-3′ 
               
               
                   
               
               
                 [SEQ ID NO: 19] 
               
               
                 (5′-ggtatctcgcgtttgtttgatgccacggttcccacaatgg-3′) 
               
               
                   
               
               
                 M7 (R388A) 39 mer agg to gcc 
               
               
                 [SEQ ID NO: 20] 
               
               
                 5′-gtactgaaaaacagtcgctccgccccagtgggaacatcgatg-3′ 
               
               
                   
               
               
                 [SEQ ID NO: 21] 
               
               
                 (5′-catcgatgttcccactggggcggagcgactgtttttcagtac-3′) 
               
               
                   
               
               
                 M8 (K412A) 53 mer aag to gcg 
               
               
                 [SEQ ID NO: 22] 
               
               
                 5′-ctcatataaaccgaagccagctgcgatggtatacttattatcatctt 
               
               
                   
               
               
                 gtg-3′ 
               
               
                   
               
               
                 [SEQ ID NO: 23] 
               
               
                 (5′-cacaagatgataataagtataccatggcagctggcttcggtttata 
               
               
                   
               
               
                 tgag-3) 
               
               
                   
               
               
                 M8+ (K409A/ K412A) 53 mer aag to gcg/ aag to gcg 
               
               
                 [SEQ ID NO: 24] 
               
               
                 5′-cttactctcgtctcatataaaccggcgccagctgcgatggtatactt 
               
               
                   
               
               
                 a-3′ 
               
               
                   
               
               
                 [SEQ ID NO: 25] 
               
               
                 (5′-ataataagtataccatcttagctggggccggtttatatgagacgag 
               
               
                   
               
               
                 agtaagg-3′) 
               
               
                   
               
               
                 M9 (K432A) 43 mer aaa to gca 
               
               
                 [SEQ ID NO: 26] 
               
               
                 5′-cttctatcaacgaaagtaccggtgcaccgcaaatggttatgtattat 
               
               
                   
               
               
                 aatc-3′ 
               
               
                   
               
               
                 [SEQ ID NO: 27] 
               
               
                 (5′-taaccatttgcggtgcaccggtactttcgttgatagaagcat 
               
               
                   
               
               
                 c-3′) 
               
               
                   
               
               
                 M10 (R460A/ K461A) 58 mer agg to gcg/ agg to gcg 
               
               
                 [SEQ ID NO: 28] 
               
               
                 5′-ccaaatgtgttctgtgatgacctgcagtgcggcgacgaataggtggc 
               
               
                   
               
               
                 ctatggcattattg-3′ 
               
               
                   
               
               
                 [SEQ ID NO: 29] 
               
               
                 (5′-caataatgccataggccacctattcgtggcggcactgcaggtcatc 
               
               
                   
               
               
                 acagaacacatttgg-3′) 
               
               
                   
               
               
                 M11 (R499A0 61 mer cgc to gcc (not include in the 
               
               
                 data) 
               
               
                 [SEQ ID NO: 30] 
               
               
                 5′-cagtagcaagggagaaaaggttc aaagtgc cgc aaaatttatga 
               
               
                   
               
               
                 gaaac ctttac atg agcctg-3′ 
               
               
                   
               
               
                 [SEQ ID NO: 31] 
               
               
                 (5′-caggctcatgtaaaggtttctcataaattttgcggcactttgaacc 
               
               
                   
               
               
                 ttttctcccttgctactg-3′) 
               
               
                   
               
               
                 M12 (R518A) 42 mer cgt to gct (not include in the 
               
               
                 data) 
               
               
                 [SEQ ID NO: 32] 
               
               
                 5′-gacgtcatcgtttatgcgtaaggctttagaagctcctactttgaaga 
               
               
                   
               
               
                 g-3′ 
               
               
                   
               
               
                 [SEQ ID NO: 33] 
               
               
                 (5′-ctcttcaaagtaggagcttctaaagccttacgcataaacgatgacg 
               
               
                   
               
               
                 tc-3′) 
               
               
                   
               
               
                 Excision assay primers: 
               
               
                 pbexl2, 
               
               
                 [SEQ ID NO: 34] 
               
               
                 GGAACAGGAGAGCGCACGAG 
               
               
                   
               
               
                 pbexr2, 
               
               
                 [SEQ ID NO: 35] 
               
               
                 GAGAGTGCACCATATATGCGGTG 
               
               
                   
               
               
                 piggyBac Exc+Int- mutant random mutagenesis 
               
               
                 library PCR primers: 
               
               
                 pGals-f, 57-mer 
               
               
                 [SEQ ID NO: 36] 
               
               
                 CTTTAACGTCAAGGAGAAAAAACCCCGGATTCTAGAA 
               
               
                   
               
               
                 CTAGTGGAT CCCCCGGGatg 
               
               
                   
               
               
                 pGals-r, 58 mer 
               
               
                 [SEQ ID NO: 37] 
               
               
                 GATGTGGGGGGAGGGCGTGAATGTAAGCGTGAC 
               
               
                   
               
               
                 ATAACTAATTACATGACTCGAGtca 
               
               
                   
               
               
                 pGals piggyBac mutants sequencing primers: 
               
               
                 [SEQ ID NO: 38] 
               
               
                 GTATTACTTCTTATTCAAATG 
               
               
                   
               
               
                 [SEQ ID NO: 39] 
               
               
                 TCAGGTTGTCTAACTCCTTCC 
               
             
          
         
       
     
         [0032]    Plasmid constructions Mammalian expression vectors of the piggyBac transposase and its mutants were constructed by PCR. The transposases ORFs (PB-f (KpnI+Kozak) GAggtaccGCCACC ATGGGTAGTTCTTTAGACGATGAG  [SEQ ID NO:40] and PB-4r (NotI, with stop) CAGgcggccgc tcaGAAACAACTTTGGCACATAT CAATATTATG  [SEQ ID NO:41] with KpnI and NotI sites) were cloned into KpnI and NotI sites of pcDNA3.1/myc-HisA vector (Invitrogen). Excision+ integration− mutant candidates were obtained by site-directed mutagenesis. 
         [0033]    The piggyBac transposon plasmids for mammalian cell integration assay contain a green fluorescent protein (Cycle 3-GFP) and blasticidin resistance (BsdR) cassette driven by a CMV promoter, flanked by 662 bp Left and 394 bp Right end sequences. 
         [0034]    Plasmids for expression of piggyBac transposase and excision +  integration −  candidates were constructed in pET22b+(EMD) (PB-112 (NdeI) CAGcat atgGGTAGTTCTTTAGACGATGAGC  [SEQ ID NO:42] and PB-2r (XhoI, no stop codon) CAGctcgag GAAACAACTTIGGCACATATCAATATTATGC  [SEQ ID NO:43] and cloned in NdeI, XhoI sites without stop codon, which will give (extra peptide: LEHEITIHHH) C-terminal His-tag. 
         [0035]    For yeast hyperactive color screening plasmid Cherry::PBTnKan construct, pYeGAP-Cherry was obtained by homologue recombination with PCR amplified PBTnKan with homologous cherry oligos: TGGCTATTATTAAAGAATTTATGAGATTTAAAGTTCATATGGAAGGTTCAGTTAACCCTAGAAAGATAGTCTGCG [SEQ ID NO:44] and CTTCATATGGTCTACCTTCACCTTCACCTTCAATTTCAAATTCATGACCATTAACCCTAGAAAGATAATCATATT [SEQ ID NO:45]. G418 colonies were picked and determined by sequencing. The PBTnKan was introduced at position 80-83 TTAA site of the Cherry gene. 
         [0036]    For making mammalian hyperactive color assay plasmid The pC3GFP::PBTnBsd. Cycle 3-GFP gene from pTracer-CMV/Bsd (Invitrogen) was site-directed mutagenized at position 72 from T to C with primer C3GFHpa CAATTCTTGTTGAATTAGATGGTGATGTTAACGGGCACAAATTTTCTGTCAGTGG [SEQ ID NO:46] and its complimentary strand, which will generate a HpaI site. The piggyBac transposon carries a blasticidin resistance gene flanked by 662 by L-end and 394 by R-end sequences with blunt HpaI ends at both 3′ and 5′ ends that was introduced by ligation. Primers with BseRI and NotI were used to PCR the EFIα promoter with the Cycle 3-GFP::PBTnBsd out and clone into pCMV/Zeo (Invitrogen). This creates the plasmid of pEF/C3-GFP::PBBsd. To12 transposon L-end 318 by (ApaI, NotI) and R-end 302 bp (Psd, Nad) were introduced by PCR Tol2 L- and R-ends as well as pCMV/Amp+pUC Ori with primers Tol2L (NotI), GAgcggccgc GTCTGACCAATTTCATATAATGTG  [SEQ ID NO:47] and Tol2R (Sbt1), GAcctgcag gCATTAGATTGTCTGTCTTATAGTTTG  [SEQ ID NO:48]. Cut with SbfI and Nod, ligated to SbfI+NotI fragment pEF/C3-GFP::PBBsd containing EF1a/C3-GFP::PBBsd. This will generate the final plasmid for generating stable cell lines by Tol2 transposition. pGalsPB/R372A/K375A, pGalsPB, pGals7PB were cloned into pGals vector at XmaI and XhoI sites. 
         [0037]    Methods 
         [0038]    Mammalian cell culture and excision and integration assay HEK293 and HeLa cells were cultured in DMEM supplemented with 5% FBS and 2 mM L-glutamine. For excision assay by PCR, about 2×10 6  HEK293 cells were transfected with 1.6 μg of transposon plasmid—pCMV/PBGB and 0.4 μg of transposase and mutant plasmids with FuGENE 6 transfection reagent (Roche Applied Science). Three days later, cells were harvested and plasmids were recovered by Hirt methods (5). The mixtures (helper, donor, repaired donor plasmids) were used as template to perform PCR with two primers against the flanking donor sides of donor plasmid (pbexl2 and pbexr2) with PCR cycles (94° C., 4 mM; 40 cycle of 94° C., 30 sec, 65° C., 30 sec, 72° C., 45 sec; 72° C., 10 min). Reaction mixture: water 30.5 μl, 10× buffer 5 μl, 10 mM dNTP 1 μl, 20 μM Pbex12, 5 μ1, 20 μM Pbexr2, 5 μ1, Hirt method recovered DNA 2 μl, High Fidelity Taq DNA polymerase 1 μl) with Expand High Fidelity PCR system (Roche). PCR products were run on 1% agarose gel. 
         [0039]    For integration assay, 3-5×10 5  HeLa cells were transfected with 1.6 μg of transposon plasmid—p CMV/PBGB and 0.4 μg of transposase and mutant plasmids with FuGENE 6 transfection reagent (Roche Applied Science). Two days later, cells were trypsinized and diluted to different dilution and continued culturing in DMEM containing 3.5 μg/ml of blasticidin for 18-21 days. Medium was changed every 2-3 days. Surviving colonies were stained with 0.2% methylene blue. Plates were scanned with scanner. 
         [0040]    Protein expression, purification and in vitro assay. The piggyBac WT and mutant proteins were expressed in BL21 codon plus/DE3 RIL strain (Stratagene) in pET22b vector (Novagen) with C-terminal His-tag. Protein purification and in vitro nick, double-strand break, hairpin opening, strand-transfer assays have been described previously (4). For hairpin opening, a 74 nt oligo containing 35 bp of PB L-end and flanking TTAA in the middle (PGB40) was synthesized (IDT) and PAGE gel purified and labeled at the 5′-end with γ- 32 P-ATP. Reactions were run as previously described and reactions were stopped with EDTA at 1, 3, 10 and 20 min. 
         [0041]    For strand-transfer assay: R-end oligos PGB18 CCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGCGTAAAATTGACGCATGTGTTTT [SEQ ID NO:49] and its complimentary strand PGB19 TTAAAACACATGCGTCAATTTTACGCATGATTATCITTAACGTACGTCACAATATGATTATCTTTCTAGGG [SEQ ID NO:50] were annealed and labeled at 5′-end with γ- 32 P-ATP on both strands. Reactions were run as previous described and reactions were stopped with EDTA at 1, 3, 10 and 20 min. 
         [0042]    Hyperactive PB/Exc + Int −  mutants screening in yeast The yeast strain BY4727 has been described previously (4). Donor plasmid pGAP-Cherry::PBTnKan (URA+) was used in this study. Random mutagenesis libraries of PB/Exc+Int− transposase gene was carried out by error-prone PCR in the presence of manganese and the resulting mutant pools were introduced into a linearized pGALS vector (TRP+) by homologous recombination in yeast. Transformant colonies were grown for three days, resuspended in water and spotted onto SC plates lacking tryptophan and uracil and with 2% galactose (SC-Trp-Ura+Gal) for transposase induction. The piggyBac excises precisely, allowing us to directly evaluate transposon excision from a gene encoding a RFP in a two-plasmid system. The transposon donor plasmid contained a yeast maximized DsRed RFP gene (9) in which the piggyBac transposon described above was flanked by TTAA at RFP by 80-83.  S. cerevisiae  BY4727 containing this donor plasmid are non-fluorescent. Galactose induction of the transposase from the helper plasmid results in precise excision and colony fluorescence that can be detected using a fluorescence microscope (Leica: M165FC). To screen for hyperactive mutants, we mutagenized the piggyBac/R372A/K375A ORF by amplification with error-prone MnCl 2  PCR with primers from the pGalS promoter and CYCI terminator region and introduced the PCR product by homologous recombination into pRS414 linearized by Xmal-Xhoi digestion into strain BY4727 containing pGAP-Cherry::PBTnKan. More than six thousand individual transformants were picked in 96 well format on SC (synthetic complete) media containing galactose. We scored transposition by looking for increased fluorescence over 1-3 days, and about 35 hyperactive candidates were found. Yeast plasmid DNAs were isolated and the mutant PB/Exc+Int− ORFs were sequenced and retested in BY4727 containing pGAP-Cherry::PBTnKan to exclude the earlier excision events. 
         [0043]    Mammalian excision assay in vivo. For generating stable cell lines containing pEFIa/cyc-3-GFP::PBTnBsd, a To12 mediated transposition was used to integrate the DNA fragment into genomic DNA of HEK293 cells and colonies were picked and analyzed. To quantitatively analyze the relative excision activity of different mutants, a color (GFP) excision assay by FACS sorting was used. A HEK293 stable cell line containing pEFIa/cyc-3-GFP::PBTnBsd, T46 was used and transfected with PB transposase and different mutant plasmids with FuGENE 6 transfection reagent (Roche Applied Science). Four days later, cells were trypsinized and FACS analyzed with BD FACSCalibur (BD Biosciences, 488 nm excitation, 509 nm emission not the best for cycle-3 GFP, should use exc 395 nm, 507 nm) to see the percentage of GFP cell population in the whole cell population. Data were analyzed with FlowJo 8.5.3. GFP positive cells were counted against whole cells. GFP cells from WT PB were used to standardize that from mutant PB/Exc+Int−. To detect fluorescent cells, it is important to pick the best filter set to optimize detection. The primary excitation peak of cycle-3 GFP is at 395 nm. There is a secondary excitation peak at 478 nm. Excitation at either of these wavelengths yields a fluorescent emission peak with a maximum at 507 nm. Note that the quantum yield can vary as much as 5- to 10-fold depending on the wavelength of light that is used to excite the GFP fluorophore. 
         [0044]    Shuffle-PCR: To generate a library of nine mutations locate at different positions of the PB gene, we used Shuffle-PCR to make different combination. The total number of possible combination is 511. To do the Shuffle-PCR, we used the same forward and reverse primers as we generated Exc+Int− random mutation library and 2:1, 1:1 and 1:2 ration of 2 different templates—PB/Exc+Int− and 7PB/Exc+Int−+M194V+D450N with low primers/template ratio. The PCR cycle is as follow: 94° C., 4 min; 100 cycles of 94° C., 10 sec, 65° C., 20 sec, 72° C., 6 sec; 10 cycles of 94° C., 30 sec, 65° C., 30 sec, 72° C., 2.5 min; 72° C., 10 min. The PCR reaction mixture was cleaned up by Qiaquick PCR purification Kit (Qiagen). The whole elution was used as templates to amplified by normal PCR reaction. The product was digested by DpnI to remove any template DNA and then gel purified from 1% agarose gel with Qiaquick gel purification Kit (Qiagen). The same procedures were used as previously to generate combination mutation library in the yeast strain BY4727 with donor plasmid pGAP-Cherry::PBTnKan has been described previously. More than 1500 individual transformants were picked in 96 well format on SC media containing galactose. We scored transposition by looking for increasing fluorescence over 1-3 days, and about 50 hyperactive candidates were selected. Yeast plasmid DNAs were isolated and the mutant combination PB/Exc+Int         ORFs were sequenced and re-tested in BY4727 containing pGAP         Cherry::PBTnKan to exclude the earlier excision events. The ones still showed hyperactive activity in retest were further cloned into pcDNA3.1/myc-HisA mammalian expression vector and examined the relative excision activity in HEK293 stable cell line containing pEFIα/cyc-3-GFP::PBTnBsd, T46. Four days later, cells were trypsinized and FACS analyzed with BD FACSCalibur (BD Biosciences) to see the percentage of GFP cell population in the whole cell population. Data were analyzed with FlowJo 8.5.3. GFP positive cells were counted against whole cells. GFP cells from WT PB were used to standardize that from mutant PB/Exc+Int−. 
         [0045]    Acknowledgement The inventor is an Investigator of the Howard Hughes Medical Institute. This work was supported by Maryland Stem Cell Research Fund. 
       REFERENCES 
       [0000]    
       
         (1) Fraser M J, Ciszczon T, Elick T, Bauser C. Precise excision of TTAA         specific lepidopteran transposons piggyBac (IFP2) and tagalong (TFP3) from the baculovirus genome in cell lines from two species of  Lepidoptera.  Insect Mol Biol. 1996, 5(2):141-51. 
         (2) Woltjen K, Michael I P, Mohseni P, Desai R, Mileikovsky M, Hamalainen R, Cowling R, Wang W, Liu P, Gertsenstein M, Kaji K, Sung H K, Nagy A. piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature. 2009, 458(7239):766-70. 
         (3) Yusa K, Rad R, Takeda J, Bradley A. Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon. Nat Methods. 2009, 6(5):363-9. 
         (4) Mitra R, Fain-Thornton J, Craig N L. piggyBac can bypass DNA synthesis during cut and paste transposition. EMBO J. 2008, 27(7):1097-109. 
         (5) Ziegler K, Bui T, Frisque R J, Grandinetti A, Nerurkar V R. A rapid in vitro polyomavirus DNA replication assay. J Virol Methods. 2004 Dec. 1; 122(1):123-7. 
         (6) Wang W, Lin C, Lu D, Ning Z, Cox T, Melvin D, Wang X, Bradley A, Liu P. Chromosomal transposition of PiggyBac in mouse embryonic stem cells. Proc Natl Acad Sci USA. 2008 Jul. 8; 105(27):9290-5. Epub 2008 Jun. 25. 
         (7) Harper A L, Skinner L M, Sudol M, Katzman M. Use of patient-derived human immunodeficiency virus type 1 integrases to identify a protein residue that affects target site selection. J Virol. 2001 August; 75(16):7756-62. 
         (8) Maertens G N, Hare S, Cherepanov P. The mechanism of retroviral integration from X-ray structures of its key intermediates. Nature. 2010 Nov. 11; 468(7321):326-9. 
         (9) Keppler-Ross S, Noffz C, Dean N. A new purple fluorescent color marker for genetic studies in  Saccharomyces cerevisiae  and  Candida albicans.  Genetics. 2008 May; 179(1):705-10. 
         (10) Yusa K, Zhou L, Li M A, Bradley A, Craig N L. A hyperactive piggyBac transposase for mammalian applications. Proc Natl Acad Sci USA. 2011 Jan. 4. [Epub ahead of print] 
         (11) Chen Y T, Furushima K, Hou P S, Ku A T, Deng J M, Jang C W, Fang H, Adams H P, Kuo M L, Ho E N, Chien C L, Behringer R R. piggyBac transposon         mediated, reversible gene transfer in human embryonic stem cells. Stem Cells Dev. 2010 June; 19(6):763-71. 
         (12) Stadtfeld M, Hochedlinger K. Without a trace? PiggyBac-ing toward pluripotency. Nat Methods. 2009 May; 6(5):329-30. 
       
     
         [0058]    The following claims are thus to be understood to include what is specifically illustrated and described above, what is conceptually equivalent, what can be obviously substituted and also what essentially incorporates the essential idea of the invention. Those skilled in the art will appreciate that various adaptations and modifications of the just-described preferred embodiment can be configured without departing from the scope of the invention. The illustrated embodiment has been set forth only for the purposes of example and that should not be taken as limiting the invention. Therefore, it is to be understood that, within the scope of the appended claims, the invention may be practiced other than as specifically described herein.