in our previous reports we have described the safety mechanisms of pmgenie-3 we engineered into the constructs to prevent the piggybac - driving promoter ( fig . 1 ) from maintaining its activity after excision of the transposon from the plasmid . specifically , this cag promoter located in the transposon should not be able to contribute to aberrant gene activation , as the remaining fragment of the pb transposase gene and the chimeric intron contain two stop codons ( uaa ) engineered into the transposon construct . however , concerns have been raised that the cag promoter once integrated into the host genome may prove problematic as it could influence expression of host genes located nearby . to address these concerns , we have performed an experiment where we designed a transposon to which we added another outward directed cag promoter - chimeric intron construct to the 5-tre end of the pmhygenie-3 transposon . hereafter this construct will be referred to as double reporter construct ( drc ) ( fig . we placed the egfp or dsred express2 genes respectively on either side of the transposon . the 5- and 3-ttaa sequences abate a kozak consensus sequence , which will initiate transcription from the chromophore genes . the egfp gene is situated immediately downstream of the cag promoter , a fragment containing the start of the hyperactive pb gene , the chimeric intron which contains the 5-tre and the ttaa pb recognition sequence . integration of the transposon into the ttaa target sequence within the host genome introduced 8 in frame stop codons within the region spanning from the end of the truncated pb gene to the start of the egfp gene . the dsred express2 gene is situated at the opposite side of the transposon and is preceded by a similar arrangement of a cag promoter , tre and chimeric intron . however , this time the chimeric intron contains the 3-tre of pb terminating with the ttaa sequence of transpositional insertion . here there are 10 in frame stop codons within the region spanning from the end of the truncated pb gene to the beginning of the dsred express2 gene . as the transposon can be inserted into dna in one of three possible codon frames , the number of stop codons between the truncated end of the pb gene and the chromophores will vary , however there will always be at least two or more , depending on the site of insertion . figure  2 . cag promoter influence on neighboring genes . to assess the potential of the piggybac - driving cag promoter to influence expression of host genes located nearby after it 's integration into the host genome we used several different vectors . ( c ) the drc plasmid contains a transposon to which we added another outward directed cag promoter - chimeric intron construct to the 5-tre end of the pmhygenie-3 transposon and egfp or dsred express2 genes on either side of the transposon . ( d ) to assess the influence of tres within this construct , we removed the cag promoters from the drc plasmid ( drc nocag ) . ( e ) finally , we also removed the tres from the drc nocag construct to generate the drc nocag notre plasmid to evaluate promoterless and tre - free expression . pmhygenie-3 with either an egfp ( pmhygenie-3 egfp ) or dsred express2 ( pmhygenie-3 dsred ) as a transgene served as controls . chromophore expression was evaluated at four different time points ( 24 , 48 , 72 or 144 h ) . the expression of the positive controls increased over time and was at a maximum at 144 h ( fig .  3 ) . to accommodate the strong egfp signal at 144 h we noticed a marked reduction in chromophore expression for the drc plasmid when compared with the positive control , with dsred expression slightly higher than egfp . to assess the influence of the cag promoter on this residual expression we removed this promoter from the drc plasmid ( dcr nocag ) ( fig . transfection with drc nocag did not result in dsred expression after 144 h , while egfp remained at levels of drc plasmid transfections . as a final modification we also removed the tres from the drc nocag construct to generate the drc nocag notre plasmid ( fig . it is not evident to us why we observed minor dsred expression for the drc construct and egfp expression from the drc nocag plasmid , however it is possible that it originates from undefined low level transcriptional activity from the plasmid backbone . it is also possible that the observed egfp expression of the cag promoterless plasmid was due to outward - directed promoter activity of the 5-tre . inward directed promoter activity has previously been reported for both the 5- and the 3-tres of pb . however , outward directed promoter activity for other transposon inverted repeat elements have been described . we would like to note that this observation of a potential outward - directed promoter activity from pb tres was not the emphasis of our experimental setup . larger scale , more focused investigations will be necessary to elucidate this observation . in summary , our results clearly show that there is a drastic reduction in chromophore expression as a consequence of the vector design , more specifically the stop codons situated between the cag promoter and the beginning of chromophore genes . 0.5  10 hek293 t cells were transfected with 400 ng of pmhygenie-3 containing either egfp ( g3 egfp ) or dsred express2 ( g3 dsred ) in its transgene cassette , drc , drc nocag or drc nocag notre . images of egfp expression followed by dsred expression were taken for each construct at 24 , 48 , 72 and 144 h. * exposure time for egfp of the pmhygenie-3 egfp at 144 h was reduced to 10% due to overexposure at original exposure time . finally , an often - overlooked but crucial parameter that influences the success and the safety of transposition for transgenesis , transfection and gene therapy is the integrity and purity of plasmids . this is of particular concern for transposase - mediated delivery of large dna fragments as it has been shown to be possible with pb ( up to 100 kb ) . for optimal plasmid manipulation procedures the best commercially available gravity - flow purification kits must be used to obtain only covalently closed circular ( supercoiled - ccc configuration ) plasmids . the desired effects after gentle lysis of the host bacterial cells are the elimination of intracellular macromolecules and the enrichment and purification of plasmid dna . even with these kits , care must be taken to assess each batch by gel electrophoresis for both purity in terms of contaminating bacterial dna and ccc - configuration . once the correct configuration plasmids as proven by electrophoresis are purified , they can then be confirmed for functionality by testing them in easy to transfect cell lines such as hek293 . additionally , we resuspend plasmids used for transgenesis research in sigma water for embryo transfer ( sigma - aldrich ) and store 10 l aliquots in a 20c defrost free freezer . in our experience , all of these parameters , if not taken care of deliberately can significantly bias the efficiency and safety of transposition . transgenesis by microinjection is a technically challenging procedure , which needs specialist for its implementation . in addition to the technical difficulties of the process , care must be taken not to shear the transgene dna during the microinjection process . this becomes of even greater importance when large circular plasmids are used during active transgenesis . pmgenie plasmids , in addition to the transposon with the transgene in between its terminal repeat elements ( tres ) , the genie plasmid dna also contains a transposase enzyme with all its regulatory elements . the transposase has to be first transcribed once the plasmid dna reaches the nucleus , then processed into mrna and finally translated into protein . the nuclear localization signal of the pb transposase must facilitate its translocation back into the nucleus . once in the nucleus , the transposase locates the tres of the transposon and initiates formation of the synaptic - complex . after excision of the transposon from the plasmid dna via the synaptic - complex , the resulting circular structure with two transposase molecules holding it together at the tres is referred to as the transposome . it is these transposase molecules in the transposome that select the target ttaa tetranucleotide sequence for transposition and finally insert the transposon into the host s genome . as can be inferred from the above description there are several distinct regions of the plasmid dna that must retain their structural integrity . nicked or linearized plasmids may impede the successful completion of the transposition process and must therefore be avoided at all costs . to accommodate this requirement for only using ccc - configuration plasmid , we have modified the traditional pni procedure and created the new te - pni method . during te - pni a 2 m internal diameter blunt end microinjection pipette this pipette has to be well lubricated with 15% polyvinyl pyrrolidone ( pvp ) in order to render it non - sticky . the diameter size of the pipette allows the unimpeded transfer of precise amounts of plasmid dna into the pronucleus without shearing it . however , the blunt ended pipette creates a difficulty for the penetration of the one - cell zygotes protective layers ( zona pellucida and plasma membrane ) , which can be overcome by the use of a piezo actuator ( prime tech , japan ) . the piezo actuators ability to act as a small pneumatic drill facilitates the traversing of the protective layers of the embryo without manual force being applied to the zygote . this prevents the constriction of the embryo and allows smooth passage through the membranes of the one cell , two pronuclear zygote . additionally , it confers control in regards to the area of the male pronucleus to be penetrated by the injection . once the plasmid has been delivered to the pronucleus , care must be exercised not to extract a nucleolus while retracting the pipette . therefore , the less sticky the microinjection pipette by pvp lubrication , the better this outcome . the flow regulation from the pipettes during the screw - controlled injection process is mediated by the small amount of mercury back - loaded into the pipettes . the mercury is subsequently positioned near the thin tip of the micropipette with an injection syringe . an air - filled space separates the mercury from the mineral oil filled plastic tube leading to the screw - controlled syringe . clockwise turns move the mineral oil forward , applying pressure to the air and in turn to the mercury . because of the very high surface tension of mercury , large displacements of the mineral oil volume brought about by the turn of the screw - controlled syringe , result in very small displacements of the volume in front of the mercury therefore allowing precise control of the injection process . the plasmid solution is drawn into the pipette by turning the screw - controlled syringe in a counter - clockwise orientation . this dna solution is then injected into the pronucleus , by turning the screw - controlled syringe in a clockwise orientation . precise quantitation of the amount of plasmid injected can be calculated with the following equation :  r length . an injection length of 65 m equals 2.04  10 l in volume . at a concentration of 10 ng/l for plasmid solution ( pmhygenie-3-rna - out , 14,542 bp ) , the total amount of dna injected is 2.04 femtograms ( fg ) or about 130 copies of plasmid per pronucleus . obviously , not all the plasmid molecules deposited into the pronucleus integrate into the genome , as we consistently find 1 , 2 , 3 or 4 monomeric transgenes integrated into the genome of the host mouse by southern blots . similar infrequent transposition observations for the sleeping beauty mechanism were made in a recent review , where all the necessary transposition components were present in excess . in fact , during hydrodynamic delivery via tail vein injection , only about one in 10,000 sleeping beauty transposons delivered to the liver actually transpose into chromatin . in summary , under ideal conditions with ccc - configuration plasmids , the efficiency of transposition relies in several parameters working optimally for successful transposon integration . the pb transposase , unlike sleeping beauty when expressed at high levels is not subject to overexpression inhibition . we have sought to overexpress the pb transposase in hek293 cells by co - transfecting a helper plasmid for the transposase together with a mouse codon bias based pmgenie-3 plasmid containing a hygromycin resistance gene in its transposon . we intended to assess if a larger amount of transposase will indeed increase transfection efficiency . after three weeks of hygromycin selection we assessed the number of stably transfected hek293 colonies and noted that , there were 30.6% more colonies formed from the helper plasmid boosted pmgenie-3 plasmid transfection , than the one where only pmgenie-3 was used . due to these findings we proceeded to construct the pmhygenie-4 plasmid , which contains two independent self - inactivating hyperactive pb transposase genes in its structure , together with an antibiotic gene free backbone for transgenesis experiments ( fig . the antibiotic free backbone construct was achieved by replacing the ampicillin resistance gene and puc ori in the backbone of the original construct for pmhygenie-4 , as previously described for pmhygenie-3-rna - out - msc . this plasmid construct contains two independent self - inactivating hyperactive pb transposase genes in its structure , together with an antibiotic gene free backbone for transgenesis experiments . the transposon cassette for genomic integration contains the transgene cassette tgc and is delimited by the 3- and 5-tres . ci represents the chimeric intron , cag the cag promoter , and rna out the antibiotic gene free selection cassette . a comparison of transfection efficiencies of pmhygenie-4 and pmhygenie-3 in hek293 cells transfection is depicted in figure  5 . both versions of pmhygenie-4 ( either containing an antibiotic gene in the backbone or rna - out ) were considerably more effective in transfecting hek293 cells than pmhygenie-3 . lt is of note that in these preliminary experiments we used the same plasmid concentrations for each transfection . as the pmhygenie-4-rna - out - egfp construct is larger than pmhygenie-3-egfp by 3,185 bp , a lower number of pmhygenie-4-rna - out - egfp molecules was transfected and we therefore expect the efficiency of the pmhygenie-4 plasmids to be even higher when equimolar concentrations of plasmids are used . 0.5  10 hek293 t cells were transfected with 400 ng of pmhygenie-3 egfp , pmhygenie-4 egfp , pmhygenie-4-rna - out egfp or pmgenie-3 -pb . transposition activity was measured by counting methylene blue stained , hygromycin - resistant colonies after a three - week selection period . we previously demonstrated that te - pni is very effective for the production of transgenic mice . the efficiencies for transgenic mice production with this method , in terms of embryos microinjected , were improved from an average of 4.6% for pni to an average of 25.6% for te - pni . in preliminary experiments we injected 13 embryos by te - pni with a plasmid solution of 3 ng / ul . all 13 embryos developed to the 2-cell stage and where then transferred to a single surrogate mother . eleven pups were born ( 85% of embryos injected ) , five of which were transgenic ( representing 38.5% of embryos injected or 45.5% of animals born ) . these transgenic rates represent an increase of 50.3% over the rate of pmhygenie-3 te - pni demonstrating the potential of pmhygenie-4 vectors . of note , in contrast to previous te - pni experiments , these results were achieved at a significantly lower plasmids concentration ( 10 ng / ul of pmhygenie-3 vs. 3 ng / ul of pmhygenie-4 ) injected into the pronucleus . in conclusion , our results lead us to suggest that our pmhygenie plasmids are effective , efficient and safe to use in a wide array of application such as cell transfections , gene therapy and transgenesis .
transposons are mobile genetic elements that are capable of self - directed excision and subsequent reintegration within the host genome . 
 transposase such as piggybac , sleeping beauty and tol2 catalyze these reactions and have shown potential as tools for the stable integration of transgenes when used in the binary plasmid mode . 
 recent modifications to the transposase and/or the terminal repeats of the transposon have increased their integration efficiency and/or specificity . 
 we recently described the development of a piggybac transposase system , the helper independent , single construct self - inactivating plasmid called genie . 
 here we describe the structure , safety and function of these transpositional vectors and their use in animal transgenesis and cell transfection .