transposon - based systems are simple and efficient transfection tools suitable for a variety of gene transfer applications . advantages over viral vectors include tolerance by the immune system , decreased preference for integration into genes , and increased cargo capacity . the sb transposon , originally reconstructed from inactivated sequences found in fish genomes , has proven to be a flexible and effective nonviral genetic tool . recently , piggybac ( pb ) , isolated from the moth trichoplusia ni , has emerged as a highly efficient gene delivery vector for numerous in vitro and in vivo applications . the typical 2-plasmid system includes both a  helper  plasmid encoding the transposase enzyme and a  donor  plasmid containing the intended integration sequence , such as a gene of interest , flanked by the transposon terminal repeat elements ( tres ) . upon entrance to the cell , the pb transposase recognizes the tres and excises the transposon from the donor plasmid . pb subsequently integrates the transposon permanently into the genome at a ttaa tetranucleotide sequence . by introducing pb tres into bacterial artificial chromosomes , it is possible to facilitate integration of large pb inserts greater than 100  kb . pb 's unprecedented cargo capacity can allow for the integration of large genes , regulatory elements , and multiple reading frames . pb has been used to generate induced pluripotent stem cells due to its ability to precisely excise its transposon from the genome without leaving a dna  footprint  . this feature allows for the removal of transgenes following complete reprogramming without leaving residual sequences . in addition , pb has been shown to be amenable to dna - binding domain fusions , allowing for targeted transposition to chromosomal locations . we have recently designed improved self - inactivating vectors that contain all transpositional machinery on a single helper - independent plasmid , termed pmgenie-3 . pmgenie-3 encodes a specialized pb transposase containing a tre within an engineered intron . upon transfection , pb recognizes the tres and subsequently excises the transposon from the plasmid , thereby truncating the transposase gene . consequentially , potentially negative genotoxic effects that may develop by the persistence of an active pb gene are eliminated . over the past 20 years , rna interference , using silencing rna molecules or short hairpin rna molecules ( shrna ) has developed into a reliable method to target and reduce the expression of specific genes . eukaryotic expression vectors have been shown to effectively drive expression of shrna in mammalian cells , however the relative ability of transposase - based delivery systems , in particular the pb transposase systems , have not yet been thoroughly assessed . telomerase is an enzymatic complex that is required for prolonged cancer cell proliferation and is present in ~90% of all human tumors . the activity of the telomerase complex is entirely dependent on expression of the catalytic component of telomerase , tert . therefore , in the present study , we sought to assess the potential of a novel pb - based vector to express shrna to specifically target tert , in human telomerase - positive cell lines . we have previously shown that the pmgenie-3 transposase - based nonviral delivery system can efficiently express genes in mammalian cells both in vitro and in vivo in transgenic mice . to assess the capacity of this system to knock down target genes using rna interference , we developed two vectors , pmgenie-3-trt1 and pmgenie-3-trt2 , to express shrna - targeting tert ( figure 1 ) . we initially compared transfection efficiency of our actively integrating pmgenie-3 to a passive , transposase - deficient pgipz shrnamir vector expressing the same tert - targeting shrna . we found the transient transfection levels ( 72 hours posttransfection ) to be enhanced ~4-fold using the pmgenie-3 vector ( figure 2a ) . furthermore , following selection with antibiotic , we observed ~5-fold enhancement of the frequency of stably transfected cells receiving pmgenie-3 based on green fluorescent protein expression ( figure 2b ) . to assess the ability of pmgenie-3-trt1 and pmgenie-3-trt2 to knock down tert , we stably transfected both hek293 cells and mcf-7 cells with these constructs . we also generated control cell lines transfected with a pmgenie-3 construct without shrna ( pmgenie-3-trt0 ) . at ~2 months postselection , we observed significant reduction in relative levels of tert mrna for both constructs in hek293 and mcf-7 ( p  0.001 ) cells ( figure 3 ) . to determine whether this level of knockdown was sufficient to have an effect on the telomerase enzymatic complex , we also measured telomerase activity in these cells . we observed significantly ( p  0.02 ) reduced levels of telomerase activity with both constructs in hek293 and mcf-7 cells , in agreement with the reduced levels of tert mrna ( figure 4 ) . these observations confirm that the nonviral pmgenie-3-tert delivery system is capable of knocking down target genes via stable expression of shrna . we next sought to determine if the reduced level of telomerase had a physiological effect in these cell lines . specifically , we measured telomere length by southern analysis of terminal restriction fragments at ~2 months posttransfection ( figure 5 ) . for both hek293 and mcf-7 cells transfected with either pmgenie-3-tert1 or pmgenie-3-tert2 , we observed a significant reduction in telomere length . for the hek293 cell line , which has relatively short initial telomere length , we noted that telomeres were approaching critical length required to maintain cell viability ( ~3 kb ) . an analysis of proliferation rate in cells transfected with the tert targeting vectors revealed a marked ( > 2-fold ) reduction in growth by ~2 months posttransfection in cells ( supplementary figure s1 ) . transposition - based gene delivery systems are appealing tools which bypass the use of viral vectors for transgene delivery . transposase / transposon vectors have been shown to have less of an affinity for transcriptional start sites , transcribed regions , and protooncogenes than lenti- or retroviruses and their ease of production at low expense make them ideal candidates to evaluate human applications of gene therapy . we have modified the pb transposon system to consist of a single self - inactivating vector for highly efficient generation of stable transformants in mammalian cells in vitro as well as in transgenic animals . due to our previous success at utilizing helper - independent pb constructs to stably transfect cell lines and animals with marker genes such as enhanced green fluorescent protein , we speculated that transgenes that affect the biological function of cells would have the same successful delivery and integration . in the present study , we have assessed the ability of our single plasmid pb vector , pmgenie-3 , to drive expression of shrna and affect the target gene expression in human cell lines . the practical utility of silent interfering rna to suppress cancer has been intensively studied over the past 10 years , however , truly effective rnai - based therapies have yet to be developed . telomerase , specifically tert , has been studied as a target for rnai technology . indeed , a tert - targeting rnai therapeutic could potentially complement other antisense targeting methods , such as using stable antisense oligonucleotides that target the telomerase rna component to dramatically reduce telomerase levels and induce cancer cell senescence or apoptosis . we show in both hek293 and mcf-7 cells that pmgenie-3 vectors designed to target the tert gene transcript , effectively knockdown tert expression and reduce telomerase activity levels . furthermore , this knockdown of telomerase was sufficient to effect the physiological function of telomerase , since in both cell lines , it was accompanied by significant telomere shortening ( p < 0.001 ) . these results suggest that the pb transposase - based delivery system is an effective technique for expressing shrna and knocking down target genes in human cell lines , including tumor cell lines . the ability of the pmgenie-3 vector to efficiently knockdown tert and reduce telomerase levels , in conjunction with the high level of long - term expression , indicative of genomic integration associated with this vector , suggest that pb transposase - based delivery systems may provide a new method to suppress tumor progression . we are presently undertaking experiments to assess the potential and effect of using pmgenie-3 to knockdown tert in tumor cells in an in vivo murine model . the results from this study have further implications for the utility of pmgenie-3 vector beyond cancer therapy , for example , to study cell aging . the use of pmgenie-3 vector to target tert and/or other genes in rodents and other laboratory animals could be a powerful way to assess the combinatorial affects of altered expression of these genes on cell aging in vitro and in vivo . the ability to express multiple genes or shrnas , from a single construct is another advantage of the pb transposase - based delivery system , as we ( data to be published elsewhere ) and others have recently demonstrated by our use of pmgenie-3 vector to efficiently derive induced pluripotent stem cellsipscs by expression of the yamanaka factors ( oct4 , sox2 , klf4 , and c - myc ) . in summary , we have shown that the pb transposase - based delivery system is effective at shrna - driven targeted gene knockdown in mammalian ( human ) cells . the ability to affect both telomerase and telomeres in a tumor cell line implies a potential utility for the pb vector in cancer therapy . pmgenie-3 is compatible with gateway recombineering cloning ( life technologies ) for simple addition of target genes into the transposon portion of the construct . the two shrna sequences were modeled after human mir-30 and were designed to target distinct regions of the tert mrna , specifically , 5-agcaagttgcaaagcat-3 ( tert1 shrna ) and 5-cgagctgctcaggtctttctt-3 ( tert2 shrna ) . the shrnas were located 3 to a bicistronic turbogfp and puromycin - coding region driven by the cytomegalovirus promoter . each cassette including shrna was excised from lentiviral pgipz shrnamir plasmids ( catalog # s rhs4430 - 101132965 and rhs4430 - 99161517 , thermo scientific , rockford , il ) via xbai and bglii ( new england biolabs , ipswich , ma ) and cloned into an empty pentr1a ( life technologies ) vector in preparation for gateway recombineering into pmgenie-3 . the hek293 and mcf-7 cell lines were grown in standard culture conditions ( 37 c , 5% co2 ) in dulbecco 's modified eagle medium for hek293 and dulbecco 's modified eagle medium f12 for mcf-7 , both in high glucose , complete media with 10% fetal bovine serum . cells were transfected using fugene 6 , ( roche , indianapolis , in ) per manufacturers ' instructions . cells were selected with puromycin ( hek293 cells with 500  ng / ml , mcf-7 cells with 300 ng / ml ) . relative levels of tert mrna were assessed by real - time polymerase chain reaction ( pcr ) using a bio - rad iq cycler . total rna was isolated from cells using trizol reagent ( invitrogen , grand island , ny ) . rna cleanup and dnase treatment was done using the rneasy mini kit ( qiagen , germantown , md ) . one microgram of total rna was reverse transcribed using the iscript advanced cdna synthesis kit for real - time quantitative pcr ( bio - rad ) . the real - time pcr assays were performed in 25 l reactions using iq sybr green supermix ( bio - rad , hercules , ca ) , 50  ng cdna , and 0.4 mol / l of forward and reverse primers . analysis was done using a myiq single - color real - time pcr detection system ( bio - rad ) . thermal cycling conditions were the following : 2 minutes at 50 c and 10 minutes at 95 c for initial denaturation , followed by 40 cycles at 95 c for 15 seconds and 54 c for 1 minute , and a dissociation step at 95 c for 15 seconds , 54 c for 30 seconds and 95 c for 15 seconds . gene relative quantification of tert was calculated as described by livak and schmittgen and normalized with the housekeeping gene hypoxanthine phosphoribosyltransferase . primers used for amplification were as follows : tert f , 5-cgtcgagctgctcaggtctt-3 and r , 5-agtgctgtctgattccaatgctt-3 : hhprt f , 5-tgacactggcaaaacaatgca-3 and r , 5-ggtccttttcaccagcaagct-3. detection of telomerase . telomerase activity was measured using the trap assay ( trapeze detection kit ; millipore , billerica , ma ) per manufacturers ' protocol . briefly , 1    10 cells per sample were lysed on ice in chaps buffer ( 30 minutes ) followed by centrifugation ( 12,000 g , 30 minutes ) . the supernatant was then used directly in trap reactions ( pcr cycling conditions : 27 cycles of 94 c/30 seconds , 60 c/30 seconds , 72 c/60 seconds ) . telomere length analysis . telomere length was assessed by southern analysis of terminal restriction fragment length as previously described .
nonviral gene delivery systems are rapidly becoming a desirable and applicable method to overexpress genes in various types of cells . 
 we have recently developed a piggybac transposase - based , helper - independent and self - inactivating delivery system ( pmgenie-3 ) capable of high - efficiency transfection of mammalian cells including human cells . in the following study , 
 we have assessed the potential of this delivery system to drive the expression of short hairpin rnas to knock down genes in human cells . 
 two independent pmgenie-3 vectors were developed to specifically target knockdown of an endogenous gene , telomerase reverse transcriptase ( tert ) , in telomerase - positive human immortalized cell lines . as compared with a transposase - deficient vector , pmgenie-3 showed significantly improved short - term transfection efficiency ( ~4-fold enhancement , 48 hours posttransfection ) and long - term integration efficiency ( ~5-fold enhancement ) following antibiotic selection . 
 we detected a significant reduction of both tert expression and telomerase activity in both hek293 and mcf-7 breast carcinoma cells transfected with two pmgenie-3 construct targeting distinct regions of tert . 
 importantly , this knockdown of expression was sufficient to abrogate telomerase function since telomeres were significantly shortened ( 34 kb , p < 0.001 ) in both tert - targeted cell lines following antibiotic selection of stable integrants . 
 together , these data show the capacity of the piggybac nonviral delivery system to stably knockdown gene expression in mammalian cells and indicate the potential to develop novel tumor - targeting therapies .