BACKGROUND
the development of rna interference  as a tool for reverse genetic studies in mammalian systems has rapidly matured.
after the seminal observation that  <dig> nucleotide, chemically-synthesized rna duplexes  are capable of targeted gene silencing in mammalian cells  <cit> , rnai has quickly become a standard technique for functional genetic analysis.
a significant advancement of this technique was the development of short-hairpin rna  expression technology  <cit> .
this strategy exploits the defined transcriptional start and termination signals of rna polymerase iii  promoters to produce a short, inverted transcript.
these stem-loop rna transcripts are then processed within the cell into functional sirnas and thereby provide a means for the stable suppression of target genes.
to this end, multiple groups have reported success in long-term silencing of target genes in engineered cell lines and mice  <cit> .
nevertheless, several limitations to the current approach remain.
the primary limitation to vector-based shrna is posed by constitutive hairpin expression.
if the shrna is directed against a gene essential to cell growth and survival, the probability of obtaining a stable line is low, and in those cell lines that survive, other factors may compensate for shrna-induced gene knockdown.
in both cases, the relevant phenotype may be obscured  <cit> .
several groups have employed inducible shrna systems to address these limitations.
one class of inducible systems co-express the tetracycline repressor  with a modified pol iii promoter containing one or more tetr operons flanking the tata-box such that transcription is blocked when the tetr is bound to the promoter.
the expression of shrna within this scenario occurs in the presence of tetracycline or related analogs  <cit> .
wiznerowicz and trono introduced a variation to the above design by fusing the tetr to krab, a transcriptional repression domain from kox <dig>  <cit> .
this fusion silences any promoter within  <dig> kb of the tetr operon.
therefore, shrna transcription from the pol iii promoter containing a tetr operon may also be monitored by the ability of the tetr-krab fusion to repress the expression of a fluorescent reporter gene driven by an additional rna polymerase ii  promoter within the same vector  <cit> .
in a different approach, several labs have developed cre recombinase-based systems in which the shrna promoter is modified to contain a 'floxed' cassette or spacer such that hairpin expression is activated only upon cre-mediated excision  <cit> .
in addition to standard or 'first generation' shrna design, it has recently been demonstrated that a primary micro-rna  scaffold can efficiently deliver a desired shrna  <cit> .
these 'second-generation' mirna mimetics can be expressed from pol ii promoters, allowing the use of established conditional expression vectors  <cit> .
each of the above classes of regulated shrna vectors has specific advantages – from the temporal and reversible control of the tetracycline-regulated systems to tissue-specific silencing of cre-lox vectors in mice.
however, a considerable limitation shared by the reported inducible shrna systems is that each system relies on a two-plasmid approach in which the shrna expression cassette is separated from the regulatory module.
to address this issue, we and others have previously reported the successful integration of a shrna/mirna expression cassette with a tetracycline dependent repressor or transactivator on a single vector  <cit> .
here we describe the development of phush , as a gateway®-compatible, conditional shrna system in which tetr expression and operon usage have been optimized on a single vector.
importantly, we compare a number of different shrna and mirna configurations in transient transfection assays and show that the phush backbone is effective at providing regulated expression from both pol ii and pol iii promoters, thereby enabling regulated shrna, mirna and protein expression.
the versatility of phush is demonstrated here using a series of in vitro and in vivo reverse genetic experiments that were designed to validate the therapeutic relevance of both novel and characterized oncology targets.
RESULTS
analysis of shrna design
as part of our effort to engineer a conditional shrna expression system, we compared hairpin stem and loop parameters to maximize knockdown efficiency.
to generate a robust, quantifiable assay, we created a transcript fusion between luciferase and melk, a ser/thr kinase that we have previously described  <cit> .
the entire melk cdna was cloned downstream of the pgl3-luc stop codon, and the resulting pgl3-melk construct was used as a reporter for shrna efficacy in transient co-transfection experiments.
we first compared several published strategies for shrna design based on two target sequences: sh <dig>  and shb .
for both target sequences, the following series of shrnas were created for comparison: n <dig> – a simple stem-loop design  <cit> , f – a 'frayed' shrna design employing artificial asymmetry  <cit> , and mi <dig> – a shrna utilizing the mir <dig> loop reported to increase cytoplasmic expression  <cit> .
the sequence of each hairpin is presented in additional file  <dig>  to compare the efficacy of each design, the resulting shrna expression constructs were co-transfected with the pgl3-melk reporter at several molar ratios and normalized to control hairpin h1-lacz.
as shown in figure 1a at a 10: <dig> ratio of shrna to pgl3-melk, luciferase expression was decreased for both shrna sequences, regardless of the hairpin design.
the standard n <dig> design and mi23-loop shrnas were highly effective, yielding 65% , 75% , 90% , and 95%  knockdown respectively.
while we noticed a modest increase in knockdown efficiency by the addition of the mir <dig> loop at a 10: <dig> ratio of shrna to pgl3-melk, there was no significant increase in efficacy with the mir <dig> loop at lower molar ratios of shrna to pgl3-melk  as had been previously reported  <cit> .
for both sh <dig> and shb, the contribution of artificial thermodynamic symmetry  by the introduction of 'frayed' ends at the 3' end of the sense strand was detrimental to hairpin efficacy, suggesting that destabilization of the shrna stem-loop in this context did not lead to increased risc loading of the antisense strand.
as there did not appear to be a reproducible improvement with modified shrnas , we have utilized a n <dig> shrna design for the remainder of this study.
over the course of our study, several labs reported robust knockdown with mirna-based vectors  <cit> .
using a luciferase transcript-fusion construct with murine p <dig> sequences cloned downstream of the pgl3-luc stop codon, we compared the 'first generation' h1-shrna  to a 'second-generation' cmv-mirna- <dig> vector  <cit> .
unlike the n <dig> shrna design, the mirna mimetic can be expressed as standard pol ii transcripts, which are then processed by the nuclear drosha, and cytoplasmic dicer endonucleases to form a 21– <dig> nt active sirna  <cit> .
to enable this comparison, we generated a vector in which a modified cmv promoter  <cit>  was used to express emerald gfp  and the mirna mimetic on the same transcript .
to compare the efficacy of a shrna versus mirna design, two effective mirnas directed against p <dig> were converted to h1-shrnas by identifying the  <dig> nt active sirna within the mirna scaffold and then converting these sequences into conventional shrna design according to the scheme presented in figure 1b.
the sequence and structure of these hairpins is presented in additional file  <dig>  although the performance of the cmv/to-mir and h1-shrna hairpins using the p53- <dig> sequence were similar, we observed increased knockdown by cmv/to-mir relative to the corresponding h1-shrna sequence for the p53- <dig> sequence .
at the lowest ratios of mir or shrna to reporter , the cmvto-mir resulted in ~65–70% knockdown of the luciferase reporter as compared to only a ~30–50% knockdown with the h1-shrna .
removing the emgfp leader from cmv-mir-p53- <dig> destroyed the knockdown efficiency of the original construct , confirming the requirement of a minimal "spacer" sequence between the transcriptional start site and the mirna sequence  <cit> .
to summarize, we have directly compared "first-generation" pol iii-shrna vectors to "second-generation" pol ii vectors using the same dicer-product sirnas.
we have found that both vector formats are functional.
however, based on our observed increase in knockdown efficiency with cmv/to-mir for the p53- <dig> sequence as compared to the corresponding h1-shrna version, there may be a slight advantage with the mirna-like systems for certain sequences under suboptimal expression conditions.
phush vector design and optimization
phush was constructed as described in the methods using the pires-puro <dig> as the backbone vector.
briefly, the human-β-actin promoter driving the tetr was subcloned into pires-puro <dig> to replace the original cmv promoter.
a modified h <dig> promoter containing a single tetr operon with a melk-targeting shrna cassette was inserted upstream to the human-β-actin promoter.
using this plasmid as the base vector, we generated two additional versions for analysis: phush-ivs in which a synthetic intron sequence between the tetr orf and the ires was removed, and phush-ivstetropt, in which the original tetr sequence within phush-ivs was replaced by a tetr expression cassette that was codon-optimized for expression in mammalian cells .
to assess the effect of these modifications on regulated gene silencing, these constructs were transfected into hek <dig> cells that were stably expressing an egfp-melk fusion protein.
after selection with puromycin, cells were cultured for five days in the presence or absence of doxycycline , and the level of egfp-melk fluorescence was scored by facs analysis.
the effectiveness of each construct to repress shrna expression was assessed by comparing the level of egfp-melk in the absence of dox to cells containing a phush construct without a hairpin .
as shown in figure 2b, all three vectors generated the same level of knockdown in the presence of dox .
however, we found that in the absence of dox , only the phush-ivstetropt pool exhibited full repression of shrna expression .
in contrast, partial repression  was obtained with phush-ivs while cells containing the base phush vector displayed a complete lack of shrna repression, as demonstrated by similar levels of egfp-melk in the presence or absence of dox .
because of the reported strong splicing donor in the 5' htlv utr  <cit> , the removal of the ivs most likely prevented a splicing event between the utr and the splice acceptor within ivs resulting in the excision of the tetr.
the above data suggests optimal dox-dependent regulation of shrna expression requires maximal tetr protein levels.
to test this hypothesis, we compared tetr expression between the original tetr construct  and the codon-optimized variant utilized for phush-ivstetropt.
as shown in figure 2c, there is a two-to-three fold increase in tetr protein levels with the codon-optimized tetr  as compared to the original construct .
therefore, we conclude that both tetr codon optimization and ivs removal are required for a regulated gene silencing system.
because of the observed sensitivity to modest changes in tetr levels, we next focused on further modifying the h <dig> promoter to ensure complete shrna repression under limiting tetr levels.
several groups have shown that increased regulation can be achieved by inserting multiple tetr operons throughout the pol iii promoter  <cit> .
to determine whether we could observe a similar enhancement of regulation, we modified the original pshuttle-h1-shrna plasmid containing a single teto <dig>  with an additional teto <dig> operon upstream of the tata box, forming a 2×-teto2-h <dig> promoter.
using pgl3-melk as a reporter, the 1×- and 2×-teto2-h <dig> promoters were co-transfected with increasing amounts of a cmv-tetropt expression plasmid in the absence of dox.
for both shrna promoter variants, maximal repression was achieved when the tetr expression construct was supplied in a 10: <dig> molar excess over the h1-shrna  while >80% knockdown was achieved in the absence of tetr .
when the tetr expression plasmid was limited to a 2: <dig> molar ratio relative to the h1-shrna plasmid, nearly maximal shrna expression was observed with the 1×-teto <dig> construct.
in contrast, shrna repression was maintained with the 2×-teto <dig> construct .
these results suggest that the use of two tetr operons enhance the ability to regulate shrna expression when tetr expression is limiting.
to explore the robustness of this system as a stably-integrated construct, we then compared the 1×-teto <dig> and 2×-teto <dig> versions of the phush vector to enable regulated silencing of melk within mouse embryonic stem  cells.
murine es cells were electroporated with phush-ivstetropt or phush-ivstetropt <dig> × teto <dig>  containing a melk-directed shrna and then selected in puromycin.
multiple clones for both versions of phush were identified exhibiting robust, regulated melk knockdown  after  <dig> h culture in the presence of dox .
to determine whether regulated shrna expression could be maintained upon differentiation, select es cell clones  were allowed to differentiate into embryoid bodies  by standard methods.
eb formation was induced and cultured in the presence or absence of  <dig> ug/ml dox for  <dig> days, and endogenous melk rna levels were quantified by qrt-pcr.
as shown in figure 3b, near maximal silencing of melk expression was observed with ebs derived from phush-ivstetropt  without dox, indicating no functional repression of shrna expression.
in contrast, ebs derived from phush-ivstetropt2×-teto <dig>  maintained a similar level of dox-dependent regulation as had been observed with the parental, non-differentiated es cells .
consistent with these results, we observed a 3-fold decrease in tetr expression upon differentiation into ebs .
although the overall level of melk knockdown is reduced within the differentiated eb derived from the 2×-teto <dig> promoter relative to the 1×-teto <dig> version , this difference is likely due to clonal variations during es cell selection rather than an inherent difference in transcriptional strength of the modified h <dig> promoter.
this is supported by the fact that the knockdown efficiency between the 1× and 2× teto <dig> constructs is the same when expressed in the absence of tetr .
in summary, the presence of multiple tetr operons within the h <dig> promoter does not significantly effect maximal shrna expression while promoting dox regulated gene silencing even under limiting tetr expression levels.
phush retroviral vectors
to enhance the stable delivery into cultured cells, the phush-ivstetropt2×-teto <dig> described above was subcloned into the pqcxip retroviral backbone to form phush-retro.
the resulting viral phush plasmid was converted to a gateway®-compatible vector to enable efficient transfer of shrna cassettes between vectors.
to compare the ability to conditionally express shrna or mirna using the retroviral backbone, the h1– <dig> × tet <dig> and cmv-to vectors containing p <dig> shrna or mirna, respectively, were introduced into the phush-retro-puro backbone by gateway® recombination .
viral supernatants of phush-retro were prepared as described in the methods and used to infect 3t <dig> cells at a multiplicity of infection of about  <dig> .
cells were selected in  <dig>  ug/ml puromycin and then cultured in the presence or absence of  <dig> ug/ml dox for four days.
interestingly, both cmv-mir-p <dig> vectors were more effective than the corresponding h1-shrna vectors .
consistent with the transient transfection results  the p53- <dig> sequence was more effective than p53- <dig>  moreover, these results indicate that inducible and stable target gene at a single copy can be achieved by employing 'second generation' cmv-mir hairpin design and are in agreement with previous reports  <cit> .
it is important to note, however, that robust silencing using the h1-shrna vectors can be achieved presumably as the result of multiple integrations per cell .
a simple strategy we have found for maximizing the level of silencing when using the h1-shrna phush vectors is to escalate the selection pressure downstream of the tetr
finally, we also provide evidence that regulated protein expression can be achieved using the phush backbone.
for this purpose, the emgfp-mir sequence of cmv-to-emgfp-mirna was replaced by egfp or egfp-melk-d150a, a previously described kinase-dead melk variant  <cit>  fused to the c-terminus of egfp.
phush protein vectors were completed by gateway® cloning, retroviral stocks were generated by standard methods, and hct <dig> cells were serially infected and selected at  <dig> ug/ml puromycin for two weeks.
the resulting stable cell lines were treated ±  <dig> ug/ml dox for  <dig> h. egfp or egfp-melk-d150a expression was analyzed by direct fluorescent imaging of a non-reducing polyacrylamide gel , and western blotted for tubulin to confirm equal loading.
the results indicate that inducible expression of the proteins can also be achieved with this vector configuration and has in fact been successfully employed to conditionally express a temperature-sensitive mutant of the gtpase dynamin- <dig>  <cit> .
we conclude that the retroviral phush vector is capable of inducible pol iii-shrna expression, pol ii-mirna expression, and pol ii-protein expression.
at present, we have generated destination phush backbones with the selectable markers as summarized in additional file  <dig>
in vivo shrna knockdown by phush
to demonstrate the utility of phush as a technology by which to interrogate the role of a target gene within relevant tumor models in vivo, we constructed a retroviral phush vector with a luciferase-directed shrna using the h1-teto <dig> promoter.
retroviral stocks were prepared as before, and luciferase-expressing svt <dig> cells were transduced and selected in  <dig> ug/ml puromycin.
we identified multiple clones that exhibited high luciferase expression without dox and significant knockdown of luciferase expression in vitro .
a single clone  expressing on average ~ <dig> relative light units per ug of total protein in the absence of dox and displaying ~60% dox induced knockdown was selected for in vivo modeling experiments.
to this end, immunodefecient nu/nu mice were injected subcutaneously with  <dig> ×  <dig> cells of svt-shluc.pgl3-b <dig>  after tumors had reached  <dig> mm <dig>  the animals were separated into the appropriate dox treatment group.
tumor growth and luciferase expression were monitored as described in methods.
as shown in figure 5a and 5b, control animals treated with sucrose water rapidly formed tumors with high luciferase expression, while those dosed with  <dig> μg/ml dox in the drinking water maintained rapid tumor growth with low luciferase expression.
these studies indicate that functional, dox regulated shrna expression can be achieved in vivo with no discernable indirect effects on tumor growth or animal health.
we then proceeded to test if inducible, shrna-mediated knockdown of reporter gene expression in an orthotopic brain tumor model could be achieved.
unlike conventional subcutaneous xenograft models that can be monitored by caliper measurement, accurate in vivo quantitation of intracranial tumor growth necessitates the use of non-invasive imaging techniques.
both mri and bioluminescence imaging have been previously established as particularly well suited to the monitoring of glioma progression in vivo  <cit> .
since dox and luciferin have been previously reported to freely cross the brain-blood barrier, we decided to test whether combining shrna knockdown and biolumenscent imaging may be a method to expedite target validation studies in these more complex tumor models.
to this end, svt2-shluc.pgl3- <dig> cells were injected intracranially and tumor growth and luciferase expression was monitored as described in methods at days  <dig>   <dig>  and  <dig> post dox treatment.
we observed dox mediated knockdown at a day  <dig> , day  <dig>  and at day  <dig>  .
tumor size at day  <dig> as quantitated by luciferase expression strongly correlated with tumor volume as measured ex-vivo by ultrasound .
representative images for both subcutaneous and orthotopic experiments are present in additional file  <dig>  these data validate the effectiveness of the phush inducible shrna system in an orthotopic mouse brain tumor model.
we next sought to combine knockdown of a relevant oncogene with bioluminescence imaging  within tumor models in vivo.
we have previously generated and characterized retroviral phush vectors with multiple b-raf-directed shrnas.
b-raf, a serine/threonine kinase, is mutated in 70% of melanomas  <cit>  and has been proposed as a candidate small molecule therapeutic target  <cit> .
we have previously provided data in support of this proposal by demonstrating the requirement of this kinase within several tumor growth models  <cit>  by employing phush to knockdown the expression of b-raf in human melanoma cell lines.
we have since continued to deplete oncogenic b-raf in multiple settings and now provide additional data to further demonstrate how this technology may be applied.
as shown in figure 6a, we were successful at generating and identifying clones with dox-dependent silencing of b-raf in a <dig> cells, a human tumor cell line derived from a malignant cutaneous melanoma.
we also observed an inhibition of b-raf signaling upon silencing, demonstrated by a decrease in phospho-erk1/ <dig> levels.
as expected, the consequence of b-raf knockdown and mapk pathway modulation is evident by a decrease in proliferation under low serum, as well as a dox-dose dependent decrease in vegf secretion in vitro .
to establish a method to non-invasively monitor braf silencing within the context of a tumor model in vivo, a lox-imv <dig> melanoma line with the phush-braf-shrna  <cit>  was re-engineered to express a luc-braf transcript fusion.
dox-regulated luciferase expression was observed in several lox-imv <dig> phush lines as indicated by the depletion of luc-b-raf fusion in vitro by a luciferase assay .
we then performed an in vivo tumor growth study by subcutaneously inoculating scid-beige mice with  <dig> ×  <dig> cells of lox-imv <dig> shbraf.pgl3-braf clone a <dig>  after tumors had reached  <dig> mm <dig>  the animals were separated into the appropriate dox treatment group, and tumor growth and luciferase expression were monitored.
as shown in additional file  <dig>  control animals treated with sucrose water rapidly formed tumors, while those dosed with  <dig> μg/ml dox had complete tumor remission according to caliper measurements of tumor volume.
we have previously demonstrated that the in vivo tumor growth of lox-imv <dig> cells, engineered to express a luciferase targeting shrna, are not affected by dox treatment  <cit> .
therefore the observed tumor remission upon dox treatment is due to braf silencing and not the result of an off-target effect of dox treatment.
animals treated with  <dig> μg/ml dox exhibited growth stasis, while those treated with  <dig> μg/ml dox resulted in an approximately 20% decrease in tumor growth relative to sucrose control animals.
by measuring luciferase expression, a robust decrease in signal was measured at  <dig> μg/ml  and a moderate decrease at  <dig> μg/ml dox  in comparison to the control or  <dig> ng/ul dox treated animals .
tumors were harvested at day  <dig> and in vivo luc-braf knockdown was confirmed in harvested tumor lysates by a luciferase assay, confirming the utility of this strategy to monitor as well as titrate target gene knockdown in vivo .
discussion
we report the development of an inducible shrna system that is delivered on a single plasmid or viral vector.
by utilizing a reporter such as the pgl3-melk construct, numerous shrnas designed against a particular gene or genes can be screened, and effective hairpins can be rapidly cloned by gateway® recombination into the appropriate phush destination vector.
"first generation" pol iii-shrna vectors, "second generation" pol ii-mirna, and protein expression vectors have been successfully developed as components for inducible expression on several backbones.
silencing is robust and titratable for both in vitro and in vivo experimental models.
the described phush system is based on the native tet repressor instead of the tetr-vp <dig> fusion protein.
because regulation is based on repression of a modified active promoter rather than the transactivation of a minimal promoter, the required components are reliably expressed as a single construct.
by combining the shrna and tetr expression cassettes on the same plasmid or virus, we have dramatically decreased the time required to generate stable cell pools or clones exhibiting the desired level of regulated rnai.
since we have not directly compared the phush vector with other inducible shrna, mirna or protein expression systems, we cannot conclude if our vector design represents a major improvement over existing systems.
however, by directly comparing the expression strategies of individual phush vector components by a variety of cell-based assays, we can conclude that a single-plasmid vector system that combines mirna expression, multiple tetr operons and a codon-optimized tetr is highly reliable for routine cell line engineering.
the ability to perform in vitro or in vivo rnai high-throughput screens  may also be possible with the phush system.
this approach is based on the delivery of a large pool of shrna or mirnas designed against thousands of genes  <cit> .
several large, vector-based rnai libraries now exist.
two are based on the first generation poliii-shrna design  <cit> , and one is based on a mir scaffold  <cit> .
recently a conditional shrna library targeting approximately  <dig>  genes and utilizing a two vector approach for inducible shrna screens has been successfully employed  <cit> .
it is therefore conceivable that that an effective single vector shrna or mirna conditional library could be based on the phush system.
moreover, this combined system could also be used for direct in vivo application of inducible rnai vectors for gene therapy studies or for inter-tumoral target validation in cancer mouse models.
by combining inducible shrna technology with bioluminescence imaging, large in vivo experiments can be performed without the use of traditional caliper measurements  <cit> .
we have previously demonstrated the utility of our approach by measuring the effect of oncogenic b-raf depletion in a mouse model of metastatic melanoma.
pulmonary tumor burden was quantified by in vivo luciferase expression without animal dissection, thereby enabling measurements of disease progression over time in the same animal  <cit> .
for the study presented in this report, endogenous b-raf depletion was tracked by engineering a luceriferase-based reporter in which b-raf was cloned downstream of the luciferase stop codon.
we demonstrate robust in vivo b-raf knockdown as detected by the decrease in luciferase signal while maintaining the reported tumor regression phenotype for endogenous b-raf knockdown in this model.
one of the primary challenges to targeted cancer therapeutics is tumor escape mediated by drug resistance as in the case of gleevec  <cit> .
inducible and reversible shrna expression systems such as phush may be utilized for tumor outgrowth or "dox cycling" experiments in which tumors regress upon shrna-mediated oncogene knockdown, acquire the ability to evade oncogene knockdown, and then progress as a resistant tumor  <cit> .
the exogenous luciferase reporter allows the integrity of regulated gene silencing to be checked, ensuring that knockdown is maintained and that tumor escape is not simply caused by the loss of regulated shrna expression.
this approach, therefore, may be suited for tumor studies to model novel mechanisms of cancer resistance as well as identify new pathways for tumorigenesis.
importantly, our approach is also effective in a mouse model for brain cancer.
several important in vivo applications for a single plasmid or viral vector system are not presented in this report.
the question remains if the system would function as a germline-integrated cassette in adult mice.
by engineering a second tet operon in the h <dig> promoter, we have achieved regulated shrna expression in embryoid bodies.
the 2×-teto <dig> h <dig> promoter exhibited total regulation of shrna expression under differentiated conditions and decreased tetr levels.
although we have carefully optimized tetr translation by codon-optimization and tetr expression by selecting a promoter associated with strong and ubiquitous expression in multiple tissue and cell types, it is unclear whether the current vector configuration will yield transgenic mice with regulated shrna expression in all tissues.
however, the potential for the direct, in vivo application of a single viral vector system for inducible shrna expression exists due to the co-integration of both the shrna and tetr cassettes.
CONCLUSIONS
the flexibility of the phush to inducibly express shrna, mirna and proteins on a variety of plasmid and viral backbones will enable rapid target validation in a diverse array of in vitro and in vivo systems.

 the development of rna interference as a tool for reverse genetic studies in mammalian systems has rapidly matured. after the seminal observation that nucleotide, chemically-synthesized rna duplexes are capable of targeted gene silencing in mammalian cells, rnai has quickly become a standard technique for functional genetic analysis. a significant advancement of this technique was the development of short-hairpin rna expression technology. this strategy exploits the defined transcriptional start and termination signals of rna polymerase iii promoters to produce a short, inverted transcript. these stem-loop rna transcripts are then processed within the cell into functional sirnas and thereby provide a means for the stable suppression of target genes. to this end, multiple groups have reported success in long-term silencing of target genes in engineered cell lines and mice. nevertheless, several limitations to the current approach remain. the primary limitation to vector-based shrna is posed by constitutive hairpin expression. if the shrna is directed against a gene essential to cell growth and survival, the probability of obtaining a stable line is low, and in those cell lines that survive, other factors may compensate for shrna-induced gene knockdown. in both cases, the relevant phenotype may be obscured. several groups have employed inducible shrna systems to address these limitations. one class of inducible systems co-express the tetracycline repressor with a modified pol iii promoter containing one or more tetr operons flanking the tata-box such that transcription is blocked when the tetr is bound to the promoter. the expression of shrna within this scenario occurs in the presence of tetracycline or related analogs. wiznerowicz and trono introduced a variation to the above design by fusing the tetr to krab, a transcriptional repression domain from kox. this fusion silences any promoter within kb of the tetr operon. therefore, shrna transcription from the pol iii promoter containing a tetr operon may also be monitored by the ability of the tetr-krab fusion to repress the expression of a fluorescent reporter gene driven by an additional rna polymerase ii promoter within the same vector. in a different approach, several labs have developed cre recombinase-based systems in which the shrna promoter is modified to contain a'floxed' cassette or spacer such that hairpin expression is activated only upon cre-mediated excision. in addition to standard or'first generation' shrna design, it has recently been demonstrated that a primary micro-rna scaffold can efficiently deliver a desired shrna. these'second-generation' mirna mimetics can be expressed from pol ii promoters, allowing the use of established conditional expression vectors. each of the above classes of regulated shrna vectors has specific advantages – from the temporal and reversible control of the tetracycline-regulated systems to tissue-specific silencing of cre-lox vectors in mice. however, a considerable limitation shared by the reported inducible shrna systems is that each system relies on a two-plasmid approach in which the shrna expression cassette is separated from the regulatory module. to address this issue, we and others have previously reported the successful integration of a shrna/mirna expression cassette with a tetracycline dependent repressor or transactivator on a single vector. here we describe the development of phush, as a gateway®-compatible, conditional shrna system in which tetr expression and operon usage have been optimized on a single vector. importantly, we compare a number of different shrna and mirna configurations in transient transfection assays and show that the phush backbone is effective at providing regulated expression from both pol ii and pol iii promoters, thereby enabling regulated shrna, mirna and protein expression. the versatility of phush is demonstrated here using a series of in vitro and in vivo reverse genetic experiments that were designed to validate the therapeutic relevance of both novel and characterized oncology targets.  analysis of shrna design as part of our effort to engineer a conditional shrna expression system, we compared hairpin stem and loop parameters to maximize knockdown efficiency. to generate a robust, quantifiable assay, we created a transcript fusion between luciferase and melk, a ser/thr kinase that we have previously described. the entire melk cdna was cloned downstream of the pgl3-luc stop codon, and the resulting pgl3-melk construct was used as a reporter for shrna efficacy in transient co-transfection experiments. we first compared several published strategies for shrna design based on two target sequences: sh and shb. for both target sequences, the following series of shrnas were created for comparison: n – a simple stem-loop design, f – a'frayed' shrna design employing artificial asymmetry, and mi – a shrna utilizing the mir loop reported to increase cytoplasmic expression. the sequence of each hairpin is presented in additional file to compare the efficacy of each design, the resulting shrna expression constructs were co-transfected with the pgl3-melk reporter at several molar ratios and normalized to control hairpin h1-lacz. as shown in figure 1a at a 10: ratio of shrna to pgl3-melk, luciferase expression was decreased for both shrna sequences, regardless of the hairpin design. the standard n design and mi23-loop shrnas were highly effective, yielding 65%, 75%, 90%, and 95% knockdown respectively. while we noticed a modest increase in knockdown efficiency by the addition of the mir loop at a 10: ratio of shrna to pgl3-melk, there was no significant increase in efficacy with the mir loop at lower molar ratios of shrna to pgl3-melk as had been previously reported. for both sh and shb, the contribution of artificial thermodynamic symmetry by the introduction of'frayed' ends at the 3' end of the sense strand was detrimental to hairpin efficacy, suggesting that destabilization of the shrna stem-loop in this context did not lead to increased risc loading of the antisense strand. as there did not appear to be a reproducible improvement with modified shrnas, we have utilized a n shrna design for the remainder of this study. over the course of our study, several labs reported robust knockdown with mirna-based vectors. using a luciferase transcript-fusion construct with murine p sequences cloned downstream of the pgl3-luc stop codon, we compared the'first generation' h1-shrna to a'second-generation' cmv-mirna- vector. unlike the n shrna design, the mirna mimetic can be expressed as standard pol ii transcripts, which are then processed by the nuclear drosha, and cytoplasmic dicer endonucleases to form a 21– nt active sirna. to enable this comparison, we generated a vector in which a modified cmv promoter was used to express emerald gfp and the mirna mimetic on the same transcript. to compare the efficacy of a shrna versus mirna design, two effective mirnas directed against p were converted to h1-shrnas by identifying the nt active sirna within the mirna scaffold and then converting these sequences into conventional shrna design according to the scheme presented in figure 1b. the sequence and structure of these hairpins is presented in additional file although the performance of the cmv/to-mir and h1-shrna hairpins using the p53- sequence were similar, we observed increased knockdown by cmv/to-mir relative to the corresponding h1-shrna sequence for the p53- sequence. at the lowest ratios of mir or shrna to reporter, the cmvto-mir resulted in ~65–70% knockdown of the luciferase reporter as compared to only a ~30–50% knockdown with the h1-shrna. removing the emgfp leader from cmv-mir-p53- destroyed the knockdown efficiency of the original construct, confirming the requirement of a minimal "spacer" sequence between the transcriptional start site and the mirna sequence. to summarize, we have directly compared "first-generation" pol iii-shrna vectors to "second-generation" pol ii vectors using the same dicer-product sirnas. we have found that both vector formats are functional. however, based on our observed increase in knockdown efficiency with cmv/to-mir for the p53- sequence as compared to the corresponding h1-shrna version, there may be a slight advantage with the mirna-like systems for certain sequences under suboptimal expression conditions. phush vector design and optimization phush was constructed as described in the methods using the pires-puro as the backbone vector. briefly, the human-β-actin promoter driving the tetr was subcloned into pires-puro to replace the original cmv promoter. a modified h promoter containing a single tetr operon with a melk-targeting shrna cassette was inserted upstream to the human-β-actin promoter. using this plasmid as the base vector, we generated two additional versions for analysis: phush-ivs in which a synthetic intron sequence between the tetr orf and the ires was removed, and phush-ivstetropt, in which the original tetr sequence within phush-ivs was replaced by a tetr expression cassette that was codon-optimized for expression in mammalian cells. to assess the effect of these modifications on regulated gene silencing, these constructs were transfected into hek cells that were stably expressing an egfp-melk fusion protein. after selection with puromycin, cells were cultured for five days in the presence or absence of doxycycline, and the level of egfp-melk fluorescence was scored by facs analysis. the effectiveness of each construct to repress shrna expression was assessed by comparing the level of egfp-melk in the absence of dox to cells containing a phush construct without a hairpin. as shown in figure 2b, all three vectors generated the same level of knockdown in the presence of dox. however, we found that in the absence of dox, only the phush-ivstetropt pool exhibited full repression of shrna expression. in contrast, partial repression was obtained with phush-ivs while cells containing the base phush vector displayed a complete lack of shrna repression, as demonstrated by similar levels of egfp-melk in the presence or absence of dox. because of the reported strong splicing donor in the 5' htlv utr, the removal of the ivs most likely prevented a splicing event between the utr and the splice acceptor within ivs resulting in the excision of the tetr. the above data suggests optimal dox-dependent regulation of shrna expression requires maximal tetr protein levels. to test this hypothesis, we compared tetr expression between the original tetr construct and the codon-optimized variant utilized for phush-ivstetropt. as shown in figure 2c, there is a two-to-three fold increase in tetr protein levels with the codon-optimized tetr as compared to the original construct. therefore, we conclude that both tetr codon optimization and ivs removal are required for a regulated gene silencing system. because of the observed sensitivity to modest changes in tetr levels, we next focused on further modifying the h promoter to ensure complete shrna repression under limiting tetr levels. several groups have shown that increased regulation can be achieved by inserting multiple tetr operons throughout the pol iii promoter. to determine whether we could observe a similar enhancement of regulation, we modified the original pshuttle-h1-shrna plasmid containing a single teto with an additional teto operon upstream of the tata box, forming a 2×-teto2-h promoter. using pgl3-melk as a reporter, the 1×- and 2×-teto2-h promoters were co-transfected with increasing amounts of a cmv-tetropt expression plasmid in the absence of dox. for both shrna promoter variants, maximal repression was achieved when the tetr expression construct was supplied in a 10: molar excess over the h1-shrna while >80% knockdown was achieved in the absence of tetr. when the tetr expression plasmid was limited to a 2: molar ratio relative to the h1-shrna plasmid, nearly maximal shrna expression was observed with the 1×-teto construct. in contrast, shrna repression was maintained with the 2×-teto construct. these results suggest that the use of two tetr operons enhance the ability to regulate shrna expression when tetr expression is limiting. to explore the robustness of this system as a stably-integrated construct, we then compared the 1×-teto and 2×-teto versions of the phush vector to enable regulated silencing of melk within mouse embryonic stem cells. murine es cells were electroporated with phush-ivstetropt or phush-ivstetropt × teto containing a melk-directed shrna and then selected in puromycin. multiple clones for both versions of phush were identified exhibiting robust, regulated melk knockdown after h culture in the presence of dox. to determine whether regulated shrna expression could be maintained upon differentiation, select es cell clones were allowed to differentiate into embryoid bodies by standard methods. eb formation was induced and cultured in the presence or absence of ug/ml dox for days, and endogenous melk rna levels were quantified by qrt-pcr. as shown in figure 3b, near maximal silencing of melk expression was observed with ebs derived from phush-ivstetropt without dox, indicating no functional repression of shrna expression. in contrast, ebs derived from phush-ivstetropt2×-teto maintained a similar level of dox-dependent regulation as had been observed with the parental, non-differentiated es cells. consistent with these results, we observed a 3-fold decrease in tetr expression upon differentiation into ebs. although the overall level of melk knockdown is reduced within the differentiated eb derived from the 2×-teto promoter relative to the 1×-teto version, this difference is likely due to clonal variations during es cell selection rather than an inherent difference in transcriptional strength of the modified h promoter. this is supported by the fact that the knockdown efficiency between the 1× and 2× teto constructs is the same when expressed in the absence of tetr. in summary, the presence of multiple tetr operons within the h promoter does not significantly effect maximal shrna expression while promoting dox regulated gene silencing even under limiting tetr expression levels. phush retroviral vectors to enhance the stable delivery into cultured cells, the phush-ivstetropt2×-teto described above was subcloned into the pqcxip retroviral backbone to form phush-retro. the resulting viral phush plasmid was converted to a gateway®-compatible vector to enable efficient transfer of shrna cassettes between vectors. to compare the ability to conditionally express shrna or mirna using the retroviral backbone, the h1– × tet and cmv-to vectors containing p shrna or mirna, respectively, were introduced into the phush-retro-puro backbone by gateway® recombination. viral supernatants of phush-retro were prepared as described in the methods and used to infect 3t cells at a multiplicity of infection of about. cells were selected in ug/ml puromycin and then cultured in the presence or absence of ug/ml dox for four days. interestingly, both cmv-mir-p vectors were more effective than the corresponding h1-shrna vectors. consistent with the transient transfection results the p53- sequence was more effective than p53- moreover, these results indicate that inducible and stable target gene at a single copy can be achieved by employing'second generation' cmv-mir hairpin design and are in agreement with previous reports. it is important to note, however, that robust silencing using the h1-shrna vectors can be achieved presumably as the result of multiple integrations per cell. a simple strategy we have found for maximizing the level of silencing when using the h1-shrna phush vectors is to escalate the selection pressure downstream of the tetr finally, we also provide evidence that regulated protein expression can be achieved using the phush backbone. for this purpose, the emgfp-mir sequence of cmv-to-emgfp-mirna was replaced by egfp or egfp-melk-d150a, a previously described kinase-dead melk variant fused to the c-terminus of egfp. phush protein vectors were completed by gateway® cloning, retroviral stocks were generated by standard methods, and hct cells were serially infected and selected at ug/ml puromycin for two weeks. the resulting stable cell lines were treated ± ug/ml dox for h. egfp or egfp-melk-d150a expression was analyzed by direct fluorescent imaging of a non-reducing polyacrylamide gel, and western blotted for tubulin to confirm equal loading. the results indicate that inducible expression of the proteins can also be achieved with this vector configuration and has in fact been successfully employed to conditionally express a temperature-sensitive mutant of the gtpase dynamin-. we conclude that the retroviral phush vector is capable of inducible pol iii-shrna expression, pol ii-mirna expression, and pol ii-protein expression. at present, we have generated destination phush backbones with the selectable markers as summarized in additional file in vivo shrna knockdown by phush to demonstrate the utility of phush as a technology by which to interrogate the role of a target gene within relevant tumor models in vivo, we constructed a retroviral phush vector with a luciferase-directed shrna using the h1-teto promoter. retroviral stocks were prepared as before, and luciferase-expressing svt cells were transduced and selected in ug/ml puromycin. we identified multiple clones that exhibited high luciferase expression without dox and significant knockdown of luciferase expression in vitro. a single clone expressing on average ~ relative light units per ug of total protein in the absence of dox and displaying ~60% dox induced knockdown was selected for in vivo modeling experiments. to this end, immunodefecient nu/nu mice were injected subcutaneously with × cells of svt-shluc.pgl3-b after tumors had reached mm the animals were separated into the appropriate dox treatment group. tumor growth and luciferase expression were monitored as described in methods. as shown in figure 5a and 5b, control animals treated with sucrose water rapidly formed tumors with high luciferase expression, while those dosed with μg/ml dox in the drinking water maintained rapid tumor growth with low luciferase expression. these studies indicate that functional, dox regulated shrna expression can be achieved in vivo with no discernable indirect effects on tumor growth or animal health. we then proceeded to test if inducible, shrna-mediated knockdown of reporter gene expression in an orthotopic brain tumor model could be achieved. unlike conventional subcutaneous xenograft models that can be monitored by caliper measurement, accurate in vivo quantitation of intracranial tumor growth necessitates the use of non-invasive imaging techniques. both mri and bioluminescence imaging have been previously established as particularly well suited to the monitoring of glioma progression in vivo. since dox and luciferin have been previously reported to freely cross the brain-blood barrier, we decided to test whether combining shrna knockdown and biolumenscent imaging may be a method to expedite target validation studies in these more complex tumor models. to this end, svt2-shluc.pgl3- cells were injected intracranially and tumor growth and luciferase expression was monitored as described in methods at days and post dox treatment. we observed dox mediated knockdown at a day, day and at day. tumor size at day as quantitated by luciferase expression strongly correlated with tumor volume as measured ex-vivo by ultrasound. representative images for both subcutaneous and orthotopic experiments are present in additional file these data validate the effectiveness of the phush inducible shrna system in an orthotopic mouse brain tumor model. we next sought to combine knockdown of a relevant oncogene with bioluminescence imaging within tumor models in vivo. we have previously generated and characterized retroviral phush vectors with multiple b-raf-directed shrnas. b-raf, a serine/threonine kinase, is mutated in 70% of melanomas and has been proposed as a candidate small molecule therapeutic target. we have previously provided data in support of this proposal by demonstrating the requirement of this kinase within several tumor growth models by employing phush to knockdown the expression of b-raf in human melanoma cell lines. we have since continued to deplete oncogenic b-raf in multiple settings and now provide additional data to further demonstrate how this technology may be applied. as shown in figure 6a, we were successful at generating and identifying clones with dox-dependent silencing of b-raf in a cells, a human tumor cell line derived from a malignant cutaneous melanoma. we also observed an inhibition of b-raf signaling upon silencing, demonstrated by a decrease in phospho-erk1/ levels. as expected, the consequence of b-raf knockdown and mapk pathway modulation is evident by a decrease in proliferation under low serum, as well as a dox-dose dependent decrease in vegf secretion in vitro. to establish a method to non-invasively monitor braf silencing within the context of a tumor model in vivo, a lox-imv melanoma line with the phush-braf-shrna was re-engineered to express a luc-braf transcript fusion. dox-regulated luciferase expression was observed in several lox-imv phush lines as indicated by the depletion of luc-b-raf fusion in vitro by a luciferase assay. we then performed an in vivo tumor growth study by subcutaneously inoculating scid-beige mice with × cells of lox-imv shbraf.pgl3-braf clone a after tumors had reached mm the animals were separated into the appropriate dox treatment group, and tumor growth and luciferase expression were monitored. as shown in additional file control animals treated with sucrose water rapidly formed tumors, while those dosed with μg/ml dox had complete tumor remission according to caliper measurements of tumor volume. we have previously demonstrated that the in vivo tumor growth of lox-imv cells, engineered to express a luciferase targeting shrna, are not affected by dox treatment. therefore the observed tumor remission upon dox treatment is due to braf silencing and not the result of an off-target effect of dox treatment. animals treated with μg/ml dox exhibited growth stasis, while those treated with μg/ml dox resulted in an approximately 20% decrease in tumor growth relative to sucrose control animals. by measuring luciferase expression, a robust decrease in signal was measured at μg/ml and a moderate decrease at μg/ml dox in comparison to the control or ng/ul dox treated animals. tumors were harvested at day and in vivo luc-braf knockdown was confirmed in harvested tumor lysates by a luciferase assay, confirming the utility of this strategy to monitor as well as titrate target gene knockdown in vivo. discussion we report the development of an inducible shrna system that is delivered on a single plasmid or viral vector. by utilizing a reporter such as the pgl3-melk construct, numerous shrnas designed against a particular gene or genes can be screened, and effective hairpins can be rapidly cloned by gateway® recombination into the appropriate phush destination vector. "first generation" pol iii-shrna vectors, "second generation" pol ii-mirna, and protein expression vectors have been successfully developed as components for inducible expression on several backbones. silencing is robust and titratable for both in vitro and in vivo experimental models. the described phush system is based on the native tet repressor instead of the tetr-vp fusion protein. because regulation is based on repression of a modified active promoter rather than the transactivation of a minimal promoter, the required components are reliably expressed as a single construct. by combining the shrna and tetr expression cassettes on the same plasmid or virus, we have dramatically decreased the time required to generate stable cell pools or clones exhibiting the desired level of regulated rnai. since we have not directly compared the phush vector with other inducible shrna, mirna or protein expression systems, we cannot conclude if our vector design represents a major improvement over existing systems. however, by directly comparing the expression strategies of individual phush vector components by a variety of cell-based assays, we can conclude that a single-plasmid vector system that combines mirna expression, multiple tetr operons and a codon-optimized tetr is highly reliable for routine cell line engineering. the ability to perform in vitro or in vivo rnai high-throughput screens may also be possible with the phush system. this approach is based on the delivery of a large pool of shrna or mirnas designed against thousands of genes. several large, vector-based rnai libraries now exist. two are based on the first generation poliii-shrna design, and one is based on a mir scaffold. recently a conditional shrna library targeting approximately genes and utilizing a two vector approach for inducible shrna screens has been successfully employed. it is therefore conceivable that that an effective single vector shrna or mirna conditional library could be based on the phush system. moreover, this combined system could also be used for direct in vivo application of inducible rnai vectors for gene therapy studies or for inter-tumoral target validation in cancer mouse models. by combining inducible shrna technology with bioluminescence imaging, large in vivo experiments can be performed without the use of traditional caliper measurements. we have previously demonstrated the utility of our approach by measuring the effect of oncogenic b-raf depletion in a mouse model of metastatic melanoma. pulmonary tumor burden was quantified by in vivo luciferase expression without animal dissection, thereby enabling measurements of disease progression over time in the same animal. for the study presented in this report, endogenous b-raf depletion was tracked by engineering a luceriferase-based reporter in which b-raf was cloned downstream of the luciferase stop codon. we demonstrate robust in vivo b-raf knockdown as detected by the decrease in luciferase signal while maintaining the reported tumor regression phenotype for endogenous b-raf knockdown in this model. one of the primary challenges to targeted cancer therapeutics is tumor escape mediated by drug resistance as in the case of gleevec. inducible and reversible shrna expression systems such as phush may be utilized for tumor outgrowth or "dox cycling" experiments in which tumors regress upon shrna-mediated oncogene knockdown, acquire the ability to evade oncogene knockdown, and then progress as a resistant tumor. the exogenous luciferase reporter allows the integrity of regulated gene silencing to be checked, ensuring that knockdown is maintained and that tumor escape is not simply caused by the loss of regulated shrna expression. this approach, therefore, may be suited for tumor studies to model novel mechanisms of cancer resistance as well as identify new pathways for tumorigenesis. importantly, our approach is also effective in a mouse model for brain cancer. several important in vivo applications for a single plasmid or viral vector system are not presented in this report. the question remains if the system would function as a germline-integrated cassette in adult mice. by engineering a second tet operon in the h promoter, we have achieved regulated shrna expression in embryoid bodies. the 2×-teto h promoter exhibited total regulation of shrna expression under differentiated conditions and decreased tetr levels. although we have carefully optimized tetr translation by codon-optimization and tetr expression by selecting a promoter associated with strong and ubiquitous expression in multiple tissue and cell types, it is unclear whether the current vector configuration will yield transgenic mice with regulated shrna expression in all tissues. however, the potential for the direct, in vivo application of a single viral vector system for inducible shrna expression exists due to the co-integration of both the shrna and tetr cassettes.  the flexibility of the phush to inducibly express shrna, mirna and proteins on a variety of plasmid and viral backbones will enable rapid target validation in a diverse array of in vitro and in vivo systems. 
we provide examples of how phush has been successfully employed to study the function of target genes in a number of cell types within in vitro and in vivo assays, including conditional gene knockdown in a murine model of brain cancer. both pol ii and pol iii promoters have been successfully combined with a second expression cassette containing a codon-optimized tetracycline repressor and selectable marker. these studies demonstrate the broad application potential of phush for conditional genetic engineering in mammalian cells. we have therefore developed phush, an inducible expression system that allows regulated expression of shrna, mirna or cdna cassettes on a single viral vector.
 conditional expression vectors have become a valuable research tool to avoid artefacts that may result from traditional gene expression studies. however, most systems require multiple plasmids that must be independently engineered into the target system, resulting in experimental delay and an increased potential for selection of a cell subpopulation that differs significantly from the parental line. we have therefore developed phush, an inducible expression system that allows regulated expression of shrna, mirna or cdna cassettes on a single viral vector.  both pol ii and pol iii promoters have been successfully combined with a second expression cassette containing a codon-optimized tetracycline repressor and selectable marker. we provide examples of how phush has been successfully employed to study the function of target genes in a number of cell types within in vitro and in vivo assays, including conditional gene knockdown in a murine model of brain cancer.  we have successfully developed and employed a single vector system that enables doxycycline regulated rnai or transgene expression in a variety of in vitro and in vivo model systems. these studies demonstrate the broad application potential of phush for conditional genetic engineering in mammalian cells. 