BACKGROUND
there is a growing awareness of the significance of small rnas in biology, which has led to increased use of small rnas as tools in biological research.
for example, micrornas  are small non-coding rnas that regulate gene expression by reducing the stability or the translation of partially complementary mrna  <cit>  and up to 30% of human genes may be regulated by mirnas  <cit> .
rnai, mediated by short double-stranded rnas , has become a powerful tool for analyzing gene function through targeted gene knock down  <cit> .
improved methods for controlled expression of small rnas in the cell will advance the study of their roles in biological processes.
mirna genes are transcribed by rna polymerase ii  and the primary transcript is processed in vivo to yield first a short hairpin, and finally a 21- <dig> nt mirna  <cit> .
synthetic sirna can be synthesized in vitro and delivered to cells by transfection.
alternatively, short rna hairpins that mimic a mirna precursor can be expressed in the cell using either plasmid or viral vectors.
the resulting transcript is processed in vivo to yield a small rna that can function as an sirna, or a mirna, inducing specific degradation of targets, similar to transfected sirna  <cit> .
for certain rnai applications, expression of short hairpins offers advantages over transient transfection of sirna.
expression vectors can be transiently transfected or integrated into the cellular genome to create stable cell lines.
the latter method provides consistent, long-term expression of the short hairpin as compared with transient transfection of sirna.
early expression vectors used the u <dig> and h <dig> rna polymerase iii  promoters which use discrete initiation and termination marks  <cit> .
constitutive pol ii promoters, such as cmv  <cit>  or ubc  <cit> , have also been used .
but in many cases, such as analysis of essential genes, a conditional expression system that can produce sirna or mirna on demand or for a limited time is required.
while pol iii promoters allow a high level of expression they are naturally constitutive.
efforts to engineer pol iii systems under drug-mediated control  have compromised either the tight repression of expression in the off state  <cit>  or the high level of expression in the on state  <cit> .
the advantage of the tet system is that expression is reversible upon drug withdrawal.
pol ii based expression systems offer better control through the use of tissue specific or conditional promoters.
a variety of regulated systems have been developed for inducible gene expression  but perhaps the most widely used are the doxycycline and ecdysone controlled systems  <cit> .
an improved version of the ecdysone-inducible approach is a two-hybrid version, also known as the rheoswitch system, which uses an artificial heterodimeric nuclear receptor for ligand-induced transcription of a gene cloned into an expression plasmid  <cit> .
two modified nuclear receptors, "rheoreceptor-1" and "rheoactivator", driven by constitutive promoters are carried on one plasmid.
the "rheoreceptor-1" is a fusion of the gal <dig> dna binding domain with a modified ecdysone receptor  ligand binding domain.
the "rheoactivator" is a fusion of the viral transcription activation domain vp <dig> with a chimeric mammalian/insect rxr ligand binding domain.
the transcription unit of interest is cloned downstream of five gal <dig> response elements  in a separate expression plasmid  <cit>  .
instead of ecdysone, a non-steroidal diphenylhydrazine compound, rsl <dig>  acts as a specific ligand that stabilizes the nuclear receptor heterodimer and activates transcription of the cloned gene of interest.
this combination of chimeric receptors with a non-steroidal synthetic ligand was designed to ensure that the expression system will not interfere with endogenous cellular pathways  <cit> .
rsl <dig>  has shown minimal effects on endogenous gene expression and cell proliferation in prostate cells  <cit>  and in hek <dig> cells  <cit> .
in addition to these systems, rheoswitch has been used to induce expression of proteins in mice, mbt- <dig> and panc <dig> carcinoma cells  <cit>  and nih3t <dig> cells  <cit> .
however, no gene silencing studies using this system have yet been published.
in this study, we used rheoswitch to produce rnas that are processed in vivo in mammalian cultured cells to generate mirnas that are functional in target gene knockdown.
RESULTS
inducible expression of mir-122
we chose mir- <dig>  an abundantly expressed mirna, as a model for inducible shrna expression.
mir- <dig> is expressed exclusively in the liver  <cit>  and plays a key role in the regulation of cholesterol and fatty acid metabolism in the adult liver  <cit> .
in hepatocarcinoma of humans and rodents, mir- <dig> has been reported to be specifically down-regulated to a significant degree  <cit> .
hepatoma cell lines expressing mir- <dig>  such as huh- <dig>  are required for the propagation and study of hepatitis c virus .
hepg <dig>  which does not express detectable mir- <dig> is resistant to hcv infection  <cit> .
recently, the first mirna-based drug has been shown to be protective against hcv infection in primates  <cit> .
these properties of mir- <dig> have made it a highly studied mirna and a prime target for therapeutics development.
to determine whether the rheoswitch expression system could be used to induce expression of short hairpin rnas, a  <dig> bp human genomic dna fragment containing mir- <dig> and its flanking dna sequence was cloned downstream of the gal <dig> binding sites in the rheoswitch expression vector pnebr-x <dig>  transient transfection of this plasmid into human embryonic kidney cells stably expressing rheoreceptor and rheoactivator  demonstrated production of mature mir- <dig> upon induction with rsl <dig> .
based on these results and additional transient transfection experiments , we constructed a cell line that carried integrated mir- <dig> expression vector  in mouse embryo fibroblasts, which also stably express the rheoswitch proteins.
we used this cell line to investigate the properties of induced mir- <dig> expression.
first, we confirmed that rsl <dig> treatment could induce the cells to produce mature mir- <dig> mirna.
northern blot analysis demonstrated that rsl1-treated cells produced the  <dig> nt guide strand, whereas the passenger strand was undetectable, indicating that the mir- <dig> primary transcript is induced and correctly processed .
in the absence of rsl <dig>  cells did not produce any detectable passenger or guide strand.
induction of the cells for  <dig> hours using different concentrations of rsl <dig> showed that the amount of accumulating mature mir- <dig> could be modulated by the concentration of the inducer .
one advantage of small molecule ligands such as rsl <dig> is the ability to diffuse into cells to rapidly induce, or turn off expression, in a dose-dependent manner  <cit> .
to study these features for mirna expression, an examination of the induction time course and on-off switching was performed.
mir- <dig> guide strand was detectable  <dig> hours after induction, its level peaked at about  <dig> hours post-induction and remained at that level after  <dig> hours, indicating rapid induction and steady levels of mirna production .
these results demonstrate that, in principle, the nih3t3-47/mir- <dig> cell line can become mir- <dig> positive or negative, depending on the presence or absence of rsl <dig> in the culture medium, mimicking huh- <dig> or hepg <dig> hepatic cells respectively in terms of mir- <dig> expression status .
we studied long-term expression and switching properties by maintaining the cells under non-induced or continuous induction conditions for several days.
non-induced cells showed no detectable mir- <dig> accumulation after  <dig> days in culture, while induced cells expressed mir- <dig> for at least  <dig> days, demonstrating tight control of the off state .
next, we tested the reversibility of the switch.
cells were treated with rsl <dig> for  <dig> hours, rsl <dig> was then withdrawn.
mir- <dig> expression was reduced over the course of the next days, reaching non-induced levels by day  <dig> .
a second induction at day  <dig> can restore expression to the fully induced levels .
these results demonstrate that induced expression of mirna using this system is sustainable and reversible, allowing control of expression of the cloned mirna.
inducible and regulated silencing
since this system proved suitable for controlled expression of mir- <dig>  we tested whether the induced mirna expression can be used in turn for controlled target gene silencing.
first, we designed a reporter-based assay using the secreted gaussia luciferase .
we had previously used a gluc-based reporter for assessing sirna potency  <cit> .
two tandem copies of a sequence complementary to the mir- <dig> guide strand were inserted into the 3' utr of a gluc reporter  .
the mir- <dig> guide strand should work, in this instance, like an sirna because it perfectly matches its target in the 3' utr of the gluc mrna.
we transfected ptk-gluc-mir <dig> or control ptk-gluc reporter into the nih3t3-47/mir- <dig> cells and measured the secreted luciferase activity after different treatments.
gluc expression was unaffected in non-induced cells, but was substantially reduced following induction of mir- <dig> expression .
consistent with the reversible mir- <dig> expression , gluc expression was restored following withdrawal of rsl <dig> , demonstrating reversible knock down of the target.
knockdown of gluc expression was target-specific since the control reporter lacking the mir- <dig> target sites was unaffected by any treatment .
since the secreted luciferase assay is non-destructive, we used the same cells to correlate the expression status of the mir- <dig> guide strand detected by northern hybridization.
consistent with the luciferase expression level, mir- <dig> was detected only under inducing conditions .
following rsl <dig> withdrawal, mir- <dig> declined over the next  <dig> hours .
since this system showed regulation of mirna output by varying the concentration of the inducer , we tested whether the downstream effects of mir- <dig> expression were also rsl <dig> concentration-dependent.
when expression of the ptk-gluc-mir <dig> reporter was assayed after treatment with increasing concentrations of rsl <dig>  a corresponding decrease in gaussia luciferase activity was observed, whereas the activity from cells transfected with the control vector produced normal amounts of luciferase .
western blot analysis confirmed that the loss of measured luciferase activity reflects the decreased gluc protein levels as a result of mirna targeting .
these results demonstrate that mir- <dig> short hairpin-mediated target silencing can be controlled in an rsl <dig> dose-dependent manner.
induced silencing of mir- <dig> target genes
we demonstrated that induced expression of the guide strand of mir- <dig>  acting as an sirna, can silence an artificial reporter gene with a perfectly matched target sequence present in its 3' utr .
to test whether the system could be used for "natural" mirna target validation we attempted to recapitulate the silencing activity of mir- <dig> through its interaction with the 3'-utr of previously identified target genes such as glycogen synthase   <cit> .
the 3' utr of gys was cloned downstream of gluc in the ptk-gluc reporter vector and the resulting construct  was transfected into the mir- <dig> expressing stable cell line nih3t3-47/mir- <dig>  upon rsl <dig> induction, gaussia luciferase activity from cells transfected with the gys reporter was reduced to 76% of control expression .
this knockdown is target sequence-specific since luciferase activity from cells transfected with ptk-gluc control plasmid with an unrelated utr was unaffected by mir- <dig> induction .
a similar reduction was observed using gluc reporter assays for another mir- <dig> target, cat <dig>  <cit>  .
in order to further test this mirna target validation methodology with an endogenous  gene target, we tested the effect of induced mir- <dig> expression on aldolase a, a validated mir- <dig> target in mouse liver,  <cit> .
we confirmed by western blot analysis that aldolase a is expressed in uninduced nih3t3-47/mir- <dig> cells .
we monitored aldolase-a protein levels by immunoblot over the course of mir- <dig> induction.
in the presence of rsl <dig> nih3t3-47/mir- <dig> cells show a gradual reduction of aldolase a protein to 75% of control in  <dig> days and 31% of control levels after  <dig> days compared to time-matched non-induced nih3t3-47/mir- <dig> control cells .
thus we achieved modulation of aldolase-a protein expression with small molecule induction of ectopic mir- <dig> expression.
these results suggest that mirna target gene validation and phenotypic analysis can be easily obtained using this inducible mirna system.
expression of inducible shrna
to test whether artificial short hairpins could be expressed and properly processed to produce a designed guide strand, different short hairpin configurations carrying the same inserted guide strand sequence were cloned in the rheoswitch expression vector pnebrx <dig> .
the sequence and structure of the short hairpins in pnebrx-sh- <dig> and pnebrx-sh- <dig> were modeled on mir- <dig> as previously described  <cit> .
in pnebrx-sh- <dig>  the short hairpin sequence is cloned directly into the mcs of the vector.
the hairpin sequence in pnebrx-sh- <dig> is the same as pnebrx-sh- <dig>  but it is inserted in the place of mir- <dig> in the  <dig> bp genomic dna fragment used above for mir <dig> expression.
pnebrx-sh- <dig> uses the structure of the mir- <dig> short hairpin but it contains the same guide strand as sh- <dig> and sh- <dig>  compensatory changes were made in the stem sequence in order to maintain a mir-122-like  structure.
plasmids encoding these short hairpins were transfected into nih3t3- <dig> cells, expression was induced by rsl <dig> and northern blot analysis using guide strand-specific probes was used to evaluate hairpin processing.
sh- <dig> produced little rna of the expected size, perhaps because the stem-loop structure was not conducive to optimal processing .
sh- <dig> produced more, suggesting that processing is more efficient if the short hairpin is surrounded by mir- <dig> genomic sequences.
sh- <dig>  which most closely mimics mir- <dig> produced the most mature guide strand .
these results suggest that the mir- <dig> stem loop structure more readily accommodates guide strand sequence variants than the mir- <dig> stem loop structure.
inducible sirna production for controlled target knock down
one hurdle in using an inducible shrna expression systems is to reliably convert a desired sirna into an inducible hairpin that can silence target genes.
this process is not always straightforward.
we tested whether the mir- <dig> backbone in the rheoswitch expression vector could be used as a platform for inducible expression of a shrna with a desired sequence for rnai silencing.
to test the system, we chose a previously described sirna directed against a  <dig> nt sequence of firefly luciferase   <cit> .
based on our previous results , we replaced the  <dig> nt guide strand of mir- <dig> in pnebrx-mir- <dig> with the fluc sirna guide strand in a structure similar to sh- <dig> .
because the published  guide strand sequence was  <dig> nt long, four nucleotides shorter than the mir- <dig> guide strand, we added four nucleotides  to the 3' end of the fluc guide strand in order to maintain the structure of the mir- <dig> short hairpin .
the mir- <dig> stem contains a bulge, so we designed one short hairpin, fluc-shm, with an internal mismatch in order to mimic the mir- <dig> structure.
a second fluc short hairpin, fluc-sh was designed with a perfectly complementary stem structure .
additionally, based on our northern blot results with sh- <dig> , we tested whether surrounding the fluc short hairpin with mir- <dig> genomic flanking sequence has an effect on its silencing properties.
therefore, we inserted both the mismatched and perfectly complementary fluc short hairpins into the rheoswitch expression vector either surrounded by genomic sequences  or directly cloned into the mcs of the vector .
the fluc short hairpin plasmids or an equivalent mir- <dig> short hairpin plasmid  were co-transfected with an fluc reporter plasmid into rheoswitch cells and compared for induced knockdown of firefly luciferase.
all of the fluc short hairpin designs were effective in reducing luciferase activity upon rsl <dig> induction  while the control mir- <dig> had no effect on the luciferase reporter activity.
the stem-mismatched short hairpins , which more closely mimic the mir- <dig> structure, were slightly more effective in knocking down the fluc reporter than the perfectly matched hairpin designs .
the surrounding dna context had no significant effect on the knockdown obtained by either fluc short hairpin, i.e., short hairpins flanked by genomic sequences were neither more nor less effective than those flanked by the mcs of the vector .
taken together these results provide guidance in designing shrna expression constructs.
short hairpins based on the mir- <dig> stem loop structure can produce more mirna guide strand than short hairpins based on the mir- <dig> structure .
if the short hairpin was not readily processed, as was the case with the mir-30-like sh- <dig>  addition of flanking sequence increased the processing efficiency .
neither sh- <dig> nor sh- <dig> produced as much mirna as the mir- <dig> short hairpin or the mir-122-like sh- <dig>  it seems probable that sh- <dig> produced more mature mirna than sh- <dig> and sh- <dig> because it is a variant of the mir- <dig> short hairpin, rather than because of the genomic dna that flanks it .
the designs with the bulge in the stem , mimicking the structure of mir- <dig>  were slightly more effective in target knockdown than those perfectly complementary .
the target knockdown results support the conclusion that in determining the processing efficiency and silencing effectiveness in the structure of the short hairpin is more important than the sequence context in which it is transcribed, and the mir- <dig> stem-loop structure is a favorable vehicle for short hairpin expression.
CONCLUSIONS
we have shown that in addition to controlled protein expression, the rheoswitch ligand-inducible system allows regulated expression of short hairpin rna.
by varying the dose of rsl <dig>  the rna expression level can be modulated, and upon rsl <dig> withdrawal, expression is turned off.
this feature is important when studying biological phenomena resulting from down-regulation, but not elimination, of gene function.
expression can be turned on and off repeatedly, allowing additional control for studying a range of experimental states, an advantage when studying essential genes.
short hairpins can be expressed and the rna processed to yield mirna  or sirna .
the expressed mirnas function as expected in target knockdown using endogenous targets, such as aldolase-a, or reporter-3' utr targets , facilitating mirna target validation assays.
we explored whether novel guide strand sequences, such as those based on an sirna, can be incorporated into the mir- <dig> short hairpin, expressed and processed to yield functional small rnas.
our experiments suggest that the mir- <dig> backbone can be adapted for inducible sirna expression.
it has been shown that multiple shrnas, directed at multiple targets, can be expressed from a single transcription unit  <cit> .
the rheoswitch system accommodates long transcription units, unlike pol iii systems that require short transcripts.
this suggests that it may be possible to build a rheoswitch expression vector with two or more shrnas in tandem.

 there is a growing awareness of the significance of small rnas in biology, which has led to increased use of small rnas as tools in biological research. for example, micrornas are small non-coding rnas that regulate gene expression by reducing the stability or the translation of partially complementary mrna and up to 30% of human genes may be regulated by mirnas. rnai, mediated by short double-stranded rnas, has become a powerful tool for analyzing gene function through targeted gene knock down. improved methods for controlled expression of small rnas in the cell will advance the study of their roles in biological processes. mirna genes are transcribed by rna polymerase ii and the primary transcript is processed in vivo to yield first a short hairpin, and finally a 21- nt mirna. synthetic sirna can be synthesized in vitro and delivered to cells by transfection. alternatively, short rna hairpins that mimic a mirna precursor can be expressed in the cell using either plasmid or viral vectors. the resulting transcript is processed in vivo to yield a small rna that can function as an sirna, or a mirna, inducing specific degradation of targets, similar to transfected sirna. for certain rnai applications, expression of short hairpins offers advantages over transient transfection of sirna. expression vectors can be transiently transfected or integrated into the cellular genome to create stable cell lines. the latter method provides consistent, long-term expression of the short hairpin as compared with transient transfection of sirna. early expression vectors used the u and h rna polymerase iii promoters which use discrete initiation and termination marks. constitutive pol ii promoters, such as cmv or ubc, have also been used. but in many cases, such as analysis of essential genes, a conditional expression system that can produce sirna or mirna on demand or for a limited time is required. while pol iii promoters allow a high level of expression they are naturally constitutive. efforts to engineer pol iii systems under drug-mediated control have compromised either the tight repression of expression in the off state or the high level of expression in the on state. the advantage of the tet system is that expression is reversible upon drug withdrawal. pol ii based expression systems offer better control through the use of tissue specific or conditional promoters. a variety of regulated systems have been developed for inducible gene expression but perhaps the most widely used are the doxycycline and ecdysone controlled systems. an improved version of the ecdysone-inducible approach is a two-hybrid version, also known as the rheoswitch system, which uses an artificial heterodimeric nuclear receptor for ligand-induced transcription of a gene cloned into an expression plasmid. two modified nuclear receptors, "rheoreceptor-1" and "rheoactivator", driven by constitutive promoters are carried on one plasmid. the "rheoreceptor-1" is a fusion of the gal dna binding domain with a modified ecdysone receptor ligand binding domain. the "rheoactivator" is a fusion of the viral transcription activation domain vp with a chimeric mammalian/insect rxr ligand binding domain. the transcription unit of interest is cloned downstream of five gal response elements in a separate expression plasmid. instead of ecdysone, a non-steroidal diphenylhydrazine compound, rsl acts as a specific ligand that stabilizes the nuclear receptor heterodimer and activates transcription of the cloned gene of interest. this combination of chimeric receptors with a non-steroidal synthetic ligand was designed to ensure that the expression system will not interfere with endogenous cellular pathways. rsl has shown minimal effects on endogenous gene expression and cell proliferation in prostate cells and in hek cells. in addition to these systems, rheoswitch has been used to induce expression of proteins in mice, mbt- and panc carcinoma cells and nih3t cells. however, no gene silencing studies using this system have yet been published. in this study, we used rheoswitch to produce rnas that are processed in vivo in mammalian cultured cells to generate mirnas that are functional in target gene knockdown.  inducible expression of mir-122 we chose mir- an abundantly expressed mirna, as a model for inducible shrna expression. mir- is expressed exclusively in the liver and plays a key role in the regulation of cholesterol and fatty acid metabolism in the adult liver. in hepatocarcinoma of humans and rodents, mir- has been reported to be specifically down-regulated to a significant degree. hepatoma cell lines expressing mir- such as huh- are required for the propagation and study of hepatitis c virus. hepg which does not express detectable mir- is resistant to hcv infection. recently, the first mirna-based drug has been shown to be protective against hcv infection in primates. these properties of mir- have made it a highly studied mirna and a prime target for therapeutics development. to determine whether the rheoswitch expression system could be used to induce expression of short hairpin rnas, a bp human genomic dna fragment containing mir- and its flanking dna sequence was cloned downstream of the gal binding sites in the rheoswitch expression vector pnebr-x transient transfection of this plasmid into human embryonic kidney cells stably expressing rheoreceptor and rheoactivator demonstrated production of mature mir- upon induction with rsl. based on these results and additional transient transfection experiments, we constructed a cell line that carried integrated mir- expression vector in mouse embryo fibroblasts, which also stably express the rheoswitch proteins. we used this cell line to investigate the properties of induced mir- expression. first, we confirmed that rsl treatment could induce the cells to produce mature mir- mirna. northern blot analysis demonstrated that rsl1-treated cells produced the nt guide strand, whereas the passenger strand was undetectable, indicating that the mir- primary transcript is induced and correctly processed. in the absence of rsl cells did not produce any detectable passenger or guide strand. induction of the cells for hours using different concentrations of rsl showed that the amount of accumulating mature mir- could be modulated by the concentration of the inducer. one advantage of small molecule ligands such as rsl is the ability to diffuse into cells to rapidly induce, or turn off expression, in a dose-dependent manner. to study these features for mirna expression, an examination of the induction time course and on-off switching was performed. mir- guide strand was detectable hours after induction, its level peaked at about hours post-induction and remained at that level after hours, indicating rapid induction and steady levels of mirna production. these results demonstrate that, in principle, the nih3t3-47/mir- cell line can become mir- positive or negative, depending on the presence or absence of rsl in the culture medium, mimicking huh- or hepg hepatic cells respectively in terms of mir- expression status. we studied long-term expression and switching properties by maintaining the cells under non-induced or continuous induction conditions for several days. non-induced cells showed no detectable mir- accumulation after days in culture, while induced cells expressed mir- for at least days, demonstrating tight control of the off state. next, we tested the reversibility of the switch. cells were treated with rsl for hours, rsl was then withdrawn. mir- expression was reduced over the course of the next days, reaching non-induced levels by day. a second induction at day can restore expression to the fully induced levels. these results demonstrate that induced expression of mirna using this system is sustainable and reversible, allowing control of expression of the cloned mirna. inducible and regulated silencing since this system proved suitable for controlled expression of mir- we tested whether the induced mirna expression can be used in turn for controlled target gene silencing. first, we designed a reporter-based assay using the secreted gaussia luciferase. we had previously used a gluc-based reporter for assessing sirna potency. two tandem copies of a sequence complementary to the mir- guide strand were inserted into the 3' utr of a gluc reporter. the mir- guide strand should work, in this instance, like an sirna because it perfectly matches its target in the 3' utr of the gluc mrna. we transfected ptk-gluc-mir or control ptk-gluc reporter into the nih3t3-47/mir- cells and measured the secreted luciferase activity after different treatments. gluc expression was unaffected in non-induced cells, but was substantially reduced following induction of mir- expression. consistent with the reversible mir- expression, gluc expression was restored following withdrawal of rsl, demonstrating reversible knock down of the target. knockdown of gluc expression was target-specific since the control reporter lacking the mir- target sites was unaffected by any treatment. since the secreted luciferase assay is non-destructive, we used the same cells to correlate the expression status of the mir- guide strand detected by northern hybridization. consistent with the luciferase expression level, mir- was detected only under inducing conditions. following rsl withdrawal, mir- declined over the next hours. since this system showed regulation of mirna output by varying the concentration of the inducer, we tested whether the downstream effects of mir- expression were also rsl concentration-dependent. when expression of the ptk-gluc-mir reporter was assayed after treatment with increasing concentrations of rsl a corresponding decrease in gaussia luciferase activity was observed, whereas the activity from cells transfected with the control vector produced normal amounts of luciferase. western blot analysis confirmed that the loss of measured luciferase activity reflects the decreased gluc protein levels as a result of mirna targeting. these results demonstrate that mir- short hairpin-mediated target silencing can be controlled in an rsl dose-dependent manner. induced silencing of mir- target genes we demonstrated that induced expression of the guide strand of mir- acting as an sirna, can silence an artificial reporter gene with a perfectly matched target sequence present in its 3' utr. to test whether the system could be used for "natural" mirna target validation we attempted to recapitulate the silencing activity of mir- through its interaction with the 3'-utr of previously identified target genes such as glycogen synthase. the 3' utr of gys was cloned downstream of gluc in the ptk-gluc reporter vector and the resulting construct was transfected into the mir- expressing stable cell line nih3t3-47/mir- upon rsl induction, gaussia luciferase activity from cells transfected with the gys reporter was reduced to 76% of control expression. this knockdown is target sequence-specific since luciferase activity from cells transfected with ptk-gluc control plasmid with an unrelated utr was unaffected by mir- induction. a similar reduction was observed using gluc reporter assays for another mir- target, cat. in order to further test this mirna target validation methodology with an endogenous gene target, we tested the effect of induced mir- expression on aldolase a, a validated mir- target in mouse liver,. we confirmed by western blot analysis that aldolase a is expressed in uninduced nih3t3-47/mir- cells. we monitored aldolase-a protein levels by immunoblot over the course of mir- induction. in the presence of rsl nih3t3-47/mir- cells show a gradual reduction of aldolase a protein to 75% of control in days and 31% of control levels after days compared to time-matched non-induced nih3t3-47/mir- control cells. thus we achieved modulation of aldolase-a protein expression with small molecule induction of ectopic mir- expression. these results suggest that mirna target gene validation and phenotypic analysis can be easily obtained using this inducible mirna system. expression of inducible shrna to test whether artificial short hairpins could be expressed and properly processed to produce a designed guide strand, different short hairpin configurations carrying the same inserted guide strand sequence were cloned in the rheoswitch expression vector pnebrx. the sequence and structure of the short hairpins in pnebrx-sh- and pnebrx-sh- were modeled on mir- as previously described. in pnebrx-sh- the short hairpin sequence is cloned directly into the mcs of the vector. the hairpin sequence in pnebrx-sh- is the same as pnebrx-sh- but it is inserted in the place of mir- in the bp genomic dna fragment used above for mir expression. pnebrx-sh- uses the structure of the mir- short hairpin but it contains the same guide strand as sh- and sh- compensatory changes were made in the stem sequence in order to maintain a mir-122-like structure. plasmids encoding these short hairpins were transfected into nih3t3- cells, expression was induced by rsl and northern blot analysis using guide strand-specific probes was used to evaluate hairpin processing. sh- produced little rna of the expected size, perhaps because the stem-loop structure was not conducive to optimal processing. sh- produced more, suggesting that processing is more efficient if the short hairpin is surrounded by mir- genomic sequences. sh- which most closely mimics mir- produced the most mature guide strand. these results suggest that the mir- stem loop structure more readily accommodates guide strand sequence variants than the mir- stem loop structure. inducible sirna production for controlled target knock down one hurdle in using an inducible shrna expression systems is to reliably convert a desired sirna into an inducible hairpin that can silence target genes. this process is not always straightforward. we tested whether the mir- backbone in the rheoswitch expression vector could be used as a platform for inducible expression of a shrna with a desired sequence for rnai silencing. to test the system, we chose a previously described sirna directed against a nt sequence of firefly luciferase. based on our previous results, we replaced the nt guide strand of mir- in pnebrx-mir- with the fluc sirna guide strand in a structure similar to sh-. because the published guide strand sequence was nt long, four nucleotides shorter than the mir- guide strand, we added four nucleotides to the 3' end of the fluc guide strand in order to maintain the structure of the mir- short hairpin. the mir- stem contains a bulge, so we designed one short hairpin, fluc-shm, with an internal mismatch in order to mimic the mir- structure. a second fluc short hairpin, fluc-sh was designed with a perfectly complementary stem structure. additionally, based on our northern blot results with sh-, we tested whether surrounding the fluc short hairpin with mir- genomic flanking sequence has an effect on its silencing properties. therefore, we inserted both the mismatched and perfectly complementary fluc short hairpins into the rheoswitch expression vector either surrounded by genomic sequences or directly cloned into the mcs of the vector. the fluc short hairpin plasmids or an equivalent mir- short hairpin plasmid were co-transfected with an fluc reporter plasmid into rheoswitch cells and compared for induced knockdown of firefly luciferase. all of the fluc short hairpin designs were effective in reducing luciferase activity upon rsl induction while the control mir- had no effect on the luciferase reporter activity. the stem-mismatched short hairpins, which more closely mimic the mir- structure, were slightly more effective in knocking down the fluc reporter than the perfectly matched hairpin designs. the surrounding dna context had no significant effect on the knockdown obtained by either fluc short hairpin, i.e., short hairpins flanked by genomic sequences were neither more nor less effective than those flanked by the mcs of the vector. taken together these results provide guidance in designing shrna expression constructs. short hairpins based on the mir- stem loop structure can produce more mirna guide strand than short hairpins based on the mir- structure. if the short hairpin was not readily processed, as was the case with the mir-30-like sh- addition of flanking sequence increased the processing efficiency. neither sh- nor sh- produced as much mirna as the mir- short hairpin or the mir-122-like sh- it seems probable that sh- produced more mature mirna than sh- and sh- because it is a variant of the mir- short hairpin, rather than because of the genomic dna that flanks it. the designs with the bulge in the stem, mimicking the structure of mir- were slightly more effective in target knockdown than those perfectly complementary. the target knockdown results support the conclusion that in determining the processing efficiency and silencing effectiveness in the structure of the short hairpin is more important than the sequence context in which it is transcribed, and the mir- stem-loop structure is a favorable vehicle for short hairpin expression.  we have shown that in addition to controlled protein expression, the rheoswitch ligand-inducible system allows regulated expression of short hairpin rna. by varying the dose of rsl the rna expression level can be modulated, and upon rsl withdrawal, expression is turned off. this feature is important when studying biological phenomena resulting from down-regulation, but not elimination, of gene function. expression can be turned on and off repeatedly, allowing additional control for studying a range of experimental states, an advantage when studying essential genes. short hairpins can be expressed and the rna processed to yield mirna or sirna. the expressed mirnas function as expected in target knockdown using endogenous targets, such as aldolase-a, or reporter-3' utr targets, facilitating mirna target validation assays. we explored whether novel guide strand sequences, such as those based on an sirna, can be incorporated into the mir- short hairpin, expressed and processed to yield functional small rnas. our experiments suggest that the mir- backbone can be adapted for inducible sirna expression. it has been shown that multiple shrnas, directed at multiple targets, can be expressed from a single transcription unit. the rheoswitch system accommodates long transcription units, unlike pol iii systems that require short transcripts. this suggests that it may be possible to build a rheoswitch expression vector with two or more shrnas in tandem. 
using modified nuclear receptors in a two hybrid format and a synthetic ligand, the rheoswitch system allows rapid and reversible induction of mrna expression. methods for cell delivery of sirnas include transient transfection of synthetic sirnas and expression of sirnas in the form of short hairpins using constitutive rna polymerase iii promoters. stable cell lines were obtained, resulting in heritable, consistent and reversible expression of mir- a significant advantage over transient transfection. based on these results, obtained with a microrna we adapted the method to produce a desired sirna by designing short hairpins that can be accurately and efficiently processed. a short hairpin encoding mir- cloned into the expression vector was correctly processed to yield mature mirna upon induction with ligand and the amount of mirna produced was commensurate with the concentration of ligand. we present an inducible mammalian expression system that allows for the conditional expression of short hairpin rnas that are processed in vivo to generate mirnas or sirnas. to study the biological function of mirnas, and to achieve sustained or conditional gene silencing with sirnas, systems that allow controlled expression of these small rnas are desirable.
 to study the biological function of mirnas, and to achieve sustained or conditional gene silencing with sirnas, systems that allow controlled expression of these small rnas are desirable. methods for cell delivery of sirnas include transient transfection of synthetic sirnas and expression of sirnas in the form of short hairpins using constitutive rna polymerase iii promoters. systems employing constitutive rna polymerase ii promoters have been used to express mirnas. however, for many experimental systems these methods do not offer sufficient control over expression.  we present an inducible mammalian expression system that allows for the conditional expression of short hairpin rnas that are processed in vivo to generate mirnas or sirnas. using modified nuclear receptors in a two hybrid format and a synthetic ligand, the rheoswitch system allows rapid and reversible induction of mrna expression. we evaluated the system's properties using mir- as a model mirna. a short hairpin encoding mir- cloned into the expression vector was correctly processed to yield mature mirna upon induction with ligand and the amount of mirna produced was commensurate with the concentration of ligand. mir- produced in this way was capable of silencing both endogenous target genes and appropriately designed reporter genes. stable cell lines were obtained, resulting in heritable, consistent and reversible expression of mir- a significant advantage over transient transfection. based on these results, obtained with a microrna we adapted the method to produce a desired sirna by designing short hairpins that can be accurately and efficiently processed.  we established an inducible expression system with a mir- backbone that can be used for functional studies of mirnas and their targets, in heterologous cells that do not normally express the mirna. additionally we demonstrate the feasibility of using the mir- backbone to express shrna with a desired sirna guide strand for inducible rnai silencing. 