We have designed a conditional expression construct for miRNA expression, pCAG-EGFP/RFP-miRNAint (Figure ). This construct initially expresses EGFP. When exposed to Cre recombinase, the loxP-flanked EGFP gene is excised, allowing the CAG promoter to drive the expression of the miRNA and the marker RFP. This construct will be useful for generating conditional silencing in transgenic mice and will have several advantages compared with the currently available transgenic silencing constructs. For example, the constitutive Pol III or Pol II promoter-based constructs are inefficient in generating transgenic animals with high levels of knockdown [7
]. Although, by screening for transfected ES cells with high levels of knockdown or using lentiviral delivery, transgenic mice with hypomorphic gene functions have been generated [10
], these technologies are more complex and more costly than pronuclear injection. Furthermore, it will be difficult to obtain transgenic lines with a high degree of gene silencing if one targets an essential gene, because founders with high levels of shRNA or miRNA expression cannot survive. While this problem might be partially circumvented by expressing miRNA using a tissue-specific promoter [15
], the conditional approach that we have designed here will be more versatile and a superior solution to this problem. By crossing with different Cre-expression lines, one can induce specific gene silencing in many different tissues from a single line of mice made with this conditional construct. Transgenic silencing mice can be made with conditional constructs using pronuclear injection method [2
] or using more complex methods such as ES cell screening or lentiviral delivery. In addition, compared with the inducible transgenic RNAi constructs that have been reported in the literature, our conditional construct has the following advantages: It expresses EGFP before induction of the miRNA, thus affording a convenient screen for transgenic lines with desirable expression pattern. When crossed with a Cre driver transgenic line, the coexpression of RFP and miRNA will provide a convenient and precise indication as to in which cell type gene silencing is occurring.
While developing this construct, we compared the effects of placing the pre-miRNA in an intron with the effects of placing the pre-miRNA directly in the 3' UTR of the RFP gene. Our results indicate that placing the pre-miRNA in an intron does not increase the miRNA expression (Figure ) but enhances the marker protein RFP expression (Figure ). This conclusion agrees with a previous report, where a pre-miRNA was placed in a 5' intron in the EGFP gene [45
]. Our analysis, however, further revealed that the intron enhances the RFP gene expression by two effects. First, intron can directly enhance the protein gene expression (compare bar h with bar g in Figure ). Second, the pre-miRNA sequence placed in the 3'-UTR exerts an inhibitory effect on the protein gene expression, and this effect can be removed with the elimination of the pre-miRNA sequence by the intron splicing (Figure ). These findings justify the inclusion of the intron in our conditional miRNA expression construct.
How the pre-miRNA placed in the 3'-UTR inhibits the RFP gene expression and why intron placement of the pre-miRNA eliminates this inhibition remain unknown. One model, as has been proposed [45
], suggests that cropping of the pre-miRNA from the 3'-UTR by Drosha and DGCR8 complex led to a cleavage in the pri-miRNA, resulting in a separation between the 5'-Cap and 3'-poly A and the degradation of the residue mRNA, thereby reducing the RFP expression. By placing the pre-miRNA in an intron, the cropping of the pre-miRNA might occur in the spliced intron lariat. Alternatively, the cropping of pre-miRNA might occur before splicing, but this might not affect splicing [46
]. In either case, the integrity of the mRNA will be protected, thus enhancing the RFP expression.
Changes in the RFP expression are consistent with the above models. When the pre-miRNA was present in the 3'-UTR, the RFP expression was decreased (compare bars a, c and e with bar g in Figure ) and this decrease was prevented by inhibition of Drosha (Figure ); when the pre-miRNA was placed in the intron, the RFP expression was enhanced (compare bars a, c and e with bars b, d, and f, respectively) and inhibition of Drosha did not affect the RFP expression (Figure ). However, at the mRNA level, the presence of pre-miRNA in the 3'-UTR caused only a slight decrease (compare lanes 1, 5 and 7 with lane 2 in Figure ); and placing the pre-miRNA in an intron caused a relatively small increase (compare lanes 1, 5 and 7 with lanes 4, 6 and 8). These changes are not sufficient to account for the changes in the RFP expression, thus suggesting that protection of mRNA from Drosha/DGCR8 processing is not the only mechanism whereby the intron placement of the pre-miRNA increases the RFP expression. From our data, it is clear that most of the enhancing effect of RFP expression is derived from the intrinsic properties of the intron, rather than protecting the mRNA from Drosha/DGCR8 cropping.
Our data also show that the presence of a miRNA in an intron does not interfere with intron splicing because the level of mature mRNA produced from the construct with intron alone was not higher than those from the constructs with intron containing pre-miRNA (Figure , compare lane 3 with lanes 4, 6 and 8). Conversely, intron containing the pre-miRNA does not affect the miRNA processing because constructs with or without an intron produced similar levels of miRNA (Figure ). Thus, intron splicing and miRNA processing do not interfere with each other in a miRNA-containing intron. This conclusion agrees with a previous study [46
]. However, our data also revealed that some pre-miRNA can dampen the intron-enhanced RFP expression (e.g. compare bar b with bar h in Figure ), possibly by interfering with the translation-enhancing effect of the intron. This observation indicates that the levels of the reporter gene expression cannot be used to compare the expression levels of different miRNA because the miRNAs may affect the reporter gene expression differently. Nevertheless, the reporter expression levels may be used as an indicator of the same miRNA expression levels in different cell populations, provided that the miRNA processing machinery is not saturated.
Based on our analysis, several improvements in our construct design is worth noting. First, we use a single promoter to drive both the reporter and the miRNA expression. This is advantageous compared with a commonly used design in Pol III-driven shRNA constructs, which places a reporter expression cassette on the same linear sequence with the shRNA expression cassette [12
]. Because the synthesis of the reporter and the shRNA are driven by independent promoters, the reporter expression does not necessarily represent the shRNA expression and the degree of silencing. This is especially worrisome in transgenic animals, where a genomic insertion locus may influence different promoters differently. Our construct will not have this problem because the expression of the reporter and the miRNA is driven by a single promoter. Second, the intron-placement of pre-miRNA enhances the reporter expression, which improves from constructs that place the miRNA directly in the 3' UTR of the reporter [22
] (Figure ). Third, our placement of the intron in the 3' UTR makes our construct easily adaptable if one desires to use other reporters. The RFP in our construct can be easily replaced by any other commonly used reporter. This is advantageous compared with the construct using an in-frame intron within the open reading frame [25
], where the reporter cannot be easily replaced.