A crucial breakthrough occurred with the report that siRNAs could be expressed as shRNA from pol III promoters cloned into plasmids [4
]. The two pol III promoters most commonly used are H1 and U6 (both human and mouse). These promoters are characterized by their compact size (less than 400 bp), and by the fact that all sequences required for promoter function are upstream of the +1 transcriptional start site. Pol III promoters have ubiquitous expression and efficiently express short RNAs. Thus, they are ideally suited to express short hairpins RNAs. ShRNAs to be driven by the H1 promoter can begin with any base, but the stronger U6 promoter requires a G as its first base [12
]. In our experience the U6 promoters (especially mU6) generates slightly better knockdown effect than H1 promoter. Nevertheless, there are some indications that U6 promoters might be more toxic in some tissues (e.g. bone marrow) probably due to their robust transcription capacity. Our preferred shRNA design consists of a 19–23 nucleotide sense sequence that is identical to the target sequence in the mRNA to be downregulated, followed by a 9 bp loop [4
] and an antisense 19–23 nucleotide sequence. A stretch of 5 T’s provides a pol III transcriptional termination signal. The total length of the silencing cassette is ~350 bp. Thus, when this construct is expressed, a short 21–23 base pair hairpin is formed; the loop is digested by Dicer and the resulting siRNA triggers degradation of the mRNA target ().
Fig. 2 Scheme depicting the RNA interference pathway. A pol III promoter drives expression of the silencing cassette and produces a hairpin, which is exported into the cytoplasm, processed by Dicer and assembled into the RISC complex. Recognition of the target (more ...)
Ideally, a silencing lentiviral vector would contain both a marker gene such as EGFP or an antibiotic resistance gene and the shRNA silencing cassette. Previous experience in our lab indicates that the position and precise combination of elements in a lentiviral vector construct can have unforeseen effects both on the viral titer and the efficiency of expression of both the marker and the silencing cassette. We have designed two different versions of lentiviral silencing vectors that differ both in the position of the silencing cassette and in the cloning strategy required to construct them ().
a) 3′ LTR double shRNA cassette design. b) Gateway single shRNA cassette design.
The first design involved a lentiviral vector carrying GFP cassette as a marker gene and an additional silencing cassette inserted into a unique restriction site in the 3′LTR [13
]. Upon transduction of target cells by the lentiviral particles, reverse transcriptase generates a viral cDNA, which then stably integrates into the host genome. During this process, the 5′LTR is generated from the 3′LTR. Thus, cloning of the silencing cassette into the 3′LTR results in duplication of the cassette and doubling of the shRNA expression levels in every transduced cell (). This feature is important since following transduction of primary cells and especially live animals, single integrations can be common and might not be sufficient for robust silencing.
In a second design the silencing cassette is inserted in the middle of the viral vector genome using a gateway cloning strategy (). While this design does not result in duplication of silencing cassette it can be use to generate a CRE recombinase induced knockdown phenotype [15
The effectiveness of a particular siRNA is largely unpredictable and presumably reflects both mechanistic constraints of the RNAi pathway and accessibility of the target sequence within the tertiary structure of the target mRNA. A number of algorithms have been developed to predict effective siRNA sequences [16
] and many of them are available [17
In general, the target sequence should be 19 to 23 bases long, but lengths of up to 29 bases have been reported [19
]. Longer targets should be avoided, as longer dsRNA molecules can trigger a PKR response [20
]. Targets can be directed to 5′UTR, ORF or 3′UTR of the target mRNA as desired.
The discovery that miRNAs can act as siRNAs and vice versa [21
] had lead to development of a new type of shRNAs – miRsiRNAs. These molecules are generated by changing the stem sequence of an endogenous miRNA into a given siRNA while maintaining the extra stem sequences, loop and 5′ and 3′ sequences. Processing of miRNAs require two maturation steps, nuclear processing of pri-miRNA into pre-miRNA require recognition and cleavage of 5′ and 3 extra stem sequences by Drosha/DGCR8 complex and cytoplasmic processing of pre-miRNA into mature miRNA require recognition and cleavage of loop sequences by Dicer (reviewed by [23
]). Since mammalian miRNAs are naturally transcribe by RNA pol II promoters, any tissue specific and drug-regulated pol II promoter can transcribe miRsiRNAs [24
]. Many miRNAs are initially expressed as a poly cistronic transcript containing several individual miRNAs and in some cases an additional protein-coding region. This finding enables the expression of several miRsiRNAs and a marker gene under one pol II promoter [25
]. This is an extremely desirable attribute since it allowes the combination of regulated expression with complex silencing of several genes driven by one promoter. Nevertheless, RNA pol II promoters are significantly weaker than pol III promoters and achieving sufficient knockdown is more demanding in compare to the robust transcription of shRNA by pol III promoter.