RNA interference (RNAi) has been increasingly used for reverse genetics. Both pol III and pol II promoters have been used to synthesize short hairpin RNA (shRNA) for knockdown of gene expression in mammalian cells and animals. Compared with gene knockout approaches, RNAi has the advantage of being simple, quick, and low cost. Here we describe a method that enhances this advantage where knockdown of expression of multiple genes in the same cells is required. A tetracycline-regulated pol II promoter construct allows the expression of up to three shRNA genes that have been cloned into introns of a transcript bearing green fluorescent protein (GFP) coding sequences. This method may be used to establish stable knockdown cell lines and may also prove useful for investigating gene-gene interactions in transgenic animals.
RNA interference (RNAi) has become a powerful tool in reverse genetics. Sustained RNAi can be achieved with the application of short hairpin RNA (shRNA) synthesized by either an RNA polymerase III (pol III) or an RNA polymerase II (pol II) promoter (1). Pol III promoters have been widely used to synthesize shRNA in culture and in viral vectors. However, they lack temporal and spatial control in vivo, and their successful use in transgenic animals has been limited (2). This contrasts with a wide array of pol II promoters that confer properties ranging from ubiquitous expression to cell- and time-specific expression in transgenic animals. Pol II is also the endogenous promoter for expression of microRNA (miRNA) (3,4,5) and thus may mimic the action of miRNA when it is used to express shRNA in vivo. Furthermore, pol II can express multiple endogenous miRNA genes in the same transcript (5,6). In some circumstances, expression of multiple shRNAs that are capable of knocking down multiple genes simultaneously can accelerate investigations of gene interactions. We combined these advantages in a pol II-shRNA system and built an inducible construct that can inducibly express multiple shRNAs and silence expression of multiple genes.Materials and Methods Plasmid Construction
Constructs that express enhanced green fluorescent protein (EGFP) driven by human ubiquitin C (UbC) promoter have been described previously (7). For construction of tetracy-cline-regulatable UbC promoters, the tetracycline-responsive element (TRE) was inserted into two sites: one within the promoter region and the other in the first noncoding exon of the human UbC gene. To insert the TRE in the promoter, a restriction site for XhoI was introduced at a position 5 nucleo-tides (nt) downstream of the TATA box by changing the sequence 5′-…TATATAAGGACGCGCCGG…-3′ to 5′-…TATATAAGGACGCtCgaG…-3′. To insert the TRE in the first exon, an XhoI site was created at a position 26 nt downstream from the transcription start site by changing the sequence 5′-…GGGTCGCGGTTCTT…-3′ to 5′-…GGGTCtCGagTCTT…-3′. One or two TRE sites (5′-TCCCTATCAGTGATAGAGA-3′) were then inserted at the XhoI site (Figure 1A). The insertions destroyed the XhoI sites.
The self-regulatable UbC vector (Figure 2A) was built in two steps (1). The EGFP coding sequence of the UbC-EGFP vector was replaced with the coding sequence for a tetracy-cline-controlled transcriptional silencer (tTS) that was PCR-cloned from pTeT-tTS vector (Clontech Laboratories, Mountain View, CA, USA). Two restriction sites for XhoI and NotI were included in the downstream primer and introduced after the insertion (2). The newly constructed vector (UbC-tTS) was sequentially digested with restriction enzymes XhoI and NotI. The internal ribosome entry site (IRES) of the encephalomyocarditis virus and the EGFP coding region were released from the pIRES2-EGFP vector (Clontech Laboratories) by digestion with XhoI and NotI and then ligated into the UbC-tTS to generate the UbC-tTS-IRES-EGFP (Figure 2A).
The constructs for simultaneous expression of multiple shRNAs were constructed from UbC-TRE4 (Figure 1) in the following steps. The fifth intron of the human actin gene with a 5-nt flanking sequence at each end was chemically synthesized and inserted at the EcoRV site, which is 10 nt from the 3′ end of the first intron of the human ubiquitin C gene. The EcoRV site was destroyed by this insertion. The fourth intron of human actin gene with a 5-nt flanking sequence was chemically synthesized and inserted at the XhoI site (inactivated by insertion) downstream of the EGFP coding region. The individual hairpins were chemically synthesized and inserted sequentially into the EcoRI site within the first intron of the human ubiquitin C gene, the PstI site (introduced during the chemical synthesis) in the fifth intron of the human actin gene, and the XhoI site (also introduced during the chemical synthesis) inside the fourth intron of the human actin gene (Figure 3A). Each inserted hairpin was verified by sequencing. The hairpin targeting sequences were: human sod1 gene, 5′-GCCGAUGUGUCUAUUGAAG AU-3′; mouse Sod2 gene, 5′-GCCA AGGGAGAUGUUACAACU-3′; and mouse Dj-1 gene, 5′-GCAGU GUAGCCGUGAUGUAAU-3′.