Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells

Abstract

RNA interference (RNAi) was first recognized in Caenorhabditis elegans as a biological response to exogenous double-stranded RNA (dsRNA), which induces sequence-specific gene silencing. RNAi represents a conserved regulatory motif, which is present in a wide range of eukaryotic organisms. Recently, we and others have shown that endogenously encoded triggers of gene silencing act through elements of the RNAi machinery to regulate the expression of protein-coding genes. These small temporal RNAs (stRNAs) are transcribed as short hairpin precursors (approximately 70 nt), processed into active, 21-nt RNAs by Dicer, and recognize target mRNAs via base-pairing interactions. Here, we show that short hairpin RNAs (shRNAs) can be engineered to suppress the expression of desired genes in cultured Drosophila and mammalian cells. shRNAs can be synthesized exogenously or can be transcribed from RNA polymerase III promoters in vivo, thus permitting the construction of continuous cell lines or transgenic animals in which RNAi enforces stable and heritable gene silencing.

Figure 1

Short hairpins suppress gene expression in Drosophila S2 cells. (A) Sequences and predicted secondary structure of representative chemically synthesized RNAs. Sequences correspond to positions 112–134 (siRNA) and 463–491 (shRNAs) of Firefly luciferase carried on pGL3-Control. An siRNA targeted to position 463–485 of the luciferase sequence was virtually identical to the 112–134 siRNA in suppressing expression, but is not shown. (B) Exogenously supplied short hairpins suppress expression of the targeted Firefly luciferase gene in vivo. Six-well plates of S2 cells were transfected with 250 ng/well of plasmids that direct the expression of firefly and Renilla luciferase and 500 ng/well of the indicated RNA. Luciferase activities were assayed 48 h after transfection. Ratios of firefly to Renilla luciferase activity were normalized to a control transfected with an siRNA directed at the green fluorescent protein (GFP). The average of three independent experiments is shown; error bars indicate standard deviation. (C) Short hairpins are processed by the Drosophila Dicer enzyme. T7 transcribed hairpins shFfL22, shFfL29, and shFfS29 were incubated with (+) and without (−) 0–2-h Drosophila embryo extracts. Those incubated with extract produced ∼22-nt siRNAs, consistent with the ability of these hairpins to induce RNA interference. A long dsRNA input (cyclin E 500-mer) was used as a control. Cleavage reactions were performed as described in Bernstein et al. (2001a).

Figure 2

Short hairpins function in mammalian cells. HEK 293T, HeLa, COS-1, and NIH 3T3 cells were transfected with plasmids and RNAs as in Figure 1 and subjected to dual luciferase assays 48 h posttransfection. The ratios of firefly to Renilla luciferase activity are normalized to a control transfected with an siRNA directed at the green fluorescent protein (GFP). The average of three independent experiments is shown; error bars indicate standard deviation.

Figure 3

siRNAs and short hairpins transcribed in vitro suppress gene expression in mammalian cells. (A) Sequences and predicted secondary structure of representative in vitro transcribed siRNAs. Sequences correspond to positions 112–134 (siRNA) and 463–491 (shRNAs) of firefly luciferase carried on pGL3-Control. (B) In vitro transcribed siRNAs suppress expression of the targeted firefly luciferase gene in vivo. HEK 293T cells were transfected with plasmids as in Figure 2. The presence of non-base-paired guanosine residues at the 5′ end of siRNAs significantly alters the predicted end structure and abolishes siRNA activity. (C) Sequences and predicted secondary structure of representative in vitro transcribed shRNAs. Sequences correspond to positions 112–141 of firefly luciferase carried on pGL3-Control. (D) Short hairpins transcribed in vitro suppress expression of the targeted firefly luciferase gene in vivo. HEK 293T cells were transfected with plasmids as in Figure 2.

Figure 3

siRNAs and short hairpins transcribed in vitro suppress gene expression in mammalian cells. (A) Sequences and predicted secondary structure of representative in vitro transcribed siRNAs. Sequences correspond to positions 112–134 (siRNA) and 463–491 (shRNAs) of firefly luciferase carried on pGL3-Control. (B) In vitro transcribed siRNAs suppress expression of the targeted firefly luciferase gene in vivo. HEK 293T cells were transfected with plasmids as in Figure 2. The presence of non-base-paired guanosine residues at the 5′ end of siRNAs significantly alters the predicted end structure and abolishes siRNA activity. (C) Sequences and predicted secondary structure of representative in vitro transcribed shRNAs. Sequences correspond to positions 112–141 of firefly luciferase carried on pGL3-Control. (D) Short hairpins transcribed in vitro suppress expression of the targeted firefly luciferase gene in vivo. HEK 293T cells were transfected with plasmids as in Figure 2.

Figure 3

siRNAs and short hairpins transcribed in vitro suppress gene expression in mammalian cells. (A) Sequences and predicted secondary structure of representative in vitro transcribed siRNAs. Sequences correspond to positions 112–134 (siRNA) and 463–491 (shRNAs) of firefly luciferase carried on pGL3-Control. (B) In vitro transcribed siRNAs suppress expression of the targeted firefly luciferase gene in vivo. HEK 293T cells were transfected with plasmids as in Figure 2. The presence of non-base-paired guanosine residues at the 5′ end of siRNAs significantly alters the predicted end structure and abolishes siRNA activity. (C) Sequences and predicted secondary structure of representative in vitro transcribed shRNAs. Sequences correspond to positions 112–141 of firefly luciferase carried on pGL3-Control. (D) Short hairpins transcribed in vitro suppress expression of the targeted firefly luciferase gene in vivo. HEK 293T cells were transfected with plasmids as in Figure 2.

Figure 4

Transcription of functional shRNAs in vivo. (A) Schematic of the pShh1 vector. Sequences encoding shRNAs with between 19 and 29 bases of homology to the targeted gene are synthesized as 60–75-bp double-stranded DNA oligonucleotides and ligated into an EcoRV site immediately downstream of the U6 promoter. (B) Sequence and predicted secondary structure of the Ff1 hairpin. (C) An shRNA expressed from the pShh1 vector suppresses luciferase expression in mammalian cells. HEK 293T, HeLa, COS-1, and NIH 3T3 cells were transfected with reporter plasmids as in Figure 1, and pShh1 vector, firefly siRNA, or pShh1 firefly shRNA constructs as indicated. The ratios of firefly to Renilla luciferase activity were determined 48 h after transfection and represent the average of three independent experiments; error bars indicate standard deviation.

Figure 4

Transcription of functional shRNAs in vivo. (A) Schematic of the pShh1 vector. Sequences encoding shRNAs with between 19 and 29 bases of homology to the targeted gene are synthesized as 60–75-bp double-stranded DNA oligonucleotides and ligated into an EcoRV site immediately downstream of the U6 promoter. (B) Sequence and predicted secondary structure of the Ff1 hairpin. (C) An shRNA expressed from the pShh1 vector suppresses luciferase expression in mammalian cells. HEK 293T, HeLa, COS-1, and NIH 3T3 cells were transfected with reporter plasmids as in Figure 1, and pShh1 vector, firefly siRNA, or pShh1 firefly shRNA constructs as indicated. The ratios of firefly to Renilla luciferase activity were determined 48 h after transfection and represent the average of three independent experiments; error bars indicate standard deviation.

Figure 5

Dicer is required for shRNA-mediated gene silencing. HEK 293T cells were transfected with luciferase reporter plasmids as well as pShh1-Ff1 and an siRNA targeting human Dicer either alone or in combination, as indicated. The Dicer siRNA sequence (TCA ACC AGC CAC TGC TGG A) corresponds to coordinates 3137–3155 of the human Dicer sequence. The ratios of firefly to Renilla luciferase activity were determined 26 h after transfection and represent the average of three independent experiments; error bars indicate standard deviation.

Figure 6

Stable shRNA-mediated gene silencing of an endogenous gene. (A) Sequence and predicted secondary structure of the p53 hairpin. The 5′ shRNA stem contains a 27-nt sequence derived from mouse p53 (nucleotides 166–192), whereas the 3′ stem harbors the complimentary antisense sequence. (B) Senescence bypass in primary mouse embryo fibroblasts (MEFs) expressing an shRNA targeted at p53. Wild-type MEFs, passage 5, were transfected with pBabe-RasV12 with control plasmid or with p53hp (5 μg each with FuGENE; Roche). Two days after transfection, cells were trypsinized, counted, and plated at a density of 1 × 10⁵/10-cm plate in media containing 2.0 μg/mL of puromycin. Control cells cease proliferation and show a senescent morphology (left panel). Cells expressing the p53 hairpin continue to grow (right panel). Photos were taken 14 d posttransfection.