A versatile approach to multiple gene RNA interference using microRNA-based short hairpin RNAs
© Zhu et al; licensee BioMed Central Ltd. 2007
Received: 01 June 2007
Accepted: 30 October 2007
Published: 30 October 2007
Effective and stable knockdown of multiple gene targets by RNA interference is often necessary to overcome isoform redundancy, but it remains a technical challenge when working with intractable cell systems.
We have developed a flexible platform using RNA polymerase II promoter-driven expression of microRNA-like short hairpin RNAs which permits robust depletion of multiple target genes from a single transcript. Recombination-based subcloning permits expression of multi-shRNA transcripts from a comprehensive range of plasmid or viral vectors. Retroviral delivery of transcripts targeting isoforms of cAMP-dependent protein kinase in the RAW264.7 murine macrophage cell line emphasizes the utility of this approach and provides insight to cAMP-dependent transcription.
We demonstrate functional consequences of depleting multiple endogenous target genes using miR-shRNAs, and highlight the versatility of the described vector platform for multiple target gene knockdown in mammalian cells.
The discovery of RNA interference (RNAi) and its use as an experimental tool has heralded a new era in functional genomics [1, 2]. The ability of short interfering RNA (siRNA) to perturb expression of any gene target highlights the enormous potential of this technique. However, siRNA delivery into primary cells, cell lines that are difficult to transfect and delivery to specific cell types in vivo remains a key technical issue.
We report the development of a versatile system which permits the knockdown of multiple target genes from a single transcript, and we show efficient depletion of endogenous pairs of signaling genes in the RAW264.7 murine macrophage-like cell line. Importantly, we demonstrate the absolute requirement for multi-gene depletion to observe phenotypes in processes dependent on proteins with redundant isoforms.
Results and discussion
Using our multi-miR-shRNA expression platform, we demonstrate potent knockdown of up to three genes in transient expression studies (Fig. 1d–f) and three different pairs of endogenous genes after viral infection of a murine macrophage cell line (Fig. 3b–e). This approach has great potential for experimental strategies or clinical applications requiring depletion of multiple gene targets in cells with low transduction efficiency. It should be noted however, that the presence of increasing numbers of miR-shRNA cassettes in a viral transcript leads to a gradual reduction in viral titer (see Additional file 2), likely due to Drosha cleavage of the viral transcript during packaging. Despite this titer reduction, we were able to achieve viral titers of >1 × 106 pfu/ml from unconcentrated supernatants of lentivirus expressing three miR-shRNAs, well within the practical range for infection of most intractable cells.
We have developed an flexible cloning platform for generation of plasmid vectors and viruses expressing miR-shRNAs against multiple target genes. The ability to express multiple engineered miR-shRNA cassettes from a single transcript has been previously reported [5, 11], and recent reports have shown that it can also be used to improve knockdown efficiency by expressing multiple miR-shRNA against the same target gene [22, 23] and to promote multi-gene knockdown [22–24]. However, Refs.22 and 24 used intron-based expression of miR-shRNA cassettes from plasmids introduced into easily transfected cell lines, which is not compatible with the viral based approaches required with less tractable cell systems. To our knowledge, our data are the first to demonstrate functional consequences of depleting multiple endogenous target genes in mammalian cells using miR-shRNA where multi-gene knockdown is necessary to observe a phenotype (Fig. 4). We believe that the versatility of the vectors we describe here make them a valuable resource to the research community. Although we clone shRNAs into our entry vectors using BfuAI compatible linkers, we include Xho I and Eco RI cloning sites in the flanking miR30 sequence to allow subcloning of miR-shRNAs from popular whole genome libraries [2, 7] into our plasmids (Fig. 2b). Any shRNA subcloned by either of these methods is then compatible for concatenation in a multi-miR-shRNA transcript (Fig. 1c).
Availability and requirements
In accordance with the policy of the Alliance for Cellular Signaling (AfCS), all the vectors described in this study will be readily available through the American Type Culture Collection. Details will be provided at http://www.signaling-gateway.org/data/plasmid/.
8-Br-cAMP and 8-pCPT-cAMP were purchased from Calbiochem.
Entry vectors for cloning miR-shRNA downstream of various promoters were created by excision of the U6 promoter from the pEN_hUmiRc2 plasmid  by SalI digest and ligation of SalI-flanked PCR products of the β-actin, CMV, EF1, MSCV LTR and Ubi-c promoters. Cloning of gene specific miR-shRNA into these entry vectors was by generation of BfuAI-compatible linkers as described previously . For the lentiviral destination vectors, the Ubi-c promoter and GFP cDNA were removed from the pDSL_hpUG vector (ATCC# 10326371, AfCS# L06DDLHPUGXA) and replaced with cassettes including an IRES sequence followed by either GFP, truncated human CD4, neomycin, hygromycin, puromycin or zeocin. The pDS_FBneo retroviral destination vector was created by insertion of an attR site containing Gateway cassette (Invitrogen) into the multiple cloning site of the pFB-Neo plasmid (Stratagene). For the FBneo vectors containing fluorescent proteins, the attR cassette and fluorescent protein were subcloned as a single fragment into pFB-Neo from the mammalian expression plasmids described below. The FBhyg retroviral vectors were prepared in the same fashion after excision of the neomycin gene from pFB-Neo by ClaI/RsrII digest and insertion of the hygromycin resistance cDNA. The CMV promoter-containing destination vectors for mammalian co-expression of miR-shRNA with GFP derivatives were created by insertion of an attR site containing Gateway cassette (Invitrogen) into the multiple cloning site of the pEGFP-N1, pECFP-N1 and pEYFP-N1 vectors (Clontech). The pDS_X-mCherry vector was created in the same fashion from pcDNA-mCherry (derived from pRSET-B-mCherry, generously provided by Dr. Roger Tsien, University of California San Diego). The EF1 promoter versions of the GFP, CFP and YFP constructs were created in the same way after replacement of the CMV promoter by EF1 in the initial plasmids. The retroviral and lentiviral constructs containing various miR-shRNA (Additional file 3) were all created by LR recombination as described below.
Cloning and validation of miR-shRNAs
Design and cloning of miR-shRNAs has been described previously . Briefly, sequences were selected using the RNAi Codex algorithm , with 2–4 independent miR-shRNAs chosen for each target gene. Gene-specific sequences were cloned as BfuAI-compatible oligonucleotide linkers into the pEN_CmiRc2 vector (Additional files 1 and 3). The efficacy of the miR-shRNAs was validated by co-expression with YFP fusions of their cognate target cDNAs in HEK293 cells as previously described . The YFP fusion constructs used for these experiments were obtained from the recently described AfCS plasmid collection ; pEX_EF1_YFP-Arrestin2; AfCS barcode A08XF063A1TK; atcc id 10374002, pEX_EF1_YFP-Grk2; AfCS barcode A08XK093A1TK; atcc id 9830392, pEX_EF1_YFP-Grk5; AfCS barcode A08XK213A1TK; atcc id 9891141, pEX_EF1_YFP-Gbeta2; AfCS barcode A08XF075A1TK; atcc id 9891160, pEX_EF1_YFP-PKACa; AfCS barcode A08XP020A1TK; atcc id 9891081 and pEX_EF1_YFP-PKACb; AfCS barcode A08XK074A1TK; atcc id 9830324. The most potent miR-shRNA identified by this method targeted the following murine sequences: arrestin 2; TCTCATAGAGCTTGACACCAAT, arrestin 3; TGCGGCTTATCATCAGAAAGGT, G-protein coupled receptor kinase 2; ACCGAGGAGAAGTGACCTTTGA, G-protein coupled receptor kinase 5; AGGCGGCAGCATCAAAGCAATT, G-protein β2; TGCTCATGTATTCCCACGACAA, Pka Cα; AGCCTATCCAGATCTATGAGAA and Pka Cβ; AAGGTTGTTAAGCTGAAGCAAA.
Creation of constructs with multiple miR-shRNA cassettes
Constructs with multiple miR-shRNA cassettes were created by ligating SpeI/PstI fragments containing the desired downstream shRNA into an XbaI/PstI cut recipient plasmid containing the desired upstream shRNA(s) (Fig. 1c). Loss of the SpeI and XbaI sites permitted cloning of additional miR-shRNA via the same approach. The resulting constructs were validated by digestion with BsrGI, which has recognition sites in the attL1 and attL2 regions.
Subcloning of miR-shRNA cassettes to different expression platforms
Constructs were designed to permit subcloning of miR-shRNA cassettes to different expression platforms by Gateway recombination. For lentiviral expression options, this involved direct LR recombination of the promoter-containing entry vector to any one of six lentiviral destination vectors with bicistronic expression of different markers or drug selection genes (Additional files 1 and 3). The destination vectors for either retroviral or mammalian plasmid expression have existing promoters, so for these platforms the promoter was removed from the entry vector prior to LR recombination. Entry vector promoters were removed either by BamHI/SpeI or DraI/SpeI digest (see Fig. 2a) and the vector backbone was religated after treatment with T4 DNA polymerase to flush the DNA termini. Promoter removal was confirmed in recombinant clones by BsrGI digest. Retroviral vectors for this study were created by LR recombination of miR-shRNA cassettes to pDS_FBneo (Additional files 1 and 3).
Generation of viruses and creation of stable RAW264.7 cell lines
Production of lentiviruses and creation of stable lentiviral RAW cells was carried out as previously described . Retrovirus was generated by transfection of the Phoenix 293T Amphotropic packaging cell line with miR-shRNA-expressing FBneo retroviral vectors. Briefly, Phoenix cells were cultured at 37°C, 5% CO2 in 100 mm tissue culture dishes (Corning) using DMEM (Gibco Invitrogen), 10% FBS (Gemini), 2 mM glutamine (Gibco Invitrogen), and 100 U/ml Penicillin with 100 μg/ml streptomycin (Gibco invitrogen). 10 μg per dish of retroviral vector diluted in Opti-MEM (Gibco Invitrogen) was transfected using Lipofectamine 2000 reagent (Invitrogen). Around 16 hours post-transfection, the medium on the Phoenix cells was replaced. Viral supernatant was harvested at 48 hours post-transfection, spun at 1200 rpm for 5 minutes and filtered through a 0.45 μm filter. Polybrene was added to a final concentration of 16 μg/ml, and equal volumes of viral supernantant were placed onto RAW264.7 cells plated in 6 well tissue culture plates (Corning) 2 hours prior to virus addition. Final polybrene concentration was 8 μg/ml. The medium on the RAW264.7 cells was replaced around 16 hours post-infection. 48 hours post-infection, RAW264.7 cells were re-seeded onto 100 mm dishes, and infected cells were selected using 500 μg/ml Geneticin (Gibco Invitrogen).
Assessment of mRNA and protein expression levels
Knockdown of endogenous target mRNA and protein in stable cell lines was assessed from samples collected at least 14 days after viral infection, with at least one assessment carried out at >21 days. Detailed procedures for assessment of mRNA by quantitative real-time PCR (qRT-PCR) and protein by western blot have been described previously . Sense and antisense amplification primers and probe primer sequences (where applicable) were: Arr2; 5'-GACTCCAGTAGACACCAATCTCAT-3'; 5'-ATCTGTTGTTGAGGTGCGGAG-3'; 5'-TexasRed-CGGTGCCGTCATCCTCTTCGTCCT-BHQ2-3', Arr3; 5'-GCCGACATTTGCCTCTTCAG-3'; 5'-CTAGGAGACACCTGGTCATCTTG-3'; 5'-TexasRed-CGCAGTACAAGTGTCCCGTGGCTC-BHQ2-3', Grk2; 5'-CCAGGAACTGTACCGCAACTT-3'; 5'-GGTCTGTTTCAGCATTGATGGTAT-3'; 5'- TexasRed-CTCTGCCACCACCTGTTGCCACCG-BHQ2-3', Grk5; 5'-ACGGTACCCTCTCACCAGAC-3'; 5'-TTTATTCGGTGGTTACAACTGGTC-3'; 5'-TexasRed-AGAAGTCAGCCTCCAGAACCGCCA-BHQ2-3', PkaCα; 5'-CTCCCACCCTCCAAACTGTC-3'; 5'-GACAGGGTCAGTTGGCTACC-3'; 5'-FAM-ACCCTCCCCAAACACCCTCCTCAC-BHQ1-3', Pka Cβ; 5'- GATGGAATGTCTTGTCAGCATGG-3'; 5'-TCGTCCAGGAGTCCTCACTG-3'; 5'-TexasRed-AGGCGCTCTGACTCACTGCTGCAT-BHQ2-3', Nr4a1; 5'-ACAGCTTGGGTGTTGATGTTCC-3'; 5'-GCCATGTGCTCCTTCAGACAG-3', Nr4a2; 5'-ACAGTTTAAAAGGCCGGAGAGG-3'; 5'-GTCATTGCCGGATTGGAATCG-3', Ctla2α; 5'-TGTGGCTTGACTGGTAAC-3'; 5'-CAGCATCATTCCTCTCATAC-3', Ctla2β; 5'-GCATTGTCTTGGGAGTCTTC-3'; 5'-ACAGTGTTGATCTTATATGAGTTC-3' and the β-actin reference; 5'-TCCATGAAATAAGTGGTTACAGGA-3'; 5'-CAGAAGCAATGCTGTCACCTT-3'; 5'-HEX-TCCCTCACCCTCCCAAAAGCCACC-BHQ1-3'. The following antisera were used; anti-YFP (BD Clontech #8371-2), anti-Pka Cα (Cell Signaling Technology, #4782), anti-Pka Cβ (Santa Cruz Biotechnology, sc-904) and Arr2 (A1CT #140; generously provided by Dr. Robert Lefkowitz, Duke University).
Phosphoprotein detection by western blot
A detailed protocol for detection of phosphoproteins in extracts from stimulated RAW cells can be found on the AfCS website  (PP00000177 and PP00000181). Following this protocol, RAW cells were stimulated for 10 min with 100 μM 8Br-cAMP or 8pCPT-cAMP, and phospho-VASP levels were assessed using an antibody from Cell Signaling Technology (#3111).
We are grateful to colleagues in the AfCS for discussions and technical advice and to Lucas Cheadle for technical assistance. We thank Linda Holloway, Jason Cooper and Unice Na at the ATCC for assistance in making the plasmids described in this study available to the research community. This work was supported by contributions from public and private sources, including the NIGMS Glue Grant Initiative (U54 GM062114). A complete listing of the Alliance for Cellular Signaling (AfCS) sponsors can be found on the AfCS website .
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