How do you make shRNA-expressing viruses for function screening?

Most people use standard cloning procedures when trying to insert shRNA templates into lentiviral vectors, i.e. anneal a pair of long oligos with sticky ends and ligate the dsDNA into a linearized plasmid with compatible overhangs. However, since typical lentiviral vector plasmids have terminal repeats and are relatively large, when ligated to hairpin sequence-containing shRNA templates, recombination often occurs inside bacteria that results in smaller plasmids. This problem is common for cloning shRNA or other unstable DNA pieces into viral vectors. This cloning issue is further compounded by the fact that it is difficult to sequence any shRNA template region because the hairpin may block the progress of the DNA polymerase used in sequencing, sometimes requiring several repeats under different sequencing conditions, incurring high costs charged by sequencing service providers.

To deal with these aspects of the cloning difficulties, particularly for the purpose of increasing cloning efficiency RNAi-based screening, we compared three different strategies

First, we built a smaller shRNA cloning vector to clone and sequence shRNA templates prior to transferring to lentiviral vectors. This smaller vector does not have a severe recombination problem and is easier to sequence in the hairpin-containing region. After an initial round of cloning with this new vector, we further improved it by inserting an XbaI and a NheI site between the BamHI and SpeI insertion sites, so that any plasmid preparations can be screened for recombinants by a simple XbaI or NheI digest before sequencing. After cloning into this intermediate vector, the shRNA expression cassette can be transferred into the lentivirus vectors with some flanking viral sequences so that the insert size will be around 1kb.

Second, we developed a novel DNA preparation procedure after realizing that DNA damage during miniprep of vector plasmids and gel purification of vector fragment increased recombination of these constructs, which were already less stable than usual due to hairpin structures. This procedure of DNA preparation avoids UV or guanidium exposure, which can cause nicks on double-stranded DNA and facilitate recombination. This new procedure relies on purifying DNA through surface-binding to regular reaction tubes treated with a proprietary reagent (SurfaceBind Purification). The process simply requires adding a proprietary, guanidium-free binding buffer to the DNA, which has been processed in a specially coated tube (eppendorf or thin-wall PCR tube), and purifying directly in the same tube. Vectors prepared this way indeed provide more colony counts and a higher percentage of correct constructs as shown by our test runs. The procedure also requires less time and the purified DNA can be dissolved in volumes as small as a few microliters.

Third, to enable truly high throughput shRNA screening (i.e. looking for effective RNAi reagents), we further tested and adapted a ligationless cloning protocol that can be handled by a liquid handler almost entirely. In order to increase throughput, we designed a drastically different procedure that could bypass ligation and sequencing altogether before functional tests. Briefly, DNA molecules that would provide enhanced recombination were created by one round of PCR, purified directly in the surface bind PCR reaction tubes (any template DNA would be removed with DpnI enzyme that cuts non-PCR DNA), pooled, and transformed in bacteria directly. DNA plasmids from transformed bacteria can be used for lentivirus packaging, bypassing sequencing at the initial screening stage, and choose single colonies for sequencing only after a shRNA sequence shows promise in functional assays. This is based on the fact that such cloning rarely has any background colonies, and that among all oligos (if using the correct grade of oligos from validated suppliers) inserted this way, a good portion encodes the correct sequence.

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Tags: lentivirus packaging, lentiviurs shRNA, RNAi, RNAi screening, shRNA, shRNA cloning, shRNA lentivirus packaging, shRNA library

Generate mouse and human iPS cells with transfected mature miRNAs

In last week’s blog we discussed generation of induced pluripotent stem cells (iPSCs) with miRNAs expressed from lentivirus. To take it a step further, synthetic, mature miRNAs can be used to avoid the use of viral vectors. Sure enough, Miyoshi et al. published a paper online a few days ago showing that by transfecting 6 miRNAs at 48 hour intervals, they were able to create iPSCs from mouse and human somatic cells. The efficiency is comparable to retrovirus-mediated OSKM factor over-expression (Yoshida et al.), and therefore lower than lentivirus-mediated miR302/369 expression (Anokye-Danso et al.).

In the study of using mature miRNA for obtaining iPSCs, the researchers transfected miRNAs mir200c, mir302s and mir-369 into tissue cultured cells and achieved reprogramming results. Interestingly, only mir302s are common between this study and that with lentivirus-mediated miRNAs by Anokye-Danso et al. There is no current explanation as to why mir-367, which was shown to be required by Anokye-Danso et al., did not seem to be needed in the mature miRNA transfection experiments. Perhaps a level of redundancy among miRNAs, combined with their broad target range and relatively low specificity, allow some of the miRNAs to be interchangeable when used for reprogramming.

Finally, neither of these two recent miRNA-iPSCs works was the first to demonstrate that miRNAs can initiate or facilitate reprogramming. As early as 2008, Lin et al. showed that mir302s could induce pluripotency in a dose-dependent manner by using tet-induced lentivirus expression. They further illustrated that the underlying mechanism is likely through mir302s’ regulation of epigenetic regulators AOFs and other similar factors.

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Tags: Anokye-Danso, iPS cells, iPSCs, mir-367, mir-369, mir200c, mir302s, Miyoshi et al., no foot print iPSCs, pluripotency

Dealing with Interferon Response When Doing RNAi

Off-target effects are a major problem when using RNA interference (RNAi) to silence genes in mammalian systems. One potential source of off-target effects, by either transfected siRNA duplexes or transcriptionally expressed shRNAs, is the inadvertent activation of the interferon response. There are several steps that can be taken to deal with this problem.

Delivery
Interferon response is more likely when high levels of siRNA are used; it is important to transfect the minimum amount of the siRNA duplex that gives rise to a specific RNAi response, as assessed by the level of expression of the target mRNA and/or protein. The level of stable shRNA expression achieved by using lentiviral or retroviral vectors is comparatively modest. Unless very high levels of shRNA expression are achieved, for example, by using highly transfectable cells and a very efficient shRNA expression plasmid, nonspecific activation of the innate immune response are less likely to be induced.

Design
Previous work has shown that the interferon response is induced by dsRNAs of ?30 bp in length and that perfect dsRNAs of as little as 11 bp in length can produce a weak induction. One possible approach to solving the problem of nonspecific activation of the cellular interferon response is to design the siRNA duplex or shRNA precursor so that it does not contain any stretches of perfect dsRNA of ?11 bp.

Detection
If activation of the interferon response remains a concern, it is possible to routinely check for this effect during the course of an RNAi experiment. Analyzing the level of expression of an interferon-response gene, such as oligoadenylate synthase-1 (OAS1), interferon-stimulated gene-54 (ISG54), and guanylate-binding protein (GBP), in the transfected or transduced cells by northern blot or RT- PCR assays are commonly used.

Can there be any more convenient alternative method for checking interferon response? One potentially useful product could be HiTiter™ pre-packaged lentiviruses that would have a fluorescent protein (mTFP1, mWasabi, or the brightest FP in lanYFP) under the control of an ISRE (IFN-stimulated response element) or GAS (IFN gamma-activating sequence)*. This could be another group of Product-on-Demand type of reagents, meaning that we will have the design ready, but only to produce them upon ordering. This way the cost to us and the price to customers can be kept at minimum.

*The expression of the interferon-stimulated genes (ISGs) is induced by the type I interferons IFN-alpha and IFN-beta. A cis-acting element (TAGTTTCACTTTCCC, nucleotides -101 to -87) has been identified in its promoter of one of these genes, ISG54. This element is responsible for the inducible expression of the ISG54 gene and is referred to as IFN-stimulated response element. The human guanylate-binding (GBP) gene is induced by INF-gamma in fibroblasts within 15 minutes of treatment. An IFN gamma-activating sequence (GAS) has been identified in the GBP promoter (nucleotides -123 to -103). To create the interferon reporters, we would insert five direct repeats of this ISRE and/or four direct repeats of this GAS upstream of the basic promoter element (TATA box) and mWasabi GFP gene of the Allele’s patented pLico lentiviral plasmid backbone.

It should be noted, however, that simple transfection of cells with expression plasmids can induce low-level activation of the interferon response, presumably owing to the presence of cryptic convergent promoters that cause the expression of low levels of dsRNA. In general, very low-level activation of the interferon response, that is, activation that exerts a global inhibitory effect on protein translation of less than twofold, is unlikely to be a problem as long as the specificity of any observed phenotype is fully confirmed.

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Tags: dsRNA, expressed shRNA, interferon, miRNA, off-target, RNA interference, RNAi, RNAi toxicity, siRNA

RNAi Therapy Mediated by Linear DNA Cassettes

RNA interference (RNAi) has been demonstrated to be a powerful tool to silence gene expression. Therapies based on RNAi are being developed in numerous application areas at fast paces. Although in basic research both expressed and synthetic double-stranded RNA molecules are broadly used to induce gene silencing, synthetic small interfering RNAs (siRNAs) are deemed easier to deliver in preclinical and clinical studies. Compared to synthetic siRNAs, DNA cassettes that express small hairpin RNA (shRNA), microRNA (miRNA), or strands of siRNAs have advantages of prolonged effects.

RNAi-expressing DNA cassettes have been incorporated into viral and non-viral vectors for delivery. Viral vectors for RNAi carry the same risks as those for gene therapies, and are currently not the method of choice for human therapies. Non-viral DNA molecules, often in the form of plasmids, can be easily created and reproduced, but their efficacy is hindered by delivery barriers at the tissue, cell, and the nucleus levels. These difficulties are in part due to the plasmids’ large size, presence of antibiotic resistance genes, and immuresponse-generating CpG islands created in bacteria during propagation.

One way to alleviate these difficulties with non-viral DNA vectors for RNAi is to use linear DNA cassettes. Linear DNAs traverse nucleopores efficiently. The DNA molecules can be conveniently produced by PCR reactions without going through production in bacteria, avoiding DNA modifications such as CpG motifs and the need for replication origin or drug-resistance genes. Linear DNA encompassing a promoter, coding region, and poly(A) signals has been used for protein production. Similarly, by incorporating a miRNA cassette into linear transcription unit driven by a Pol II promoter was used to express RNAi for inhibiting HBV (Chattopadhyay et al. (2009). There are now available technologies and commercial services (e.g. Vandalia Research, Inc.) to produce therapeutic grade linear DNA by specialized PCR reactions.

Allele Biotech’s patents on DNA-expressed RNAi provide a platform for highly express shRNA or siRNA from a DNA molecule as short as fewer than 200 basepairs, potentially more suitable for large scale production, and even more efficient transduction trough tissue, cell membrane, and nuclear pores than the large linear cassettes used by Chattopadhyay et al. A set of experiments similar to the cited HBV studies could quickly lead to the validation of a possibly the most effect way yet for RNAi therapeutics.

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Tags: Allele RNAi, gene silencing, miRNA, non viral vector, PCR DNA manufacture, pol II promoter, pol III promtoer, RNA interference, RNAi patent, RNAi therapeutics, RNAi therapy, shRNA, siRNA, viral vector

Allele Custom Services for Drug Screening Companies

Many target discovery and validation programs can benefit from RNA interference, fluorescent proteins, stem cells, and viral delivery systems. However, applications of these technologies require special reagents and laboratory know-how. Even when available, many generic reagent kits are not tailored for your particular needs in screening or validation.

At Allele, we accelerate your discovery efforts with custom RNAi screening, fluorescence based assays, and cell model development services.

1) Our RNAi platform, based on our patented shRNA/miRNA technologies, use DNA linear template, plasmid, lentivirus, retrovirus, or baculovirus vectors that prompt cells to endogenously express RNAi. As a result, our screens offer advantages over synthetic siRNAs:
• Higher levels of consistency
• Greater delivery and gene silencing efficiencies
• Accessibility to difficult-to-transfect cells, including primary cells
• Potential for inducible RNAi expression
• More persistent silencing with shRNA under Allele’s own IP–you may not need to license siRNA patents!

2) Fluorescent proteins (FPs), which can span the entire visual spectrum, have become some of the most widely used genetically encoded tags. Genes encoding FPs alone or as fusions to a protein of interest may be introduced to cells by a number of different methods, including simple plasmid transfection or viral transduction. Allele Biotech is one of a few companies that develop and improve FPs through fundamental research. We have so far achieved:
• The brightest cyan and green FPs, true monomers for minimum artifact or cytotoxicity
• The brightest yellow and red FPs from lancelet, only FPs from vertebrate
• mTFP1 as the best FRET donor by 3 independent reports
• Photoconvertible FPs for super imaging or kinetic labeling
• Delivery on plasmid, retrovirus, lentivirus, baculovirus vectors

3) As a major advancement in the stem cell field, it has recently been shown that mouse and human differentiated cells may be reprogrammed into stem-like, pluripotent cells by the introduction of defined transcription factors. These induced stem cells (iPSCs) provide unprecedented resources of cells of different differentiation stages for functional testing and drug screening. Allele Biotech develops and provides state-of-the-art reagents in convenient forms for iPSC production
• iPS factors carried on lentivirus, retrovirus, baculovirus for different cell types
• Availability in combination with fluorescent proteins under own IP, and drug resistant genes
• 4-in-1 or 2-in-1 effective use of iPS factors on one viral vector
• Feeder cells of human origin expressing factors essential for stem cell culturing

4) Introduction of protein factors, miRNA, promoter-reporter, and virtually any other genetic element of interest via the most efficient viral packaging systems.
• Introducing protein-FP fusion, promoter-FP reporter, photoactivatable factors for cell-based assays
• Introducing critical factors for cell immortalization
• Episomal or integrated expression using baculoviral vectors
• High throughput, systematic expression of whole class of molecules in any type of cell
• High titer viral packaging at low cost for delivery to animal tissues

In addition, the Allele team can provide custom-designed assays that can be used for assaying enzyme activities in almost any pathway, such as the EGF pathway, TNF response/apoptosis pathway, nuclear receptors, etc. We utilize technically advanced methods to provide our partners with advantages over alternative methods or other services.

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Tags: cell assays, cell based assays, cell culture, drug screening, FP assays, FRET, iPS cells, kinase assasy, pathway, RNAi