Sigma shrna design tool

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FEBS Letters Ying, M. Stem Cells 29 1 Which shRNA reagent is best for you? Pooled shRNA libraries A pooled lentiviral screen can be performed to identify genes that regulate cellular responses and signaling pathways, or to discover novel gene functions. Moreover, inducible knockdown allows the investigation of genes that would be lethal or otherwise poorly tolerated if constitutively knocked down.

Lentiviral inducible shRNA vectors are readily available, but unfortunately the process of cloning, screening, and testing shRNAs can be time-consuming and expensive.

Therefore, we sought to refine a popular vector Tet-pLKO-Puro and streamline the cloning process with efficient protocols so that researchers can more efficiently utilize this powerful tool.

Our primary modification was to shrink the stuffer region, which allows vector purification via polyethylene glycol precipitation thereby avoiding the need to purify DNA through agarose. In addition, we generated EZ-Tet-pLKO vectors with hygromycin or blasticidin resistance to provide greater flexibility in cell line engineering.

Furthermore, we provide a detailed guide for utilizing these vectors, including shRNA design strategy and simplified screening methods.

Notably, we emphasize the importance of loop sequence design and demonstrate that the addition of a single mismatch in the loop stem can greatly improve shRNA efficiency. Our aim was twofold: first, to take a very useful shRNA vector and make it more amenable for molecular cloning and, secondly, to provide a streamlined protocol and rationale for cost-effective design, cloning, and screening of shRNAs.

With this knowledge, anyone can take advantage of this powerful tool to inducibly knockdown any gene of their choosing. The benefits of siRNA include commercially available RNA oligos which can be transfected into cells for quick and efficient knockdown.

However, siRNA becomes less useful when working with cell types with low transfection efficiency or in experiments that require prolonged gene knockdown [ 1 ]. Though not as simple to use as siRNA, shRNA can avoid concerns of low transfection efficiency and temporary knockdown by using retroviral delivery and selection for stable genomic integration [ 2 — 4 ]. Lentiviral shRNA vectors are popular due to their ability to infect nearly any cell type and integrate into the genome of both dividing and non-dividing cells.

The shRNAs were not all functionally validated but were given a computationally calculated score for predicated efficiency and specificity.

Together, these modifications allow transcription of shRNA upon the addition of tetracycline, or its analogue doxycycline Dox , to sequester TetR and relieve repression at the TetO [ 5 , 6 ]. This vector combines the benefits of lentiviral delivery and inducible gene knockdown, providing many advantages over siRNA or constitutive shRNA. By combining inducible vectors with the list of candidate shRNA sequences from the RNAi consortium it is now possible to induce knockdown of nearly any gene in virtually any cell type.

In an effort to improve this tool even further we made some modifications to make it more amenable for cloning. Furthermore, we establish clear and efficient protocols for designing and cloning shRNAs into the vector.

In addition, we demonstrate the importance of loop design including using a single mismatch to improve shRNA efficiency. With our modified vector EZ-Tet-pLKO and a detailed description for designing and cloning shRNAs, we aim to make it easy for anyone to quickly adopt and utilize this tool.

Additionally, we generated matching vectors with mammalian selection markers for hygromycin Hygro or blasticidin Blast resistance Fig.

The smaller stuffer makes it possible to purify cut vector by size-selective DNA precipitation with polyethylene glycol PEG. Together, the combination of vector modifications and utilization of PEG precipitation provides a simplified method for preparing cut vector.

Vector maps and PEG purification. Developing functional shRNA constructs often requires testing many targeting sequences; therefore, a process for designing shRNAs quickly and efficiently is quite valuable. Targeting sequences were selected as described in the methods section and used to generate sense and antisense shRNA oligos. The antisense oligo bottom strand is a reverse complement of the sense oligo with complementary overhangs.

Without a mismatch, a 6 nt palindrome loop is predicted to collapse to a 4 nt loop and shift the targeting sequence by one base Fig. Creb1 and pools were selected containing the same targeting sequence with or without a single mismatch.

Probing for TetR showed that both pools were infected with the lentivirus and had similar expression levels of the lentiviral construct. A similar test was performed using cells containing the sh. Creb1 construct and produced similar results Fig. Thus, when designing shRNA sequences it is crucial to consider not only the targeting sequence, but also a mismatch in the loop stem.

Upper strand is sense oligo, lower strand is anti-sense oligo. Colors correlate to calculated likelihood of the depicted pairing. See methods for details on prediction tool. TetR was probed on a separate gel. After ligation of vector and shRNA oligos the DNA must be transformed into competent bacteria and colonies must be screened. PCR product was visualized by agarose gel electrophoresis, which produced clearly identifiable bands for true clones and background colonies Fig.

Screening techniques. Asterisks indicate corresponding bands in Fig. Additionally, clones can be further validated by restriction enzyme RE digest screening, which requires a miniprep step to isolate plasmid DNA. As a way to simplify and improve the RE screening process, we recommend a SpeI site for loop design Fig. Thus, the combination of colony-PCR as a cheap and quick primary screen and SpeI-based digest as a secondary screen creates a streamlined process for identifying positive shRNA clones.

Cells were infected with lentivirus and pools were selected with puromycin. We performed a titration with Dox 0. Furthermore, the target protein can be recovered after removal of Dox. Cells with sh. Dox was removed and samples were harvested over a recovery time course Fig. Recovery of protein began four days after removal of Dox. Specificity is constrained by genome evolution--since many genes are part of extensive gene families, targeting a specific family member can be difficult.

Furthermore, functionally distinct genes share many motifs with underlying nucleic acid sequence similarity. Our knowledge of transcript structure and variants is still very incomplete as well. For all these reasons and more, we construct several shRNAs for each transcript with the expectation of getting a range of knockdown efficiencies across the set and at least a few which knockdown effectively.

Because of the high sequence identity among different transcripts from the same gene, the majority of the shRNAs target all known transcript variants. Candidates with no off-target matches receive a specificity factor "bonus" value of 1. Furthermore, if the candidate is a perfect match to a majority of the current transcripts of the target gene its specificity factor is 1. Cecconi F, D'Amelio M. Lingor, P. Netherlands: Springer Available from: link.

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