RNAi Technology
RNA interference, or RNAi, is a process that sequence-specifically destroys mRNA, causing null or hypomorphic phenotypes. RNAi provides an excellent technology platform for gene expression and gene function studies in many different models, including Drosophila, C. elegans, and mammalian cell systems. RNAi allows researchers to fully or partially suppress the expression of a specific gene, allowing targeted gene knockout and gene knockdown.
Small interfering RNAs, or siRNAs, are short RNA molecules of 19 to 22 nucleotides in length. The siRNAs are generated via cleavage of dsRNA templates by DICER, an RNAse III ribonuclease. The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) and unwound into single-stranded siRNAs. Next, the single-stranded siRNAs guide the RISC complex to the target mRNAs for destruction, causing RNA interference. Depending on the amount of siRNA expressed and its inhibitory efficiency, expression of the target gene can be either completely blocked or measurably suppressed. This allows researchers to determine and study the function of genes, particularly the genes that are lethal upon complete knockout. GenScript's Tet-on and other inducible siRNA expression vectors provide the fine degree of controlled RNAi necessary to these delicate experiments.
GenScript vector-based siRNA technology is an adaptation of GenScript gene synthesis technology to the rise of RNA interference (RNAi) as a powerful tool for gene function analysis and drug target validation. The GenScript siRNA technology package includes siRNA design, siRNA vectors, and custom siRNA construction. GenScript siRNA technology has several advantages over synthetic siRNA methods as follows:
- It produces highly reliable siRNA constructs at minimal cost.
- Its products are more stable and last longer.
- It frees scientists from the time-consuming and difficult task of siRNA vector construction.
Vector-based siRNA Technology:
Short siRNA molecules can be prepared either by traditional RNAi methods, which involve the use of synthetic RNA duplexes consisting of two unmodified 21-oligonucleotide molecules annealed together, or by transcription driven by RNA polymerase promoters. The two most critical factors in determining the effectiveness of RNAi experiments are the ability of the siRNA sequence to silence the specific target mRNA and the efficiency with which the siRNA construct or expression vector can be transfected into the cells.
The direct transfection of chemically synthesized siRNA duplexes into cells, originally demonstrated by Rockefeller University's Tuschl Lab, is currently the most popular approach. However, the success of this technique is heavily dependent on the ability of the model cell system to undergo transfection and to sustain the RNAi effect. Also, the noncontinuous presence of siRNA in the cell renders this technique less feasible for long-term studies. This issue, like many other drawbacks of direct siRNA transfection, is completely sidestepped in the case of DNA-vector-based siRNA.
There are several ways to induce RNAi into a live host, but only three of them have consistently shown themselves effective in vivo: direct delivery of active siRNA constructs into animal tissue, delivery of siRNAs through viral vectors, and delivery of RNAs through plasmid vectors. (These siRNAs are quickly processed into active siRNAs within the cell.) These methods have extended the applicability of siRNA technology to viral-based therapies and in vivo gene function experiments [1]. GenScript is proud to offer adenoviral, lentiviral, and retroviral vectors and a selection of proven siRNA plasmid vectors.
Creating knockout mice using transgenic siRNA:
The generation of knockout mouse lines, using conventional techniques, can be prohibitively expensive in both time and cost. Furthermore, many genes of interest cannot be knocked out completely without killing the mice. However, even difficult genes can be knocked down or out in live mice using transgenic siRNA [2]:
- Design several vector-based siRNA constructs.
- Test the vector-based siRNA in cell lines such as HEK293 to confirm that they downregulate the expression of the gene of interest.
- Linearize and electroporate the vector-based siRNA into ES cells. Transfected ES cell lines can be selected via drug resistance.
- Generate the transgenic mouse line using the drug-resistant ES cell lines.
Advantages of transgenic siRNA over conventional knockout technology:
- Transgenic siRNA is a time-saving and cost-effective approach to the generation of knockout mice. The time and expense needed to build siRNA constructs (three weeks and $375 per construct) is only a fraction of that of conventional methods (three months and at least $8000).
- Transgenic siRNA does not require genomic sequencing, which is essential for conventional knockout technology.
- Transgenic siRNA can generate both partial and complete knockout phenotypes, allowing for the knockout of difficult or vitally necessary genes.
Introducing siRNA into animals via other methods:
- Plasmid injection: Vector-based siRNA in plasmid form can be directly injected into certain organs [3].
- Tail vein: Vector-based siRNA can be introduced into various animals using tail vein hydrodynamic injection [4].
- Liposome formulation: Vector-based siRNA can be formulated into cationic liposomes and introduced into animals via intravenous injection [5].
References:
- Haibin Xia, Qinwen Mao, Henry L. Paulson, Beverly L. Davidson. siRNA-mediated gene silencing in vitro and in vivo. Nat Biotechnol. 2002 Oct; 20 (10): 1006-1010.
- Kunath T, Gish G, Lickert H, Jones N, Pawson T, Rossant J. Transgenic RNA interference in ES cell-derived embryos recapitulates a genetic null phenotype. Nat Biotechnol. 2003 May; 21 (5): 559-561.
- Giladi H, Ketzinel-Gilad M, Rivkin L, Felig Y, Nussbaum O, Galun E. Small interfering RNA inhibits hepatitis B virus replication in mice. Mol Ther. 2003 Nov; 8 (5): 769-776.
- Gratsch TE, De Boer LS, O'Shea KS. RNA inhibition of BMP-4 gene expression in postimplantation mouse embryos. Genesis. 2003 Sep; 37 (1): 12-17.
- Sorensen DR, Leirdal M, Sioud M. Gene silencing by systemic delivery of synthetic siRNAs in adult mice. J Mol Biol. 2003 Apr 4; 327 (4): 761-766.
miRNAs, due to their low abundance, are difficult to isolate and once isolated, these 18-24 nt RNAs have to purified, ligated to 5' and 3' adapter sequences and after RT-PCR cloned into vectors and sequenced. In place of this, computational analysis from genomic sequences is often used as a valuable tool that complements cloning. New miRNAs are identified in this manner that should correlate with computational analysis. At GenScript, we offer you an alternate way to clone your sequences of interest with our vector-based miRNA technology. In addition to our existing siRNA construction and the entire spectrum of siRNA vectors that we offer, we have launched a miRNA construction service in the vector of your choice. We can assist you with the construction of the gene sequence that you have chosen and provided us, that can be then cloned into any of our miRNA cloning vectors. The resulting miRNA expressing plasmid is purified and ready to transfect and can be used for a variety of applications such as quantitation using Northern blotting, dot blotting, RNAse protection assay, primer extension analysis, Invader assay and quantitative PCR.
Selected Publications Citing GenScript's siRNA Technology:
- Man Li, De-Shu Shang, Wei-Dong Zhao, Li Tian, Bo Li, Wen-Gang Fang, Li Zhu, Shu-Mei Man, and Yu-Hua Chen. Amyloid β Interaction with Receptor for Advanced Glycation End Products Up-Regulates Brain Endothelial CCR5 Expression and Promotes T Cells Crossing the Blood-Brain Barrier. J. Immunol. May 2009; 182(9): 5778 - 5788
- Xin Ge, Horace H Loh, and Ping-Yee Law. ?-Opioid receptor cell surface expression is regulated by its direct interaction with RibophorinI. Mol. Pharmacol. Mar 2009
- Rajarshi Sengupta, Silvia Burbassi, Saori Shimizu, Silvia Cappello, Richard B. Vallee, Joshua B. Rubin, and Olimpia Meucci. Morphine Increases Brain Levels of Ferritin Heavy Chain Leading to Inhibition of CXCR4-Mediated Survival Signaling in Neurons. J. Neurosci. Feb 2009; 29(8): 2534 - 2544
- Nicole B. Bryan, Andrea Dorfleutner, Yon Rojanasakul, and Christian Stehlik. Activation of Inflammasomes Requires Intracellular Redistribution of the Apoptotic Speck-Like Protein Containing a Caspase Recruitment Domain. J. Immunol. Mar 2009; 182(5): 3173 - 3182
- Xiangdong Wang, Ning Yang, Luqin Deng, Xin Li, Jing Jiang, Yujun Gan, and Stuart J. Frank. Interruption of Growth Hormone Signaling via SHC and ERK in 3T3-F442A Preadipocytes upon Knockdown of Insulin Receptor Substrate-1. Mol. Endocrinol. Apr 2009; 23(4): 486 - 496
- Mridula Rewal, Rachel Jurd, T. Michael Gill, Dao-Yao He, Dorit Ron, and Patricia H. Janak. α4-Containing GABAA Receptors in the Nucleus Accumbens Mediate Moderate Intake of Alcohol. J. Neurosci. Jan 2009; 29(2): 543 - 549
- Dympna Harmey, Anthony Smith, Scott Simanski, Carole Zaki Moussa, and Nagi G. Ayad. The anaphase promoting complex induces substrate degradation during neuronal differentiation. J. Biol. Chem. Feb 2009; 284(7): 4317 - 4323
- Thomas D. Helton, Takeshi Otsuka, Ming-Chia Lee, Yuanyue Mu, and Michael D. Ehlers. Pruning and loss of excitatory synapses by the parkin ubiquitin ligase. PNAS. Dec 2008; 105(49): 19492 - 19497
More details please check at siRNA Selected Publications
