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siRNA 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 [1]. 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.

DICER cuts viral RNA into 22-nucleotide strips

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.

DNA-vector-based siRNA technology, in contrast, involves cloning a small DNA insert of about 70 bp into a commercially available or custom vector. This vector can be transfected into the cell, where the DNA insert expresses a short hairpin RNA. The hairpin RNA is rapidly processed by the cellular machinery into double-stranded siRNA. The use of plasmids allows researchers to use highly reliable GenScript inducible promoters.

Fig 1. DICER cuts viral RNA into 22-nucleotide strips.

Key Benefits of Vector-Based siRNA:

  • Stronger in vivo inhibitory effects:Vector-based siRNA is more effective in vivo than synthetic siRNA and can work with tissue-specific promoters.
  • Stable transfection: Vector-based siRNA allows the researcher to obtain a stable cell line and observe the long-term effects of RNAi [2-5].
  • Easy to handle: Vector-based siRNA is delivered in plasmid form, which is more stable and easier to handle than synthetic siRNA.
  • Renewable: Vector-based siRNA can be regenerated on-site.
  • Less costly: Vector-based strategies are less costly in high-throughput applications because they can be regenerated on-site.
  • Wide applications: Vector-based siRNA facilitates the formation and use of stable cell lines, inducible systems, knockout mouse lines [6], and fluorescence monitoring of transfection efficiency.
  • Inducible expression: Vector-based siRNA allows the researcher to establish inducible systems using inducible vectors.
  • Gene-therapy compatibility: Viral vector-based siRNA can be used to infect primary cell lines for gene therapy purposesb [7,8].
  • Easy removal of untransfected cells: Vectors can be equipped with antibiotic resistance genes, allowing the easy removal of untransfected cells.

Selected Publications Citing GenScript's siRNA Technology:

  1. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411: 494-498
  2. Yu JY, DeRuiter SL, Turner DL. (2002) RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells Proc Natl Acad Sci U S A 99(9):6047-6052
  3. Brummelkamp TR, Bernards R, and Agami R. (2002) A system for stable expression of short interfering RNAs in mammalian cells Science 296: 550-553
  4. Jacque JM, Triques K, and Stevenson M. (2002) Modulation of HIV-1 replication by RNA interference Nature 418: 435-438
  5. Sui G, Soohoo C, Affar el B, Gay F, Shi Y, Forrester WC, and Shi Y. (2002) A DNA vector-based RNAi technology to suppress gene expression in mammalian cells Proc Natl Acad Sci USA 99(8): 5515-5520
  6. Kunach T, Gish G, Lickert H, Jones N, Pawson T, and Rossant J. (2003) Transgenic RNA interference in ES cell-derived embryos recapitulates a genetic null phenotype Nature Biotechnology 21:559-561
  7. Shen C, Buck AK, Liu X, Winkler M, and Reske SN. (2003) Gene silencing by adenovirus-delivered siRNA FEBS Lett 539(1-3):111-114
  8. Barton GM, and Medzhitov R. (2002) Retroviral delivery of small interfering RNA into primary cells Proc Natl Acad Sci U S A 99(23):14943-14945

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