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Recombinant Dicer efficiently converts large dsRNAs into siRNAs suitable for gene silencing


Stanford Reference:

02-028


Abstract


RNA interference (RNAi) is a powerful method for silencing the expression of specific genes in diverse plant and animal cells. RNAi is mediated by 21-23 nucleotide small interfering RNAs (siRNAs), which are produced in vivo when Dicer, an RNase III-family enzyme, cleaves larger double-stranded RNAs (dsRNAs). siRNAs, but not large dsRNAs (>30 base pairs), can be used to silence gene expression in commonly used mammalian cell lines. Unlike the larger dsRNAs, siRNAs do not initiate a non-specific, cytotoxic response. Here we present a technology that introduces a cost-effective and time efficient method for generating siRNAs. Recombinant Dicer is used to cleave large dsRNAs (>500 base pairs) into a complex pool of siRNAs (d-siRNAs) in vitro. The d-siRNAs are subsequently used to specifically and efficiently silence gene expression in mammalian cell lines.

Stage of Development:

Numerous exogenous and endogenous genes have been silenced with a high degree of specificity in a variety of cultured mammalian cells. Cell models include: HEK 293 cells, HeLa cells, NIH 3T3 cells, and RBL cells. Genes suppressed include: Green Fluorescent Protein and other fluorescent proteins, Firefly Luciferase, Renilla Luciferase, ATR, CaMKII-a, CaMKII-b, B-Raf, Cdc25C, Cdk1, Cdk2, cyclin A2, cyclin B1, cyclin B2, cyclin E1, Mad2, Mre11, PKC-e, PKC-d. The specificity has been tested using d-siRNAs to silence pairs of genes with a high degree of nucleotide identity. For example, the phosphatase Cdc25C has been suppressed to 90% of normal levels without affecting the expression levels of the closely related family member Cdc25A. Furthermore cyclin B1 has been silenced to nearly 99% of the normal levels without any decrease in the most closely related gene cyclin B2; the converse is also true. Other examples include the cyclin dependent kinases, Cdk1 and Cdk2, the protein kinase C family members, PKC-e and PKC-d, and two Ca2+/calmodulin dependent kinases, CaMKII-a and CaMKII-b. In each case silencing is very specific since these gene pairs share a high degree of nucleotide identity and only the targeted gene is suppressed. At this point we believe that any given gene can be specifically silenced without any appreciable off-target or nonspecific effects.

Continuing Research:

Currently we are testing the specificity further by comparing the silencing and microarray profiles attained with d-siRNAs to that of a particular chemically synthesized siRNA that has been shown to induce high levels of off-target effects. We are also testing specificity with reconstitution experiments; if d-siRNA-resistant cDNAs abrogate the phenotype when co-transfected with the d-siRNAs, silencing is free of off-target and nonspecific effects. In addition we are testing other nonspecific effects associated with cytotoxic responses such as the interferon response and general inhibition of translation.

In a biological context:

...We are using d-siRNAs to analyze the function of various proteins in regulation of somatic cell mitosis.

...We are determining how many genes can be silenced at one time, 1, 5, or 10, etc.

...We are attempting to make mammalian somatic cells genetically tractable; the function of gene products is being tested in a nearly null background by silencing the endogenous gene and reconstituting with a d-siRNA-resistant cDNA.

...We are also beginning to systematically characterize nearly 2400 signaling proteins from a loss of function perspective.

...We are assessing the possibilities to use d-siRNAs in a split-pool functional screen. We are evaluating the potency of gene suppression of one gene in the presence of d-siRNAs targeting 5, 10, 25, 50, or 100 other genes.

...We are assessing the potency of d-siRNAs in Xenopus laevis oocytes and embryos as well as Zebrafish embryos, two systems where the potential of d-siRNA-mediated gene suppression has not yet been realized.


Applications


  • · The loss-of-function phenotype of any gene in many mammalian cell culture models and vertebrate animal systems can be determined using d-siRNAs.
  • · Large-scale, high-throughput functional screening can be accomplished using d-siRNAs.
  • · d-siRNAs can be used in a high-throughput fashion to validate the genetic targets of drugs and small molecules.
  • · d-siRNAs may be used for therapeutic applications.

Advantages


  • ·Simple: siRNAs can be produced by a variety of methods: chemical synthesis, by in vitro transcription from a short DNA template, or by transfection of DNA expression construct that gives rise to a siRNA or short-hairpin-RNA (shRNA) in vivo. The advantage of using d-siRNAs instead of the currently available siRNAs is dependent on which method is used to generate the siRNA, but in general using d-siRNAs is advantageous because they are comprised of a pool of siRNAs. In certain instances, individual siRNAs evoke a cytotoxic response thus another potent sequence must be determined. The complexity of the d-siRNA pool avoids this issue.
  • ·Cost effective: With our technology, for any single gene at least 300 different d-siRNAs can be produced from a ~600 base pair dsRNA at a cost equal to that required to produce an in vitro transcribed siRNA or in vivo encoded siRNA or shRNA. In addition, this cost is a few orders of magnitude less expensive than a chemically synthesized siRNA. The current price of a single chemically synthesized siRNA is around $500. The major difference in cost lies in the fact that when single siRNAs are used three to eight siRNAs may be required to attain a high probability of silencing any particular gene.
  • ·Time efficient: d-siRNAs can be generated in two days and the phenotype can be assayed in less than one week.
  • ·Improved gene silencing: To determine the target region of a single siRNA an educated guess must be made, but d-siRNAs obviate the need to guess. Although the rules for what makes a potent siRNA are better characterized there is a substantial chance that any single 21-nucleotide region selected from the mRNA will be ineffective in initiating mRNA cleavage or inhibiting translation. d-siRNAs target a large region (at least 500 base pairs) thereby improving the chances of silencing a gene because if there is secondary structure or protein binding in the target region silencing will not be as efficient with a single siRNA.
  • ·Improved scale-up: Using d-siRNAs for gene silencing, scales up well for studying gene function in a high-throughput, large-scale fashion (e.g. screening of cDNA libraries).Whereas, any method using a single siRNA does not scale up well because one or more siRNA would need to be designed and generated for each gene in the genome. d-siRNAs could be generated from a cDNA library either from individual cDNAs or from pools of cDNAs, and only one pool of d-siRNAs needs to be generated for efficient gene silencing making this approach less expensive and more efficient than using single siRNAs.
  • ·More selective: Each siRNA within the complex pool of d-siRNAs is ~1/300th the concentration thereby decreasing the likelihood of off-target effects. Furthermore, d-siRNAs will cause destruction of a large portion of the targeted mRNA decreasing the chances of generating a truncated protein. Single siRNAs cause cleavage at one site and the resulting N-terminal truncated protein may act as a dominant negative or constitutively active protein rather than as a true protein-null.
  • ·Unbiased: d-siRNAs can be generated from unknown sequences providing a means to study uncharacterized gene products or genomic elements. In a therapeutic setting, very little information about the genome of the pathogen (e.g. virus or parasite) must be known. Furthermore, in the case rapidly mutating virions the genomic material could be repeatedly isolated from the infected cells, and converted into d-siRNAs avoiding the complications of potency involved with mismatches between the siRNA and sense RNA.

Publications



Innovators & Portfolio


  • James Ferrell   
  • Jason Myers   

Patent Status



Date Released

 6/7/2002
 

Licensing Contact


Sara Nakashima, Licensing Associate
650 725 9115 (Direct)
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Related Keywords


RNA interference (RNAi)   functional genomics   research tool: reagent   screening: HTS   siRNA   research tool   screening   
 

   

  

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S02-028 Recombinant Dicer efficiently converts large dsRNAs into siRNAs suitable for gene silencing