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Brief History of RNAi

In 1990, Jorgensen and colleagues tried to enhance the colour of petunias by introduction of addtional pigment-producing genes into petunia. To their suprise, instead of the expected deep purple color, many of the flowers appeared patterned white or complete white. They named the phenomenon observed as "co-suppression".

At the same time, Alexander R. vean der Krol also observed similar phonotype when they introduced genes involved in flower pigmentation about quarter of plants demonstrated reduced floral pigmentatin, accompanies by dramatic reduction of expression of both the introduced gene and the homologous endogenous gene.

Guo and colleague produced the loss-of-function or gene-knockout phenotype in the offspring of the injected C. elegans by direct injection of antisense siRNA (par-1 gene) and a similar inhibitory effect on par-1 gene function was elicited by the injection sense siRNA. Cell 81:611, 1995

In 1998, Fire A and colleagues reported that double-strand RNA was at least tenfold potent as a silensing trigger than were antisense or sense RNA alone.

In 2001, 21 to 23 Nucleotide fragments hence siRNA from the dsRNA was demonstrated to guide mRNA cleavage Zamore P and colleagues.

Elbashir SM et al first reported that Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.

Martinez J et al published the results demonstrating that Single-stranded antisense siRNAs are also effective to silence genes (5'-phosphorylated).

2001 - Dicer, which can produce putative guide RNAs was discovered. Dicer is a member of the RNase III family of nucleases that specifically cleave double-stranded RNAs, and is evolutionarily conserved in worms, flies, plants, fungi and mammals. The enzyme has a distinctive structure, which includes a helicase domain and dual RNase III motifs. Dicer also contains a region of homology to the RDE1/QDE2/ARGONAUTE family that has been genetically linked to RNAi.

Bernstein E, Caudy AA, Hammond SM, Hannon GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature. 2001 Jan 18;409(6818):363-6.

2002 - shRNA: Driven in part by their desire for a longer-lasting alternative to small RNAs, Hannon and his Cold Spring Harbor Laboratory colleagues tried using RNAs folded over like hairpins to quash the function of specific genes. They were inspired to try these short hairpin RNAs (shRNAs) by their finding, in collaboration with Dutch worm researcher Ronald Plasterk, that some genes naturally regulate other genes-through RNAi-by coding for just such hairpin-shaped pieces of RNA.

Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, Conklin DS. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev. 2002 Apr 15;16(8):948-58.

2002 - Silencing in murine cells: Paddison PJ, Caudy AA, Hannon GJ. Stable suppression of gene expression by RNAi in mammalian cells. Proc Natl Acad Sci U S A. 2002 Feb 5;99(3):1443-8.

2003 - In vivo p53 silencing. The new study indicates that stable suppression of deleterious genes by RNAi--in which adult stem cells are isolated, modified ex vivo, and then re-introduced into the affected individual--might be an effective strategy for treating human disease.

The study, published in Nature Genetics (online edition February 3/ print edition March 2003), focussed on the role of a tumor suppressor gene called p53 in a mouse model of lymphoma. In the mouse model, forced expression of the Myc oncogene in B-cells causes the mice to develop B-cell lymphomas by 4 to 6 months of age. The scientists, led by Greg Hannon and his CSHL colleague, Scott Lowe, knew that completely deleting the p53 gene causes lymphomas to develop much sooner, and in a more aggressive, highly-invasive form, than lymphomas that develop when the p53 gene is present.

To test the effect of decreasing p53 to particular levels via RNA interference, the scientists reconstituted the blood cells of mice by first irradiating the animals to destroy their endogenous, bone marrow supply of hematopoietic stem cells, and then injected the mice with a fresh supply of hematopoietic stem cells that had been engineered through RNAi to produce low, medium, or high levels of p53.

The study showed that establishing different levels of p53 in B-cells by RNAi produces distinct forms of lymphoma. Similar to lymphomas that form in the absence of p53, lymphomas that formed in mice with low p53 levels developed rapidly (reaching terminal stage after 66 days, on average), infiltrated lung, liver, and spleen tissues, and showed little apoptosis or "programmed cell death".

In contrast, lymphomas that formed in mice with intermediate p53 levels developed less rapidly (reaching terminal stage after 95 days, on average), did not infiltrate lung, liver, or spleen tissues, and showed high levels of apoptosis. In mice with high B-cell p53 levels, lymphomas did not develop at an accelerated rate, and these mice did not experience decreased survival rates compared to control mice.

The study illustrates the ease with which RNAi "gene knockdowns" can be used to create a full range of mild to severe phenotypes (something that geneticists dream about), as well as the potential of RNAi in developing stem cell-based and other therapeutic strategies.

Hemann MT, Fridman JS, Zilfou JT, Hernando E, Paddison PJ, Cordon-Cardo C, Hannon GJ, Lowe SW. An epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo. Nat Genet. 2003 Mar;33(3):396-400.

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