2004 Fiscal Year Final Research Report Summary
RNA interference by transgenesis ; a new approach for functional genomics
| Project/Area Number |
15310143
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied genomics
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| Research Institution | RIKEN |
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Principal Investigator |
PERRY Anthony RIKEN, Laboratory of Mammalian Molecular Embryology, Team leader, 哺乳類胚発生研究チーム, チームリーダー (80360486)
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| Project Period (FY) |
2003 – 2004
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| Keywords | RNA interference / Transgenesis / shRNA / siRNA / mouse / tissue culture |
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| Research Abstract |
We wished to elicit RNA interference by transgene(tg) expression in mice. As a prelude to this, short interfering RNAs(siRNAs) were shown effectively to reduce target mRNA levels in germinal vesicle(GV) oocytes. We selected a range of targets, reproducing phenotypes previously shown to be associated with the loss of one, whilst revealing novel phenotypes for others. Target transcript depletion was confirmed by semi-quantitative reverse transcriptase PCR on single GV oocytes. We next validated DNA-directed RNAi in vectors directing expression of hairpin RNAs(shRNAs) by RNA polymerase III ; shRNA targets corresponded to tyrosinase, eGFP and leptin as outlined in our original proposal. We developed a novel, fluorescence based co-transfection assay in which shRNA-encoding constructs were cotransfected with a reporter that comprised the target open reading frame followed by an internal ribosome entry site(ires) to facilitate expression of a reporter (venus) from a bicistronic mRNA. This approach allowed us to demonstrate through loss of reporter-meditated epifluorescence that target transcript levels were markedly reduced in tissue culture, thereby validating shRNA constructs prior to their use in transgenesis. This has paved the way for use of the vectors in the ongoing production of transgenic mouse lines. We are employing the assay system to evaluate a second series of potential tgs whose expression is directed by PolII.
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