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Title: Elucidation of the Mechanism of Gene Silencing using Small Interferin RNA: DNA Hybrid Molecules

Elucidation of the Mechanism of Gene Silencing using Small Interferin RNA: DNA Hybrid Molecules The recent discovery that short hybrid RNA:DNA molecules (siHybrids) induce long-term silencing of gene expression in mammalian cells conflicts with the currently hypothesized mechanisms explaining the action of small, interfering RNA (siRNA). As a first step to elucidating the mechanism for this effect, we set out to quantify the delivery of siHybrids and determine their cellular localization in mammalian cells. We then tracked the segregation of the siHybrids into daughter cells after cell division. Markers for siHybrid delivery were shown to enter cells with and without the use of a transfection agent. Furthermore, delivery without transfection agent only occurred after a delay of 2-4 hours, suggesting a degradation process occurring in the cell culture media. Therefore, we studied the effects of nucleases and backbone modifications on the stability of siHybrids under cell culture conditions.
Authors:
Publication Date:
OSTI Identifier:OSTI ID: 900164
Report Number(s):UCRL-TR-218809
TRN: US200709%%562
DOE Contract Number:W-7405-ENG-48
Resource Type:Technical Report
Research Org:Lawrence Livermore National Laboratory (LLNL), Livermore, CA
Sponsoring Org:USDOE
Country of Publication:United States
Language:English
Subject: 59 BASIC BIOLOGICAL SCIENCES; CELL CULTURES; CELL DIVISION; DNA; GENES; MODIFICATIONS; NUCLEASES; RNA; SEGREGATION; STABILITY