Methods and constructs for evaluation of rnai targets and effector molecules
Methods and constructs for selecting double-stranded RNA molecules capable of post-transcriptional gene silencing (PTGS) or RNA interference (RNAi); and methods of selecting targets susceptible to double-stranded RNA mediated PTGS or RNAi.
1 . A method for evaluating dsRNA-mediated silencing or inhibition of a target nucleotide sequence by a selected dsRNA effector molecule in an RNAi-competent system, comprising the steps of:
a) introducing into such system:
i) a capped and polyadenylated fusion mRNA encoding both a reporter gene sequence capable of translation in said system and a sequence to be evaluated as a target for RNAi (RNAi target sequence) and
ii) a dsRNA effector molecule having an at least partially double-stranded RNA sequence, one strand of said sequence being substantially homologous to at least a portion of the RNAi target sequence; and
b) detecting the presence of the reporter gene product.
2 . A method of claim 1 wherein the RNAi target sequence is positioned within either the 5′ or 3′ untranslated region of the fusion mRNA.
3 . A method of claim 2 wherein the RNAi target sequence is positioned within the 3′ untranslated region of the fusion mRNA.
4 . A method of claim 1 wherein the reporter gene sequence encodes a chemiluminescent or fluorometric reporter.
5 . A method of claim 4 wherein the reporter gene sequence encodes a fluorometric reporter.
6 . A method of claim 5 wherein the fluorometric reporter is a green fluorescent protein (GFP).
7 . A method of claim 6 wherein the reporter is EGFP.
8 . A method of claim 1 wherein the RNAi target sequence is a sequence from a pathogen, an endogenous sequence associated with disease or pathology in a vertebrate, or a transgene desired to be modulated.
9 . A method of claim 8 wherein the pathogen is a virus, bacterium, fungus, nematode or a prion.
10 . A method of claim 9 wherein the virus is HBV, HCV, HIV, HSV, HPV, CMV, EBV, or HTLV.
11 . A method of claim 8 wherein the endogenous sequence is from TNF alpha, a cancer-associated sequence, or a host gene responsible for entry or infection by a pathogen.
12 . A method of claim 1 in which the RNAi-competent system is a cell.
13 . A method of claim 12 in which the cell is an RD cell, a Huh7 cell, or a HeLa cell.
14 . A method of claim 1 in which the fusion mRNA is expressed within the cell.
15 . A method of claim 14 in which the fusion mRNA is expressed from a plasmid.
16 . A method of claim 1 in which the fusion mRNA and the effector dsRNA are both expressed within the cell.
17 . A method of claim 16 in which both the fusion mRNA and the effector dsRNA are expressed from one or more plasmids.
18 . A capped and polyadenylated fusion mRNA encoding both a reporter gene sequence capable of translation in said system and a sequence to be evaluated as a target for RNAi (RNAi target sequence).
19 . An mRNA of claim 18 wherein the RNAi target sequence is positioned within either the 5′ or 3′ untranslated region of the fusion mRNA.
20 . An mRNA of claim 19 wherein the RNAi target sequence is positioned within the 3′ untranslated region of the fusion mRNA.
21 . An mRNA of claim 18 wherein the reporter gene sequence encodes a chemiluminescent or fluorometric reporter.
22 . An mRNA of claim 21 wherein the reporter is a green fluorescent protein (GFP).
23 . An mRNA of claim 22 wherein the reporter is EGFP.
24 . An expression construct encoding an mRNA of any of claims 18 through 23 .
25 . An expression construct of claim 24 which is a DNA plasmid.
26 . An RNAi competent cell transfected with an expression construct claim 24 .
27 . An RNAi competent cell stably transfected with an expression construct of claim 24.