USE OF POST-TRANSCRIPTIONAL GENE SILENCING FOR IDENTIFYING NUCLEIC ACID SEQUENCES THAT MODULATE THE FUNCTION OF A CELL
Described herein are methods for identifying nucleic acid sequences that modulate the function of a cell, the expression of a gene in a cell, or the biological activity of a target polypeptide in a cell. The methods involve the use of double stranded RNA expression libraries, double stranded RNA molecules, and post-transcriptional gene silencing techniques.
1 . A method for identifying a nucleic acid that modulates the function of a vertebrate cell, said method comprising the steps of:
(a) transforming a population of vertebrate cells with a double stranded RNA expression library; wherein at least two cells of said population of cells are each transformed with a different nucleic acid from said double stranded RNA expression library, wherein said transformed nucleic acid is capable of forming double stranded RNA, and wherein said transformation and said formation of double stranded RNA are carried out under conditions that inhibit or prevent an interferon response or a double stranded RNA stress response;
(b) selecting a vertebrate cell in which said nucleic acid is expressed in said cell; and
(c) assaying for a modulation in the function of said cell, wherein said modulation identifies a nucleic acid that modulates the function of said vertebrate cell.
2 . A method for identifying a nucleic acid that modulates expression of a target nucleic acid in a vertebrate cell, said method comprising the steps of:
(a) transforming a population of vertebrate cells with a double stranded RNA expression library, wherein at least two cells of said population of cells are each transformed with a different nucleic acid from said double stranded RNA expression library, wherein said transformed nucleic acid is capable of forming double stranded RNA, and wherein said transformation and said formation of double stranded RNA are carried out under conditions that inhibit or prevent an interferon response or a double stranded RNA stress response;
(b) selecting a vertebrate cell in which said nucleic acid is expressed in said cell; and
(c) assaying for a modulation in the expression of a target nucleic acid in said cell, wherein said modulation identifies a nucleic acid that modulates expression of said target nucleic acid.
3 . A method for identifying a nucleic acid that modulates the biological activity of a target polypeptide in a vertebrate cell, said method comprising the steps of:
(a) transforming a population of vertebrate cells with a double stranded RNA expression library, wherein at least two cells of said population of cells are each transformed with a different nucleic acid from said double stranded RNA expression library, wherein said transformed nucleic acid is capable of forming double stranded RNA, and wherein said transformation and said formation of double stranded RNA are carried out under conditions that inhibit or prevent an interferon response or a double stranded RNA stress response;
(b) selecting for a vertebrate cell in which said nucleic acid is expressed in said cell; and
(c) assaying for a modulation in the biological activity of a target polypeptide in said cell, wherein said modulation identifies a nucleic acid that modulates the biological activity of said target polypeptide.
4 . The method of claim 1 , wherein at most one nucleic acid is stably integrated into a chromosome of each cell.
5 . The method of claim 1 , said method further comprising: (d) identifying said nucleic acid by amplifying said nucleic acid and sequencing said amplified nucleic acid.
6 . The method of claim 1 , wherein said double stranded RNA expression library comprises cDNAs derived from said cells.
7 . The method of claim 1 , wherein said double stranded RNA expression library comprises randomized nucleic acids.
8 . The method of claim 1 , wherein said double stranded RNA expression library is a nuclear double stranded RNA expression library.
9 . The method of claim 1 , wherein said double stranded RNA expression library is a cytoplasmic double stranded RNA expression library.
10 . The method of claim 1 , wherein said cell is a mammalian cell.
11 . The method of claim 1 , wherein said nucleic acid is contained in a vector.
12 . The method of claim 11 , wherein the sense strand and the anti-sense strand of said double stranded RNA are transcribed from the same nucleic acid using two convergent promoters.
13 . The method of claim 11 , wherein said nucleic acid comprises an inverted repeat such that upon transcription said nucleic acid forms a double stranded RNA.
14 . The method of claim 1 , wherein said assaying comprises measuring an event selected from the group consisting of cell motility, apoptosis, cell growth, cell invasion, vascularization, cell cycle events, cell differentiation, cell dedifferentiation, neuronal cell regeneration, and the ability of a cell to support viral replication.
15 . The method of claim 1 , wherein said double stranded RNA is between 5 and 100 nucleotides in length, inclusive.
16 . The method of claim 1 , wherein said double stranded RNA is at least 100 nucleotides in length.
17 . The method of claim 16 , wherein said double stranded RNA is at least 250 nucleotides in length.
18 . The method of claim 17 , wherein said double stranded RNA is at least 500 nucleotides in length.
19 . The method of claim 18 , wherein said double stranded RNA is at least 1000 nucleotides in length.