Simultaneous gene silencing and suppressing gene silencing in the same cell

A process for selecting a eukaryotic cell with a desired level of silencing of a polynucleotide of interest, the process comprising;

• i) obtaining one or more cells each comprising a first exogenous polynucleotide encoding a double stranded RNA (dsRNA) molecule which comprises a first nucleotide sequence which is complementary to a region of a target RNA encoded by the polynucleotide of interest, and a second exogenous polynucleotide encoding a silencing suppressor polypeptide,
• ii) analysing the cell(s) for one or more of (a) the level of the target RNA encoded by the polynucleotide of interest, (b) the amount of the protein encoded by the target RNA, (c) the level of the dsRNA molecule or a processed RNA product thereof which comprises the first nucleotide sequence and which is capable of reducing in the cell the level of the target RNA encoded by the first polynucleotide of interest and/or the amount of a protein encoded by the target RNA when compared to a corresponding cell lacking the first exogenous polynucleotide, and (d) for a phenotype that is determined by the polynucleotide of interest,
• iii) if the cell(s) does not have the desired level of silencing of the polynucleotide of interest, substituting one or both of the exogenous polynucleotides with an alternate polynucleotide(s) and analysing the resultant cell(s) for the desired level of silencing,
• iv) if necessary, repeating step iii) until the desired level of silencing of the polynucleotide of interest is obtained, and
• v) selecting a cell with the desired level of silencing,
• wherein each exogenous polynucleotide is operably linked to one or more promoters that are capable of directing expression of the polynucleotides in the cell.

A process for selecting a silencing suppressor which is compatible with a double-stranded RNA (dsRNA) molecule of interest, comprising the steps of

• i) obtaining one or more eukaryotic cells each of which comprises (a) a first exogenous polynucleotide encoding the dsRNA molecule which comprises a first nucleotide sequence which is complementary to a region of a target RNA encoded by a first polynucleotide of interest, and (b) a second exogenous polynucleotide encoding a candidate silencing suppressor polypeptide which may be compatible with the dsRNA molecule, wherein each exogenous polynucleotide is operably linked to one or more promoters that are capable of directing expression of the polynucleotide in the cell,
• ii) analysing the cell(s) for one or more of (a) the level of the target RNA encoded by the polynucleotide of interest, (b) the amount of the protein encoded by the target RNA, (c) the level of the dsRNA molecule or a processed RNA product thereof which comprises the first nucleotide sequence and which is capable of reducing in the cell the level of the target RNA encoded by the first polynucleotide of interest and/or the amount of a protein encoded by the target RNA when compared to a corresponding cell lacking the first exogenous polynucleotide, and (d) for a phenotype that is determined by the polynucleotide of interest,
• iii) if the cell(s) does not have a desired property, substituting the second exogenous polynucleotide in the cell with an alternate polynucleotide(s) which encodes a candidate compatible silencing suppressor and repeating step ii), and
• iv) if necessary, repeating step iii) until a cell(s) with the desired property is identified, thereby selecting the silencing suppressor which is compatible with the dsRNA molecule.