Reverse genetics using non-endogenous pol i promoters

Abstract

Expression of a transgene is driven in a host cell using a pol I promoter which is not endogenous to an organism from the same taxonomic order from which the host cell is derived.

1 . A host cell comprising at least one expression construct encoding a viral RNA molecule, wherein expression of the viral RNA molecule from the construct is controlled by a pol I promoter which is not endogenous to the host cell's taxonomic order. 
     
     
         2 . A cell having at least one endogenous pol I promoter which control(s) expression of endogenous rRNA and at least one non-endogenous pol I promoter which control(s) expression of a viral RNA or the complement thereof. 
     
     
         3 . A method for producing a recombinant virus, comprising a step of growing the cell of  claim 1  under conditions where the viral RNA molecule is expressed in order to produce virus. 
     
     
         4 . A method of preparing a virus, comprising steps of: (i) producing a recombinant virus by the method of  claim 3 ; (ii) infecting a culture host with the virus obtained in step (i); (iii) culturing the host from step (ii) to produce further virus; and (iv) purifying virus obtained in step (iii). 
     
     
         5 . A method for preparing a vaccine, comprising steps of (a) preparing virus by the method of  claim 4  and (b) preparing vaccine from the virus. 
     
     
         6 . The cell of  claim 1 , wherein the pol I promoter is a primate pol I promoter and the cell is a non-primate cell. 
     
     
         7 . The cell of  claim 1 , wherein the pol I promoter is a non-canine pol I promoter and the cell is a canine cell. 
     
     
         8 . The cell of  claim 7  wherein the pol I promoter is a human pol I promoter and the cell is a canine cell. 
     
     
         9 . The cell of  claim 8  wherein the cell is an MDCK cell. 
     
     
         10 . The cell of  claim 9  wherein the MDCK cell is cell line MDCK 33016 (DSM ACC2219). 
     
     
         11 . The cell of  claim 1 , wherein the cell includes at least one bidirectional expression construct 
     
     
         12 . The cell of  claim 1 , wherein the expression construct is an expression vector or a linear expression construct. 
     
     
         13 . The cell of  claim 1 , wherein the virus is a segmented virus. 
     
     
         14 . The cell of  claim 1 , wherein the virus is a non-segmented virus. 
     
     
         15 . The cell of  claim 1 , wherein the virus is a negative-strand RNA virus. 
     
     
         16 . The cell of  claim 15 , wherein the virus is influenza virus.