Expression of heterologous genes according to a targeted expression profile

Abstract

A gene trap vector comprises a DNA construct containing an expression unit of an internal ribosome binding site (IRES) coupled to a heterologous gene sequence; this expression unit is used in gene trap protocols to obtain expression of the heterologous gene in the host.

1 . A method of inserting a gene of interest into an endogenous gene in a mouse embryonic stem cell such that the gene of interest is expressed in said mouse embryonic stem cell, wherein said method comprises transforming the mouse embryonic stem cell with a trap vector comprising
 a heterologous gene coding sequence: 5′ X-A-P-B-Q-C-Y-3′ wherein:
 X and Y are substantially homologous with separate sequences of the endogenous gene, and are of sufficient length to undergo homologous recombination with the endogenous gene so as to insert the A-P-B-Q-C elements into the host cell's genome; 
 P is an internal ribosome entry site (IRES); 
 Q is the gene of interest 
 A, B, and C are each independently at least one of a linker sequence and a covalent bond; 
 a polyadenylation site is located 3′ (downstream) of Q; 
 a splice acceptor site is located 5′ (upstream) of P; and 
 wherein the heterologous gene coding sequence lacks a promoter element. 
   
     
     
         2 . A mouse embryonic stem cell comprising a heterologous gene code sequence inserted by the method of  claim 1 . 
     
     
         3 . A descendant of the mouse embryonic stem cell according to  claim 2 , wherein the descendant has inherited the inserted heterologous gene coding sequence. 
     
     
         4 . An animal comprising a heterologous gene coding sequence inserted by the method of  claim 1 . 
     
     
         5 . A descendant of an animal according to  claim 4 , wherein the descendant has inherited the inserted heterologous gene coding sequence. 
     
     
         6 . A method of inserting a heterologous gene coding sequence into an endogenous gene in a mouse embryonic stem cell genome and expressing said heterologous gene coding sequence, comprising the step of transforming the mouse embryonic stem cell with a random gene trap vector comprising a DNA construct, wherein the heterologous gene coding sequence lacks a promoter, and comprises the sequence:
 5′ A-P-B-Q-C 3′   
       in which
 P is an internal ribosome entry site (IRES); 
 Q is the heterologous gene sequence, and 
 A, B and C are, separately, optional linker sequences; 
 
       wherein the DNA construct further comprises a polyadenylation signal at the 3′ (downstream) end of Q and a splice acceptor site located 5′ (upstream) of Q. 
     
     
         7 . A method according to  claim 6  where the heterologous gene coding sequence is randomly inserted into an endogenous gene so that transcription of the heterologous gene coding sequence is directed by the host regulatory elements of the endogenous gene. 
     
     
         8 . A method according to  claim 6  in which the splice acceptor permits functional integration of the heterologous gene coding sequence into an intron sequence. 
     
     
         9 . A method according to  claim 6  further comprising the step of identifying cells expressing the heterologous gene coding sequence. 
     
     
         10 . A method according to  claim 9  wherein the construct also comprises a gene encoding a selectable marker and the method comprises selecting cells that express the selectable marker. 
     
     
         11 . A mouse embryonic stem cell comprising a heterologous gene coding sequence inserted by the method of  claim 6 . 
     
     
         12 . A descendant of the mouse embryonic stem cell according to  claim 11 , wherein the descendant has inherited the inserted heterologous gene coding sequence. 
     
     
         13 . A mouse comprising a cell according to  claim 12 . 
     
     
         14 . A descendant of a mouse according to  claim 13 , wherein the descendant has inherited the inserted heterologous gene coding sequence. 
     
     
         15 . A DNA construct comprising the sequence:
 5′ A-P-B-Q-C 3′   
       in which
 P is an internal ribosome entry site (IRES); 
 Q is a heterologous gene sequence; and 
 A, B and C are, separately, optional linker sequences; 
 
       wherein the DNA construct further comprises a polyadenylation signal at the 3′ (downstream) end of Q and a splice acceptor site located 5′ (upstream) of Q. 
     
     
         16 . A DNA construct according to  claim 15  in which the splice acceptor permits functional integration of the heterologous gene into an intron sequence. 
     
     
         17 . A DNA construct according to  claim 15  in which the construct also comprises a gene encoding a selectable marker to facilitate selection of cells containing a heterologous gene that has been inserted into an endogenous gene. 
     
     
         18 . A method according to  claim 6 , wherein the construct also comprises a gene encoding antibiotic resistance, and the method comprises selecting cells that express the antibiotic resistance. 
     
     
         19 . A DNA construct according to  claim 15 , wherein the construct additionally comprises a gene encoding antibiotic resistance. 
     
     
         20 . A mouse comprising a heterologous gene coding sequence inserted by the method of  claim 1 . 
     
     
         21 . A descendant of the mouse according to  claim 20 , wherein the descendant has inherited the inserted heterologous gene coding sequence. 
     
     
         22 . A cell comprising a heterologous gene coding sequence inserted according to the method of  claim 15 . 
     
     
         23 . A descendant of the cell according to  claim 22 , wherein the descendant has inherited the inserted heterologous gene coding sequence. 
     
     
         24 . A mouse comprising a heterologous gene coding sequence inserted according to the method of  claim 15 . 
     
     
         25 . A descendant of the mouse according to  claim 24 , wherein the descendant has inherited the inserted heterologous gene coding sequence. 
     
     
         26 . A cell comprising an inserted heterologous gene coding sequence inserted into a target endogenous gene in a eukaryotic cellular host cell genome by transforming the host cell with a vector comprising a DNA construct, wherein the DNA construct comprises the elements:
 5′ X-A-P-B-Q-C-Y 3′   
       in which
 X and Y are substantially homologous with separate sequences from the target endogenous gene and are of sufficient length to undergo homologous recombination with the host cell genome so as to insert the A-P-B-Q-C elements into the host cell genome; 
 P is an internal ribosome entry site (IRES); 
 Q is the heterologous gene coding sequence; and 
 A, B, and C are, separately, linker sequence or a covalent bond. 
 
     
     
         27 . A descendant of the cell of  claim 26 , wherein the descendant has inherited the inserted heterologous gene coding sequence. 
     
     
         28 . A mouse comprising an inserted heterologous gene coding sequence inserted into a target endogenous gene in a eukaryotic cellular host cell genome by transforming the host cell with a vector comprising a DNA construct, wherein the DNA construct comprises the elements:
 5′ X-A-P-B-Q-C-Y 3′   
       in which
 X and Y are substantially homologouse with separate sequences from the target endogenous gene and are of sufficient length to undergo homologous recombination with the host cell genome so as to insert the A-P-B-Q-C elements in to the host cell genome; 
 P is an internal ribosome entry site (IRES); 
 Q is the heterologous gene coding sequence; and 
 A, B, and C are, separately, linker sequence or a covalent bond. 
 
     
     
         29 . A descendant of the mouse of  claim 28 , wherein the descendant has inherited the inserted heterologous gene coding sequence. 
     
     
         30 . A cell according to  claim 26 , wherein the construct also comprises a gene encoding a selectable marker. 
     
     
         31 . A mouse according to  claim 28 , wherein the construct also comprises a gene encoding a selectable marker.